Germinal Center B-cell Subtype Research Articles

Genomic Stability, DNA Repair and the SKP1-CUL1-F-Box Complex in HIV Associated Diffuse Large B-Cell Lymphoma

Introduction: Previously we demonstrated that HIV associated diffuse large B-cell lymphomas (DLBCL) of Germinal Center B-cell subtype (GCB) are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes, particularly Fanconi anemia (FA) and homologous recombination (HR) genes compared to their HIV negative counterparts (Maguire et al, 2019). It is unknown if this signature is unique to the GCB subtype or if it is also observed in Activated B-cell (ABC) and Unclassified (UNC) DLBCL. Herein we validate our HIV associated GCB DLBCL findings in a larger independent cohort and report for the first time the aberrational profile of molecularly profiled HIV associated ABC DLBCL compared to their HIV negative counterparts respectively. Methods: An independent cohort of 353 DLBCL FFPE cases was assembled, including 125 HIV-DLBCL samples. All samples underwent hematopathological review to confirm diagnosis and determine tumor content. Samples with <60% tumor content were macro-dissected before they were RNA/DNA extracted. Cell of origin (COO) profiles were assessed using the Lymph3Cx molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) like from GCB, ABC and UNC DLBCL (Mottok et al 2018). Copy number (CN) analyses were performed on samples with sufficient DNA (41 HIV neg, 32 HIV pos) using the OncoScan FFPE assay kit and the resulting data analyzed using Chromosome Analysis Suite (ChAS) v4.1 and the multisample viewer module of Reproductive Health Research Analysis Software (RHAS) v1.1. Additional digital gene expression profiling was performed using the Pan-Cancer panel (NanoString, Seattle, WA) customized to include 9 BCL2 family genes for a total of 739 genes and analyzed using the NanoString nSolver Advanced Analysis module. Results: GCB, ABC, UNC and PMBL-like proportions were 50%, 30%, 14% and 6% in HIV unassociated (HIV neg) and 50%, 23%, 25% and 2% in HIV pos tumors respectively, confirming both cohorts were GCB-enriched by molecular profiling. PMBL-like cases were excluded from further analyses. OncoScan CN analyses showed that HIV pos DLBCL were significantly less aberrant than HIV neg DLBCL (median CN per sample 0.56 vs 1.01, p<0.001). HIV pos GCB tumors were significantly less aberrant than both HIV neg GCB tumors (median CN per sample 0.50 vs 1.11, p<0.001), and HIV pos ABC tumors (median CN per sample 0.50 vs 0.81, p=0.009). HIV pos ABC tumors were also less aberrant than HIV neg ABC tumors, however, the difference was not significant overall (median CN per sample 0.81 vs 1.03, p=0.162) or for gain/amplifications (median CN per sample 0.38 vs 0.42, p=0.814), but was borderline significant for loss/deletions (median CN per sample 0.31 vs 0.47, p=0.06). GEP analyses identified Cell Cycle and DNA repair as the top two upregulated pathways in HIV pos GCB tumors compared to HIV neg GCB tumors. In contrast, Cell Cycle and DNA repair were downregulated in HIV pos ABC tumors compared to HIV neg ABC tumors. However, one gene ( RBX1) was identified as significantly upregulated in both pathways in HIV pos GCB (adjusted p-value 0.0215) and HIV pos ABC (adjusted p-value 0.0046) tumors compared to their HIV neg counterparts. A second gene ( SKP1) was also significantly upregulated in HIV pos GCB (adjusted p-value 3.94E-5) and HIV pos ABC (adjusted p-value 0.0031) tumors. Together RBX1 and SKP1 encode the two principal functional subunits of the SKP1, CUL1, F-box protein (SCF) complex which is part of the ubiquitin proteasome system (UPS) and has been reported as essential for maintaining genome and chromosome stability (Thompson et al 2021). Conclusions: These results demonstrate that HIV pos DLBCL tumors are less aberrant than their HIV neg counterparts irrespective of COO subtype. However, the degree of genomic stability differs by COO, with HIV pos GCB tumors being more genomically stable than HIV pos ABC tumors. Although both have enhanced expression of UPS related SCF genes ( RBX1, SKP1), only HIV pos GCB tumors have enhanced expression of key DNA repair genes as well including Fanconi Anemia ( BRCA2/FANCD1, FANCL) and mismatch repair ( MSH2, MSH6) genes (adjusted p-values of ≤ 0.0263). Given the reported role of SCF in genomic stability maintenance, the HIV pos COO CN differences could be due to the contributions of both DNA repair and SCF mechanisms in HIV pos GCB and mainly SCF mechanisms in HIV pos ABC tumors.

Genetic Mutations in Primary Refractory Diffuse Large B-Cell Lymphoma: Next Generation Sequencing Analysis

