Mutations at Codon 641 of Exon 16 of EZH2 in Circulating Tumor DNA: A Biomarker for Relapse in Patients With Diffuse Large B-cell Lymphoma Treated With the R-CHOP Scheme.

Background: The epigenetic regulator EZH2 is a subunit of the polycomb repressive complex 2 (PRC2), methylates H3K27, resulting in transcriptional silencing. The mutation at Y646 amino acid in the EZH2 gene is mutated in up to 40 % of B-cell lymphomas. Methods: We compared the presence of exon 16 EZH2 mutations in tumor samples and ctDNA in a prospective trial. The mutations were determined by sanger sequencing, and by ddPCR. We also evaluated the impact of these mutations on response, relapse, and survival. Results: One hundred and thirty-eight cases were included. Ninety-eight were germinal center, and twenty had EZH2 mutations. Mean follow-up (IQR 25-75) was 23 (7- 42) months. The tumor samples were considered the standard of reference. Considering the results of the mutation in ctDNA by Sanger sequencing, the sensibility (Se) and specificity (Sp) were 52 % and 99 %, respectively. After adding the ddPCR analysis, the Se and Sp increased to 95 and 100 %, respectively. After bivariate analysis, only the presence of double-hit lymphoma (p=0.04), or EZH2 mutations were associated with relapse. The median PFS (95 % Interval confidence) was 27.7 (95 % IC: 14-40) vs 44.1 (95 % IC: 40-47.6) months for the mutated vs wt patients. Conclusions: The ctDNA is usefull to analyse EZH2 mutations, which have an impact in PFS.

Double-hit lymphoma: optimizing therapy

Aggressive B-cell lymphoma is a heterogeneous entity with disparate outcomes based on clinical and pathological characteristics. While most tumors in this category are diffuse large B-cell lymphoma (DLBCL), the recognition that some cases have high-grade morphology and frequently harbor MYC and BCL2 and/or BCL6 translocations has led to their separate categorization. These cases are now considered distinct from DLBCL and are named “high-grade B-cell lymphoma” (HGBL). Most are characterized by distinct rearrangements, but others have high-grade morphological features without these and are called HGBL-not otherwise specified. Studies have demonstrated that this group of diseases leads to poor outcomes following standard rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone therapy; retrospective and recent single-arm, multicenter studies suggest they should be approached with dose-intense treatment platforms. As yet, this has not been validated in randomized trial settings due to the rarity of these diseases. In the relapsed and refractory setting, novel approaches such as anti-CD19 chimeric antigen receptor T cells and antibodies against CD19 have demonstrated high efficacy in this subgroup. Recently, genomic studies have made much progress in investigating some of the molecular underpinnings that drive their lymphomagenesis and have paved the way for testing additional novel approaches.

Primary breast double-hit lymphoma management and outcomes: a real-world multicentre experience

Background Primary breast double-hit lymphoma (PB-DHL) is a rare, highly aggressive malignancy that poses challenges regarding accurate diagnosis and selecting optimal treatment regimens. Methods We retrospectively reviewed 48 cases of patients diagnosed with PB-DHL in six academic centres between June 2014 and June 2020 in China. Study-specific data were recorded, including treatment options, therapeutic evaluation, prognostic factors and relapse patterns, and the overall survival (OS) and progression-free survival (PFS) were evaluated. Results In total, 48 patients were enrolled, with 14 patients treated with DA-EPOCH-R/MA (rituximab, dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, alternating with high-dose methotrexate and cytarabine), 18 patients treated with DA-EPOCH-R (rituximab, dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin), and 16 patients treated with R-HyperCVAD (rituximab, hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, alternating with cytarabine plus methotrexate). The overall 5-year OS and PFS rates were 41.7% (95% confidence interval [CI], 27.6–56.8%) and 37.5% (95% CI, 24.0–52.6%), respectively. Of the three treatment regimens, the 5-year OS was higher in DA-EPOCH-R/MA group than in the DA-EPOCH-R or R-HyperCVAD subgroups (57.1% vs. 38.9% vs. 31.3%; P = 0.016), as was the 5-year PFS (50.0% vs. 38.9% vs. 25.0%; P = 0.035). Autologous stem cell transplantation (ASCT) prolonged the OS and PFS compared with non-ASCT patients (5-year OS: 72.2% vs. 23.3%; P < 0.001; 5-year PFS: 72.2% vs. 16.7 %, P < 0.001). Multivariate analysis identified tumour size, risk stratification, treatment with DA-EPOCH-R/MA, breast irradiation, and ASCT as significant prognostic factors. Conclusions DA-EPOCH-R/MA is a promising regimen for PB-DHL, and breast irradiation yields complementary benefits for prognosis. ASCT significantly decreased disease relapse, providing a potential curative PB-DHL intervention and justifying ASCT as first-line therapy for young patients. More effective treatment strategies for PB-DHL patients remain encouraging.