Introduction Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of newly diagnosed adult non-Hodgkin lymphoma. About 60-70% of DLBCL patients could be cured with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), while the other patients show chemo-refractory or relapse of disease and have poor prognosis. The prognostic difference between DLBCL patients is not fully understood, and many attempts to classify DLBCL have been made. Recently, with the development of the next generation sequencing (NGS) technique, Schmitz et al. identified four prominent genetic subtypes named MCD, BN2, N1, EZB. In this study, we performed targeted deep sequencing in DLBCL patients and analyzed whether there is any genetic difference between the patients who are primary refractory disease and the patients who are not. Methods Totally, 77 patients who was diagnosed with DLBCL and treated with R-CHOP from January 2021 to April 2023 were included in this analysis. Formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained at diagnosis were archived. Genomic DNA was extracted using QIAmp DNA FFPE tissue kit according to the manufacturer's protocol. Genetic profiling included the targeted deep sequencing of 424 genes, which had been selected based on recurrent mutations from previous studies, and sequenced using an Illumina Nextseq. The threshold of mutation positivity as a variant allele frequency ≥2% for SNV/Indel and as a copy number ≥4.0(gain) for CNV. Patients were assigned as germinal center B-cell (GCB) or non-GCB subtype based on the immunohistochemistry (IHC) algorithm. BCL-2 and MYC FISH were conducted in patients with double-expressor DLBCL with non-GCB subtype. Results Median age of the patients were 66 years (24-86 years), and 49 patients (63.6%) were male. Forty-five patients (58.5%) were advanced stage. Twenty-four patients (31.2%) were GCB subtype and 51 patients (66.2%) were non-GCB subtype. Among the GCB type DLBCL patients, 10 patients were double expressor DLBCL, and there was no case of double hit lymphoma. As a result of targeted deep sequencing, 317 mutations were found in at least 1 patient, and the patients had a median number of 16 mutations (4-50). KMT2D was the most commonly detected mutation (22/77, 29.3%), followed by MYD88 (19/77, 25.3%), PCLO (19/77, 25.3%), PIM1 (19/77, 25.3%), TP53 (18/77, 24.0%). According to NGS analysis, 26 patients (33.85%) were assigned to MCD subgroup, 13 patients (16.9%) to BN2, 11 patients (14.3%) to N1, 10 patients (13.0%) to EZB, and 17 patients (17%) were not assigned to any subrgroup. Median follow-up of the patients was 13.8 months (0.4-22.8 months), and 12 patients (15.6%) relapsed and 5 patients (6.5%) died. Median PFS and OS were not reached. Among relapsed patients, 7 patients were primary refractory to R-CHOP. In primary refractory DLBCL patients, TP53 (3/7, 42.9%) and CIITA (3/7, 42.9%) were most commonly detected. CIITA is known to be associated with immune escape and treatment resistance. CIITA, RASSF54, STAT6, SYK significantly more mutated in the primary refractory patients than the other ( CIITA, 42.9% vs. 8.6%, p=0.031; RASSF54, 28.6% vs. 2.9%, p=0.039; STAT6, 28.6% vs. 1.4%, p=0.021; SYK, 28.6% vs. 2.9%, p=0.039) (Table 1). Conclusion CIITA, RASSF54, STAT6, SYK were frequently mutated in primary refractory DLBCL in this study. Because of the small sample size, however, the prognostic significance of these mutations needed to be studied in further researches.

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.

The Outcome of Tisagenlecleucel (Tisa-Cel) Vs Axicabtagene Ciloleucel (Axi-Cel) CD-19 CAR-T Cell Therapy in Relapsed/Refractory NHL, Real World Data, Single Institution Experience

ABSTRACT Background: In the Kingdom of Saudi Arabia, non-Hodgkin lymphoma (NHL) reported as 6.1%. The estimated age adjusted incidence is 6/100,000. The more advanced disease stage at presentation reaches up to 40% stage IV. The cure rate with first line of therapy (R-CHOP) reaches up to 70%. The CD 19 chimeric antigen receptor (CAR) T-cell therapy targets CD19-expressing B cells and shows efficacy against B-cell lymphomas. Objective: The registration of CAR-T therapy products at KSA was in 2020 for Tisa-Cel and 2022 for Axi-Cel. The objective is to describe the outcome of the more available CAR-T therapy products in relapsed or refractory NHL. Methods: Retrospective analysis of CAR-T therapy outcome in NHL and compare the outcome of the two products for the same patient's population, using patients' chart electronic file assessment in the tertiary institution treating over 500 new lymphoma per year. Patients: Between November 2020 and May 2023, 44 patients with relapsed or refractory NHL to ≥2 lines of therapy, Follicular lymphoma (FL) relapsed after three lines of therapy counted for four patients treated with Axi-Cel. All patients received an infusion were included in the analysis. Of the 44 patients, 54.5% were male (n=24), the median age was 51 (20-75), and ECOG was 0-3. NHL subtype includes germinal center B-cell (GCB) 52% (n=23) and activated B-cell (ABC) 38.6% (n=17); the disease stage by PET/CT scan was III (47.7%, n=21) and IV (47.7%, n=21). In Axi-Cel group, 9.1% (n=4) received CAR-T as second line for relapse refractory disease or relapse within 12 months of complete remission (CR) after 1 st line therapy. Other patients received at least two lines of therapy prior to CAR-T infusion; 65.9% received ≥ 3 lines of therapy. Seven patients (16%) relapsed after autologous stem cell transplantation (ASCT) have received CAR-T therapy Tica-Cel product (n=6). Four patients (9.1%) have controlled CNS involvement at the infusion time. All patients received lymphodepletion (LD) therapy with fludarabine and cyclophosphamide (Flu/Cy). 25% (n=11) needed bridging chemotherapy therapy before LD, radiation therapy (XRT) bridging was counted for 13 patients (29.5%). The patients' characteristics and disease in Table-1. Results: Overall response rate (ORR), complete responses (CR), partial responses (PR). The respective toxicities include cytokine release syndrome (CRS) and neurologic events namely ICANS. The best ORR was 68.2%; 43.2% of the patients had CR and 25% had PR. The disease progression (DP) was 29.5%. In Axi-Cel group, ORR, CR, PR, and DP was 84.2%, 52.6%, 31.6% and 10.5, respectively. In Tica-Cel group; ORR, CR, PR, and DP was 56%, 36%, 20% and 44%, respectively. The estimated 2 years overall survival was 75% in all patients; 89.5% and 64% in Axi-Cel and Tica-Cel, respectively (p=0.47), Figure1. The estimated overall progressive free survival (PFS) was 61.4% with 73.7% in Axi-Cel group and 52% in Tica-Cel group (p=0.2), Figure2, the best PFS was seen in GCB subtype 65.2% vs 47.1% in ABC, (p= 0.05), Figure4. In FL, PFS was 100%, the patient number is small. PFS was 62.5% in-patient with CRS compared with 50% on non-CRS patient, not statistically significant (p= 0.7), Figure3. The main toxicities post CAR-T cell infusion including cytokines release syndrome (CRS) was 90.9% with 9% grade ≥ 3. In Tica-Cel, grade CRS-IV was 9% while 0% in Axi-Cel. Neurotoxicity (NT)-ICANS was 27% with grade ≥ 3 of 13.6% in both groups (20% in Tica-Cel and 5.6% in Axi-Cel). Tocilizumab was used in 72.7% in all cohorts with higher number of patients in Axi-Cel 17/19 (98.5%). Dexamethasone (Dexa) was used in CRS in 36.4%, in Axi-Cel; 42% of patients received Dexa while 32% in Tica-Cel group. In addition, for treatment of ICANS; Dexa was needed in 31.6% of Axi-Cel patients and 20% in Tica-Cel group. Upon the latest patient's follow-up, two patients (10.5%) in Axi-Cel group and 36% (n=9) in Tica-Cel group died from DP. The outcome of both products is summarized in Table-2. Conclusions: In this single-center retrospective analysis of CAR T-cell therapy in relapsed or refractory NHL or FL in adults, the rates of durable responses were like the real-world data. The best response rate was higher in Axi-Cel group; however, the follow-up duration of disease response is shorter in Axi-Cel group. The toxicity profile is similar in both groups with a bit higher of Grade-III-IV ICANS in Tica-Cel group. Keywords NHL, FL, CAR-T, CRS, ICANS

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.

Immunohistochemical Tumor Expression Predicts Response to CD3xCD20 Bispecific Antibodies in Patients with Relapsed/Refractory B Cell Lymphoma

Introduction Several studies show that CD3xCD20 bispecific antibodies show promising results in relapsed or refractory B-cell lymphomas and may have a milder adverse effect profile compared to chimeric antigen receptor T cell therapy (CAR-T). Tumor loss of antibody target (i.e. CD19 or PD1) have shown to negatively affect treatment outcomes in CD19-CAR-T cell therapy in DLBCL, and anti-PD1 therapy in Hodgkin lymphoma. It has, to our knowledge, not been reported how loss of CD20 expression and other immunohistochemical (IHC) expression patterns affects treatment outcomes in patients treated with CD3xCD20 antibodies. Methods We collected data on all patients in Denmark who received CD3xCD20 bispecific antibodies from 2017 to 2023. Electronic health records were assessed for general baseline characteristics and response according to Lugano criteria. Pathology reports on pre- and post-CD3xCD20 therapy lymphoma samples were collected and assessed on IHC analysis of CD20, Ki67, CD10, Bcl2, Bcl6, MUM1, germinal center B-cell (GCB) subtype and non-germinal center B-cell subtypes (non-GCB) according to Hans algorithm, as well as reports on cytogenetic FISH analysis regarding MYC with BCL2 and/or BCL6 rearrangements. To determine whether an expression was weak, negative, or strong the assessment of the original pathology reports was used. Results Pre-treatment IHC assessments were available in 122 patients. These were distributed across 81 patients with DLBCL, 32 with FL and 9 with other lymphoid neoplasms (MZL, HL, CLL). Of these, 109 had a strong CD20 expression, 10 had a weak CD20 expression, while 3 had no expression of CD20. Patients with a weak CD20 expression before treatment had an overall survival (OS) rate after 12 months at 40% (95% CI, 9.5-70) compared to 69% in patients with strong CD20 expression (95% CI, 45-61) (Figure 1, p=0.08). In a univariable Cox regression, we found that negative CD20 expression and higher Ki67 was significantly associated with inferior OS (Table 1, respectively HR=2.11 and HR=1.02 (per %-change in Ki67)). Worse performance status and a higher number of previous lines of treatment were also significantly associated with inferior overall survival (respectively HR=1.85 and HR=1.20). Surprisingly, BCL6 rearrangement was significantly associated with superior OS (HR=0.46). In a multivariable cox regression using OS as the endpoint, weak and negative IHC expression of CD20 was significantly associated with inferior OS (respectively HR=3.29, 95% CI 1.32-8.23, p=0.011 and HR=6.79 95% CI 1.25-36.76, p=0.026). Positive BCL6 compared to negative was associated with increased OS (HR =0.48, 95% CI 0.24 - 0.94, p=0.032). Analyzing only patients with DLBCL (n=81), weak and negative IHC expression of CD20 were the only factors to remain significantly associated with inferior overall survival (respectively HR=3.01, 95% CI 1.10-8.28, p=0.032 and HR=7.26, 95% CI 1.35-43.35, p=0.026). BCL6 rearrangement was associated with an increased overall survival, however only borderline statistically significant (HR=0.53, 95% CI 0.26-1.08, p=0.082). With regards to treatment response weak CD20 expression, worse performance status, higher Ki67 and a higher number of prior lines of treatment was in univariable analysis significantly associated with not being in a complete remission (CR) (respectively HR=2.28, HR=1.73, HR=1.01(per %-change in Ki67), HR=1.23 (per prior line of treatment). In a multivariable cox regression only weak and negative CD20 expression remained significantly associated with not being in CR (respectively HR=2.40, 95% CI 1.1-5.25, p=0.028 and HR=5.7 95% CI 1.14-28.58, p=0.034). Of the 48 patients who had biopsy-confirmed relapse, 29 had a weak or negative IHC CD20 expression (24 with a negative expression and 5 with a weak expression). Of these 29 patients, 24 had a strong pre-treatment IHC CD20 expression. Conclusions To our knowledge, we here present the first long-term analysis of the prognostic effect of tumor immunohistochemistry properties in patients treated with CD3xCD20 bispecific antibodies. We find that CD20 expression before treatment is an important prognostic factor, as patients with lymphomas with weak CD20 expression yield significantly poorer responses and decreased overall survival. Additionally, we find that BCL6 rearrangement is associated with superior overall survival in patients treated with CD3XCD20 bispecific antibodies.