A novel CRISPR activation mouse enables modelling of aggressive lymphoma and interrogation of venetoclax resistance.

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been thoroughly exploited in this context. Here we establish a CRISPRa mouse (dCas9a-SAMKI/+) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI/KI primary lymphocytes, we induced B cell restricted genes in the T cell lineage and vice versa, demonstrating the power of this system. Next, to model double hit lymphoma (DHL), we transactivated pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Lethally-irradiated mice transplanted with these cells rapidly developed lymphomas expressing high BCL-2. Unlike standard Eµ-Myc lymphomas, BCL-2-expressing lymphomas were highly sensitive to the BCL-2 inhibitor venetoclax. Finally, we performed genome-wide activation screens in these lymphoma cells and found a dominant role for the BCL-2 family protein A1 in venetoclax resistance. This demonstrates the power of our CRISPRa model for mimicking disease and provides insights into potential resistance mechanisms towards targeted therapies.

Primary Breast Double-hit Lymphoma Management and Outcomes: A Real-world Multicentre Experience

Background: Primary breast double-hit lymphoma (PB-DHL) is a rare, highly aggressive malignancy that poses challenges regarding accurate diagnoses and selecting optimal treatment regimens. Methods: We retrospectively reviewed cases of patients diagnosed with PB-DHL in six academic centres between June 2014 and June 2020 in China. Study-specific data were recorded, including treatment options, therapeutic evaluation, prognostic factors and relapse patterns, and the overall survival (OS) and progression-free survival (PFS) were evaluated. Results: In total, 48 patients were enrolled, and the overall five-year OS and PFS rates were 41.7% (95% confidence interval [CI], 22.4–64.7%) and 37.5% (95% CI, 23.7–58.6%), respectively. Of the three treatment regimens, the five-year OS was higher in the dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R)/alternating with high-dose methotrexate and cytarabine (MA) group than in the DA-EPOCH-R or rituximab plus fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with cytarabine plus methotrexate (R-HyperCVAD) subgroups (57.1% vs. 38.9% vs. 31.3%; P = 0.016), as was the five-year PFS (50.0% vs. 38.9% vs. 25.0%; P = 0.038). Autologous stem cell transplantation (ASCT) prolonged the OS and PFS compared with non-ASCT patients (five-year OS: 72.2% vs. 23.3%; P < 0.001; five-year PFS: 72.2% vs. 16.7%, P < 0.001). Multivariate analysis identified tumour size, risk stratification, treatment with DA-EPOCH-R/MA, breast irradiation, and ASCT as significant prognostic factors. Conclusions: DA-EPOCH-R/MA is a promising regimen for PB-DHL, and breast irradiation yields complementary benefits for relapse reduction. ASCT significantly decreased disease relapse, providing a potential curative PB-DHL intervention and justifying ASCT as first-line therapy for young patients. More effective treatment strategies for PB-DHL patients remain encouraging.