YAP1-Dependent Promotion of Downstream Transcription through BRD4 Mediates Selinexor Resistance in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent subtype of non-Hodgkin lymphoma. Despite advancements in treatment, the prognosis for relapsed and/or refractory DLBCL (R/R DLBCL) patients remains unfavorable due to the development of drug resistance and disease progression. The pressing need to enhance the outcomes in R/R DLBCL patients necessitates ongoing investigations into novel therapeutic approaches. Among various exportins, the nuclear export protein exportin 1 (XPO1) solely governs the transportation of nuclear proteins harboring leucine-rich hydrophobic nuclear export signals (NES), including essential regulators like p53, RB1, and p27. Selinexor, an oral inhibitor specifically targeting XPO1, has emerged as a promising candidate in R/R DLBCL patients. Encouraging findings from a phase II clinical trial demonstrate a more favorable efficacy of Selinexor in the germinal center B-cell (GCB) subtype compared with that in other subtypes of DLBCL. Nonetheless, the clinical implementation of Selinexor has led to the emergence of acquired resistance, posing a significant challenge for medical practitioners. Efforts to overcome this resistance and establish strategies to maximize Selinexor's therapeutic potential remain of paramount importance in addressing the urgent clinical needs of R/R DLBCL patients. Methods: Characterization of Selinexor resistance targets was performed using a CRISPR/Cas9 screen transcription factors library pool at 0-day and 14-day time points after treatment. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. Synergistic treatment approach utilizing BRD4 inhibitor JQ1, known for its curative effects in AML, MM, and ALL, in combination with Selinexor were also evaluated. Mechanistic exploration of BRD4's role in Selinexor resistance was further analyzed via RNA-seq, ChIP-seq, as well as luciferase reporter experiments. Therapeutic outcomes were evaluated in R/R DLBCL patients undergoing Selinexor treatment via PET/CT imaging. Results ： BRD4, BRD7, ZMYND8, and YTHDC1 were identified as top Selinexor resistance candidate targets through CRISPR/Cas9 Screen. The knock-down of BRD4 enhanced the killing ability of Selinexor in vitro and reduced tumor burden in subcutaneously transplanted tumor mice after treatment with Selinexor in vivo in DLBCL. The combined treatment with BRD4 inhibitor JQ1 and Selinexor had a stronger ability to promote cell apoptosis, enhance DNA damage and reduce nascent DNA compared with the single drug group. Hematological and organ toxicity tests confirmed an acceptable toxicity for the combined drug regimen. ChIP-seq differential peaks and RNA-seq differential genes analysis elucidated the crucial role of YAP1's sub-cellular localization under Selinexor treatment. YAP1 nuclear translocation enhanced transcription and formed an interaction with BRD4, resulting in augmented BRD4 binding domain, and finally lead to the resistance of Selinexor. PET/CT imaging showed that one patient with high XPO1 expression achieved complete response (CR), exhibiting a sensitivity to Selinexor treatment, but the other exhibits resistance associated with a lack of XPO1 expression and BRD4-regulated acetylation. Conclusions: The nuclear translocation of YAP1 and its interaction with BRD4 mediated the transcriptional regulation and further induced resistance to Selinexor in DLBCL. BRD4 inhibitor JQ1 combined with low-dose Selinexor can mitigate blood toxicity and enhance therapeutic efficacy, achieving an improved outcome.

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.

Value of GCET1, HGAL (GCET2), and LMO2 in the Determination of Germinal Center Phenotype in Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a biologically heterogeneous disease that is classified into germinal center B-cell (GCB) and non-GCB subtypes, which are prognostically different. The Hans algorithm is the most widely used tool based on CD10, BCL6, and MUM1 expression, but some cases with the non-GCB phenotype are still known to be misclassified. In this study, we investigate the extent to which GCET1, HGAL, and LMO2 protein expressions reflect GCB phenotype together with their roles in determining the GCB phenotype of DLBCL and their contributions to the performance of the Hans algorithm. Sixty-five cases of DLBCL-not otherwise specified, 40 cases of follicular lymphoma (FL), and 19 non-GC-derived lymphoma cases were included in this study. The DLBCL cases were grouped as CD10+ (Group A) or only MUM1+ (Group B), and the remaining cases constituted the intermediate group (Group C). GCET1, HGAL, and LMO2 expressions were evaluated. In the FL group, GCET1, HGAL, and LMO2 were positive in 85%, 77.5%, and 100% of the cases, respectively. Among the non-GC-derived lymphoma cases, all three markers were negative in cases of small lymphocytic lymphoma, plasmablastic lymphoma, peripheral T-cell lymphoma, and anaplastic large cell lymphoma. GCET1 and HGAL were negative in cases of marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL). Two of the 3 MZL and 2 of the 4 MCL cases were positive for LMO2. In the DLBCL group, the number of cases with GCET1, HGAL, and LMO2 positivity was 18 (90%), 17 (85%), and 20 (100%), respectively, in Group A and 0 (0%), 2 (13.3%), and 2 (13.3%), respectively, in Group B. Considering these rates, when the cases in the intermediate group were evaluated, it was concluded that 13 cases typed as non-GCB according to the Hans algorithm may have the GCB phenotype. GCET1, HGAL, and LMO2 are highly sensitive markers for determining the germinal center cell phenotype and can increase the accuracy of the subclassification of DLBCL cases, especially for cases that are negative for CD10.

Non-germinal center B-cell subtype of pediatric diffuse large B-cell lymphoma in Japan: A retrospective cohort study.

Diffuse large B-cell lymphoma (DLBCL) is classified into two molecular subtypes according to its cell of origin: germinal center B-cell (GCB) subtype and activated B-cell/non-GCB subtype. This latter subtype shows a poorer prognosis in adults. However, in pediatric DLBCL, the prognostic impact of the subtype is yet to be clarified. This study sought to compare the prognosis between GCB and non-GCB DLBCL in a large number of cases in children and adolescents. In addition, this study intended to describe the clinical, immunohistochemical, and cytogenetic characteristics of these two molecular subtypes of DLBCL, and consider differences in the biology, frequency, and prognosis of GCB and non-GCB subtypes in pediatric versus adult DLBCL or in Japanese versus Western pediatric DLBCL patients. We selected mature B-cell lymphoma/leukemia patients for whom specimens had been submitted to the central pathology review in Japan between June 2005 and November 2019. We referred the past studies on Asian adult patients and Western pediatric patients to compare with our results. Data were obtained from 199 DLBCL patients. The median age of all patients was 10years, with 125 patients (62.8%) in the GCB group and 49 (24.6%) in the non-GCB group other than 25 cases whose immunohistochemical data were insufficient. Overall, the percentage of translocation of MYC (1.4%) and BCL6 (6.3%) was lower than in adult and Western pediatric DLBCL cases. The non-GCB group showed a significantly higher proportion of females (44.9%), a higher incidence of stage III disease (38.8%), and B-cell lymphoma 2 (BCL2)-positivity in immunohistochemistry (79.6%) compared to the GCB group; however, no BCL2 rearrangement was observed in both GCB and non-GCB groups. The prognosis did not differ significantly between the GCB and non-GCB groups. This study including a large number of non-GCB patients showed the same prognosis between GCB and non-GCB groups and suggested a difference in the biology of pediatric and adolescent DLBCL compared to adult DLBCL as well as between Asian and Western DLBCL.

Prevalence of IRF4 rearrangement in large B-cell lymphomas of the Waldeyer’s ring in adults

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin lymphoma (B-NHL) in adults. These lymphomas are classified according to gene expression profiling (GEP) into germinal center B-cell (GCB) and activated B-cell type (ABC). Recent studies have suggested new subtypes of large B-cell lymphoma, based on genetic and molecular alterations, among them is large B-cell lymphoma with IRF4-rearrangement (LBCL-IRF4). We used fluorescence in situ hybridization (FISH), GEP (using the DLBCL COO assay by HTG Molecular Inc), and next generation sequencing (NGS) to comprehensively characterize 30 cases of LBCLs located in Waldeyer’s ring in adult patients and to identify LBCL-IRF4. FISH revealed breaks of IRF4 in 2/30 cases (6.7%), BCL2 breaks in 6/30 cases (20.0%), and IGH breaks in 13/29 cases (44.8%). GEP classified 14 cases each as GCB or ABC subtype, and 2 cases remained unclassified; this was concordant with the immunohistochemistry (IHC) in 25/30 cases (83.3%). A subgrouping, based on GEP, was performed: group 1 included 14 GCB cases with the most frequent mutations in BCL2 and EZH2 in 6/14 cases (42.8%). The two cases with IRF4 rearrangement were assigned to this group by GEP and showed IRF4 mutations, supporting the diagnosis of LBCL-IRF4. Group 2 included 14 ABC cases; the most frequent mutations were CD79B and MYD88 identified in 5/14 patients (35.7%). Group 3 included 2 unclassifiable cases in which no molecular patterns were detected. Overall, LBCLs of Waldeyer’s ring in adult patients are a heterogeneous group, including LBCL-IRF4, which shares several features with cases in the pediatric population.

INCIDENTAL PRIMARY PULMONARY DIFFUSE LARGE B-CELL LYMPHOMA

INCIDENTAL PRIMARY PULMONARY DIFFUSE LARGE B-CELL LYMPHOMA

ABCL-523 Immunohistochemical (IHC) Expression of Epigenetic Regulators (EZH-2 and H3K27) as well as Cell Cycle Regulators (P16, P53 and RB) in Patients Diagnosed With Primary Diffuse Large B-Cell Lymphoma (DLBCL) and Association With Therapy Response Rates and Survival

Despite advancements in chemotherapeutics, survival rate in DLBCL remains 50% in developing countries. Transcriptomic profiling has shown that EZH-2 has a role in germinal center B-Cell (GCB) type of DLBCL, NOS. To evaluate IHC expression of epigenetic regulators and cell cycle regulators in DLBCL, NOS and correlate with cell of origin (GCB type and non-GCB type), therapy response and patient survival. A retrospective study of patients diagnosed with primary DLBCL, NOS in 2014-2015 who were followed-up until April 2022. DLBCL was classified by Han's Algorithm and IHC for EZH-2, H3K27, P16, P53, RB was done & scored semi-quantitatively. Various clinical parameters at baseline as well as treatment modalities given were also noted. Study conducted at a tertiary referral cancer care center on patients visiting the out-patient department. Patients were selected continuously from the out-patient records of the Department of Medical Oncology during 2014-2015. 37 patients were included, out of which follow-up data was available for 21 patients. Association between IHC expression and various clinical parameters as well as correlation with cell of origin, therapy response and patient survival. Out of 37 patients, 62.16% were male, with male to female ratio of 1.64. Age (48.6±14.1) ranged from 22-78 years. 15/37(40.5%) patients were diagnosed as DLBCL-GCB type. 18/37(48.6%) patients presented with at least one B-symptom. 20/37(54%) patients presented at higher stage of disease (3 or 4), while 17/37(46%) had extra-nodal involvement. Derived neutrophil-lymphocyte ratio (dNLR) was ≥ 2.2 in 13/37(35%) patients. Association of H3K27 with RB(p=0.024) and EZH-2 with P53(p=0.001) was found to be statistically significant. No other significant association was found between the various markers as well as with the cell of origin. However, association of H3K27 with response rate (RR)(p=0.012) and EZH-2 with RR(p=0.028) was found to be statistically significant. EZH-2 pathway has been described in GCB type of DLBCL. Though IHC expression contributed to a statistically significant difference in RR, the expression was not limited to GCB subtype as defined by Han's Algorithm. Therefore, further larger studies are required for validation of IHC based expression. AIIMS, Delhi.

Abstract 1755: IKS03, a CD19-targeted antibody drug conjugate with enhanced efficacy and tolerability for treatment of B-cell lymphomas

Abstract Background: Lymphoma is the most common hematological malignancy with non-Hodgkin B-cell lymphomas (NHL), including those with high unmet clinical need such as relapsed/refractory Diffuse Large B Cell Lymphoma (DLBCL), and Mantle-Cell Lymphoma (MCL), representing 90% of all lymphoma cases. CD19 is an attractive target for antibody-directed lymphoma therapies as it is expressed in most B cell malignancies, with normal tissue expression limited to B-cells thus reducing any on-target toxicity concerns. Antibody Drug Conjugates (ADCs) are the focus of intense interest as a means to provide selective tumor killing with increased efficacy and less toxicity than standard of care chemotherapies. IKS03 is an ADC comprised of an anti-CD19 antibody conjugated to a proprietary DNA-crosslinking PBD prodrug and includes a tumor-selective glucuronide-trigger technology for payload release and activation. ADC activity requires processing by beta-glucuronidase, a lysosomal enzyme often upregulated in tumor cells, while normal tissues with low levels of the enzyme are less able to process the ADC and are differentially spared. Methods: IKS03 was generated via chemo-enzymatic bioconjugation at defined sites on the antibody yielding an ADC with a drug to antibody ratio of 2. In vivo efficacy was evaluated in CD19-expressing cell line-derived xenograft models in mice including Farage (DLBCL, Germinal Centre B-cell subtype), OCI-LY10 (DLBCL, Activated B-Cell subtype), Granta-519 (MCL) and Ramos (Burkitt’s lymphoma). Activity was compared to benchmark ADCs known to have demonstrated clinical efficacy. Efficacy was also assessed in low passage DLBCL patient-derived xenograft models of known genomic profile. Toxicology studies were conducted in cynomolgus monkeys with immunophenotyping by flow cytometry included to quantify the level of normal B-cells following IKS03 administration. Results: IKS03 is highly effective in causing tumor regressions in DLBCL models at doses that are well tolerated. Complete regressions were observed with a single dose of 0.1 mg/kg in the Farage xenograft model. IKS03 is also highly active in the Granta-519 MCL model, with complete regressions observed with a single dose of 0.1 mg/kg. Tumor regression was observed in a high-grade triple-hit lymphoma PDX model with a single 0.3 mg/kg dose. IKS03 was tolerated in monkeys at the highest dose tested of 1.5 mg/kg (single dose). IKS03 cross reacts with monkey CD19 and reduced B-cell depletion was observed in relation to comparator agents, even at doses much greater (1.5 mg/kg) than that needed for complete responses in B-cell lymphoma mouse models (0.1 mg/kg). Conclusion: Preclinical data demonstrates that IKS03’s advanced ADC design results in an increased therapeutic margin compared to traditional ADCs with DNA-crosslinking payloads, with increased efficacy and decreased toxicity. Citation Format: Jenny Thirlway, Adam Lodge, Daniel J. Williamson, Justyna Mysliwy, Jutta Deckert, Xavier Chauchet, Laura Cons, Yun-Hee Park, Hyun-Min Ryu, Na Ra Han, Ho Young Song, Chul-Woong Chung, Robert J. Lutz. IKS03, a CD19-targeted antibody drug conjugate with enhanced efficacy and tolerability for treatment of B-cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1755.

The genetic deletion and protein expression of PRDM1 and its clinical implications in diffuse large B cell lymphoma: A retrospective cohort study in China

ObjectTo investigate the clinical implications of the PRDM1 deletion and PRDM1 protein expression in Chinese diffuse large B cell lymphoma (DLBCL). Materials and methodsTumor samples of 199 patients with DLBCL were obtained from the Department of Pathology of Peking University First Hospital between 2008 and 2015. The PRDM1 expression was detected by immunohistochemistry (IHC) in all samples. Among them, the PRDM1 deletion was detected in 60 samples by fluorescence in situ hybridization (FISH). The correlations between PRDM1 protein expression and PRDM1 molecular status and clinicopathological features were analyzed. ResultsImmunohistochemically, 58 (29.1%) patients were classified as the germinal center B-cell (GCB) subtype, and 141 (70.9%) patients were non-GCB subtype. PRDM1 protein was strongly expressed in 15 (7.5%) patients, weakly expressed in 67 (33.7%) patients, and negative in 117 (26.6%) patients. Heterozygous and homozygous PRDM1 deletions were observed in 28.3% (17/60) and 8.3% (5/60) of cases, respectively. The PRDM1 deletion was not significantly correlated with PRDM1 protein expression. Neither the PRDM1 protein expression nor the PRDM1 deletion was significantly associated with most clinicopathological features, including their immunophenotypes according to the Han's algorithm. However, Kaplan-Meier survival analysis showed that heterozygous and/or homozygous PRDM1 deletion but not PRDM1 expression was a poor prognostic factor in the non-GCB group. In addition, there was a positive correlation between PRDM1 and c-Myc expression. ConclusionsOur results suggested that homozygous or heterozygous PRDM1 deletion is a poor prognostic factor for non-GCB DLBCL.

Cell of Origin Based on Hans’ Algorithm as Prognostic Factor in Diffuse Large B-Cell Lymphoma: A Clinicopathologic and Survival Study

Background: Diffuse Large B-Cell Lymphoma (DLBCL) has been classified based on Cell of Origin (COO) into Germinal Center B-cell (GCB) and Non-Germinal Center B-cell (Non-GCB). The practical application of this concept by immunohistochemistry (IHC)-based subtyping with Hans’ algorithm and its role as a prognostic factor has been widely investigated with conflicting results. Moreover, the recent deeper molecular characterizations of DLBCL have been another challenges for this approach to remain as a prognostic factor. Objective: To determine the prognostic value of COO by IHC-based subtyping with Hans’ algorithm for DLBCL. Materials and Methods: This was a single-center retrospective analysis including DLBCL cases diagnosed in 2014-2019 from Dr. Sardjito Hospital, Yogyakarta, Indonesia. Hans’ algorithm with CD10, BCL6, and MUM1 were used. The two subtypes were compared in terms of clinicopathological features and 3 years overall survival. Results: Seventy patients were included in this study. Non-GCB was predominant than GCB 74.3% vs 25.7%, with the ratio of 2.8:1. No statistical difference in the clinicopathological features from both subtypes in terms of age, gender, Ann Arbor stadium, ECOG performance status, extra-nodal involvement, and LDH level. Ann Arbor stadium and ECOG showed significant prognostic value for patients outcome with multivariate analysis (OR 3.64; 95%CI 0.18-0.61; p=0.001) and (OR 2.68; 95%CI 0.11-0.77; p=0.009). Survival analysis based on COO did not show significant difference between GCB and non-GCB subtypes (log rank=0.46). Further analysis based on COO and the use of rituximab showed better OS in the two subtypes than without rituximab although not significant (log rank=0.67). Conclusion: The role of COO with Hans’ algorithm as prognostic factor in DLBCL is not confirmed in this study. Some related factors may be the addition of rituximab and the recent molecular characterization of DLBCL which covered a more subtle classification of the disease beyond GCB and non-GCB subtypes.

MUM1 Expression versus Hans Algorithm to Predict Prognosis in Indonesian Diffuse Large B-Cell Lymphoma Patients Receiving R-CHOP.

BackgroundTreatment response in diffuse large B-cell lymphoma (DLBCL) is heterogenous. The Hans algorithm (using 30% cut-offs for CD10, BCL6, and MUM1 protein expression) has been the most favored method to categorize DLBCL into germinal center B-cell (GCB) and non-GCB subtypes in order to predict prognosis. However, the algorithm’s ability to prognosticate is not always consistent.MethodsThis retrospective cohort study was conducted on DLBCL patients receiving R-CHOP therapy at Dr. Cipto Mangunkusumo Hospital, Jakarta from 2014 to 2017. We aimed to compare the prognostic value of Hans algorithm as well as the protein levels of CD10, BCL6, MUM1, and Ki67 at different cut-offs. Ninety-two patients were classified based on Hans algorithm and various proteins at different cut-off values were analyzed with regard to event-free survival at 24 months using survival analysis. The cut-off values were then compared using receiver operating characteristic curves.ResultsA significant survival difference was observed with MUM1 expression cut-off of 50% or more (log rank p = 0.035). CD10, BCL6, Ki67, and Hans algorithm showed AUCs below or near 0.5 (0.405, 0.436, 0.498, and 0.413, respectively), whereas MUM1 showed an AUC of 0.835, in predicting events within 24 months. MUM-1 cut-off of 70.5% yielded an optimal trade-off for sensitivity and specificity.ConclusionMUM1 expression of 50% or more can help predict prognosis in DLBCL patients receiving R-CHOP therapy and can be considered as for use as a single marker to predict prognosis.

Clinicopathological features and prognosis of high-grade B-cell lymphoma with MYC and bcl-2 and/or bcl-6 rearrangements

Objective: To investigate the clinicopathological characteristics and prognosis of high-grade B-cell lymphoma (HGBL) involving combined rearrangements of MYC, bcl-2 and bcl-6. Methods: A total of 1 138 cases of large B cell lymphoma (LBL) that were treated at the Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine from January 2017 to September 2020 were analyzed using fluorescence in situ hybridization (FISH) with probes against MYC, bcl-2 and bcl-6. The clinical and pathological data of the 45 patients with HGBL that had rearrangements of MYC and bcl-2 and/or bcl-6 were collected and retrospectively analyzed. Results: Among the 1 138 LBL, 45 (4.0%) cases had combined rearrangements of MYC, bcl-2 and/or bcl-6 that included 6 HGBL cases with MYC, bcl-2 and bcl-6 rearrangements, 14 HGBL cases with MYC and bcl-2 rearrangements, and 25 HGBL cases with MYC and bcl-6 rearrangements. Of these 45 patients, 29 patients were male, and 16 patients were female, aged 29 to 83 years. HGBL with MYC, bcl-2 and bcl-6 rearrangements and HGBL with MYC and bcl-2 rearrangement were reclassified as the germinal center B-cell (GCB) subtype using the Hans algorithm. HGBL with MYC and bcl-6 rearrangement were reclassified as the GCB subtype (68.0%) and the non-GCB subtype (32.0%). The vast majority of HGBL cases had a high Ki-67 proliferation index. Most HGBL patients had advanced stage disease with a high IPI score and an increased LDH level. Also, some patients had clinical features including elevated plasma β2-microglobulin levels, B symptoms, and bone marrow involvement. The IPI scores and LDH levels were significantly different between the HGBL cases with MYC, bcl-2 and bcl-6 rearrangements and the HGBL cases with MYC and bcl-6 rearrangements (P<0.05). Compared with the HGBL cases with MYC, bcl-2 and bcl-6 rearrangements, the HGBL cases with MYC and bcl-2 or bcl-6 rearrangements had a lower incidence of bone marrow involvement (P<0.05). There were no significant differences in the prognosis among HGBL cases with MYC, bcl-2 and bcl-6 rearrangements, the cases with MYC and bcl-2 rearrangements, and the cases with MYC and bcl-6 rearrangements (P>0.05). Conclusions: HGBL with MYC, bcl-2 and/or bcl-6 rearrangements are rare types of B-cell lymphoma with high degree of malignancy and have a short overall survival. To reduce misdiagnosis and improve diagnostic accuracy, it is necessary to assess the patients' clinical features and conduct histopathological, immunohistochemical and FISH analyses.

SGK1 mutation status can further stratify patients with germinal center B-cell-like diffuse large B-cell lymphoma into different prognostic subgroups.

There are over a 100 driver gene mutations in patients with diffuse large B‐cell lymphoma (DLBCL), but their clinical significance remains unclear. Here, we first analyzed the DLBCL dataset from the UK‐based Haematological Malignancy Research Network. Patients were divided into high‐ and low‐risk groups based on whether lymphoma progressed within 24 months. Genes showing significantly different frequencies between groups were selected. Survival data for patients with the selected mutant genes were analyzed. The results were validated using two other large databases to evaluate the relationship between the selected mutant genes and prognosis. The mutation frequencies of 11 genes (MYD88[L265P], SGK1, MPEG1, TP53, SPEN, NOTCH1, ETV6, TNFRSF14, MGA, CIITA, and PIM1) significantly differed between the high‐ and low‐risk groups. The relationships between these mutant genes and patient survival were analyzed. Patients who harbored SGK1 (serum and glucocorticoid‐inducible kinase 1) mutations exhibited the best prognosis. Most patients with SGK1 mutation are germinal center B‐cell (GCB) subtype. Among patients with GCB DLBCL, those harboring SGK1 mutations exhibited better prognosis than those without SGK1 mutations. Most SGK1 mutations were single‐base substitutions, primarily scattered throughout the catalytic domain‐encoding region. Multiple SGK1 mutations were identified in a single patient. Thus, SGK1 mutations are a marker of good prognosis for DLBCL and occur predominantly in the GCB subtype of DLBCL. SGK1 mutation status can further stratify patients with GCB DLBCL into different prognostic subgroups.

Immunohistochemical Characterisation of Cell of Origin in Diffuse Large B-cell Lymphoma and its Association with the Double Expressor Phenotype: A Retrospective Study

Introduction: Diffuse Large B-cell Lymphoma (DLBCL) is the most common type of Non Hodgkin Lymphoma (NHL) and is categorised into the Germinal Centre B-cell (GCB) and Activated B-cell (ABC/Non GCB) subtypes as per the Cell Of Origin (COO) model with the help of gene expression profiling/ immunohistochemistry. The non GCB subtype has been found to associate with the Double Expressor (DE) phenotype (i.e., co-expression of BCL-2 and c-myc by IHC and this association was substantiated and proven to be statistically significant in the present study. Aim: To categorise all DLBCL cases into GCB and Non GCB and further into DE and Non-DE by using Hans and Choi IHC alogrithm. Materials and Methods: A retrospective study of 50 patients was carried out with the help of archival material filed in the Department of Anatomic Pathology at Dharamshila Narayana Superspeciality Hospital, New Delhi, India from 1st January 2019 to 30th June 2020. The study was approved by Instituitional Ethics Committee (IEC). Chi-square test and Yates correction were used for statistical analysis. Results: The study cohort was divided into two Groups- group-A with nodal presentation and group-B with extranodal presentation. By using the Hans and Choi IHC algorithms, the cases were categorised into the GCB and non GCB subtypes in both the groups. The DE phenotype was determined in each case by the co-expression of c-myc (&gt;40%) and BCL-2 (&gt;50%) by IHC. By using the statistical chi-square test analysis and Yates correction, the association of DE was found to be statistically significant with non GCB type lymphomas and non DE with GCB lymphomas with p-value being 0.0005 and 0.00168, respectively. Conclusion: Due to the heterogeneity inherent in DLBCL, prediction of the DE phenotype and the COO by IHC is a sensitive tool and helps in the prognostication and therapeutic triage of patients. Hence, in all the cases diagnosed as DLBCL, a detailed morphological and IHC work up is mandatory to determine prognosis and possibly tailor therapy according to COO and DE phenotype.