High-risk Diffuse Large B-cell Lymphoma Patients Research Articles

A Bruton tyrosine kinase inhibitor-resistance gene signature predicts prognosis and identifies TRIP13 as a potential therapeutic target in diffuse large B-cell lymphoma

Bruton tyrosine kinase inhibitor (BTKi) combined with rituximab-based chemotherapy benefits diffuse large B-cell lymphoma (DLBCL) patients. However, drug resistance is the major cause of relapse and death of DLBCL. In this study, we conducted a comprehensive analysis BTKi-resistance related genes (BRRGs) and established a 10-gene (CARD16, TRIP13, PSRC1, CASP1, PLBD1, CARD6, CAPG, CACNA1A, CDH15, and NDUFA4) signature for early identifying high-risk DLBCL patients. The resistance scores based on the BRRGs signature were associated with prognosis. Furthermore, we developed a nomogram incorporating the BRRGs signature, which demonstrated excellent performance in predicting the prognosis of DLBCL patients. Notably, tumor immune microenvironment, biological pathways, and chemotherapy sensitivity were different between high- and low-resistance score groups. Additionally, we identified TRIP13 as a key gene in our model. TRIP13 was found to be overexpressed in DLBCL and BTKi-resistant DLBCL cell lines, knocking down TRIP13 suppresses cell proliferation, promotes cell apoptosis, and enhances the apoptosis effect of BTKi on DLBCL cells by regulating the Wnt/β-catenin pathway. In conclusion, our study presents a novel BRRGs signature that could serve as a promising prognostic marker in DLBCL, and TRIP13 might be a potential therapeutic target for resistant DLBCL.

Identify truly high-risk TP53-mutated diffuse large B cell lymphoma patients and explore the underlying biological mechanisms

TP53 mutation (TP53-mut) correlates with inferior survival in many cancers, whereas its prognostic role in diffuse large B-cell lymphoma (DLBCL) is still in controversy. Therefore, more precise risk stratification needs to be further explored for TP53-mut DLBCL patients. A set of 2637 DLBCL cases from multiple cohorts, was enrolled in our analysis. Among the 2637 DLBCL patients, 14.0% patients (370/2637) had TP53-mut. Since missense mutations account for the vast majority of TP53-mut DLBCL patients, and most non-missense mutations affect the function of the P53 protein, leading to worse survival rates, we distinguished patients with missense mutations. A TP53 missense mutation risk model was constructed based on a 150-combination machine learning computational framework, demonstrating excellent performance in predicting prognosis. Further analysis revealed that patients with high-risk missense mutations are significantly associated with early progression and exhibit dysregulation of multiple immune and metabolic pathways at the transcriptional level. Additionally, the high-risk group showed an absolutely suppressed immune microenvironment. To stratify the entire cohort of TP53-mut DLBCL, we combined clinical characteristics and ultimately constructed the TP53 Prognostic Index (TP53PI) model. In summary, we identified the truly high-risk TP53-mut DLBCL patients and explained this difference at the mutation and transcriptional levels.

Prognosis Prediction of Diffuse Large B-Cell Lymphoma in 18F-FDG PET Images Based on Multi-Deep-Learning Models.

Diffuse large B-cell lymphoma (DLBCL), a cancer of B cells, has been one of the most challenging and complicated diseases because of its considerable variation in clinical behavior, response to therapy, and prognosis. Radiomic features from medical images, such as PET images, have become one of the most valuable features for disease classification or prognosis prediction using learning-based methods. In this paper, a new flexible ensemble deep learning model is proposed for the prognosis prediction of the DLBCL in 18F-FDG PET images. This study proposes the multi-R-signature construction through selected pre-trained deep learning models for predicting progression-free survival (PFS) and overall survival (OS). The proposed method is trained and validated on two datasets from different imaging centers. Through analyzing and comparing the results, the prediction models, including Age, Ann abor stage, Bulky disease, SUVmax, TMTV, and multi-R-signature, achieve the almost best PFS prediction performance (C-index: 0.770, 95% CI: 0.705-0.834, with feature adding fusion method and C-index: 0.764, 95% CI: 0.695-0.832, with feature concatenate fusion method) and OS prediction (C-index: 0.770 (0.692-0.848) and 0.771 (0.694-0.849)) on the validation dataset. The developed multiparametric model could achieve accurate survival risk stratification of DLBCL patients. The outcomes of this study will be helpful for the early identification of high-risk DLBCL patients with refractory relapses and for guiding individualized treatment strategies.

PET-based radiomic feature based on the cross-combination method for predicting the mid-term efficacy and prognosis in high-risk diffuse large B-cell lymphoma patients.

This study aims to develop 7×7 machine-learning cross-combinatorial methods for selecting and classifying radiomic features used to construct Radiomics Score (RadScore) of predicting the mid-term efficacy and prognosis in high-risk patients with diffuse large B-cell lymphoma (DLBCL). Retrospectively, we recruited 177 high-risk DLBCL patients from two medical centers between October 2012 and September 2022 and randomly divided them into a training cohort (n=123) and a validation cohort (n=54). We finally extracted 110 radiomic features along with SUVmax, MTV, and TLG from the baseline PET. The 49 features selection-classification pairs were used to obtain the optimal LASSO-LASSO model with 11 key radiomic features for RadScore. Logistic regression was employed to identify independent RadScore, clinical and PET factors. These models were evaluated using receiver operating characteristic (ROC) curves and calibration curves. Decision curve analysis (DCA) was conducted to assess the predictive power of the models. The prognostic power of RadScore was assessed using cox regression (COX) and Kaplan-Meier plots (KM). 177 patients (mean age, 63 ± 13 years,129 men) were evaluated. Multivariate analyses showed that gender (OR,2.760; 95%CI:1.196,6.368); p=0.017), B symptoms (OR,4.065; 95%CI:1.837,8.955; p=0.001), SUVmax (OR,2.619; 95%CI:1.107,6.194; p=0.028), and RadScore (OR,7.167; 95%CI:2.815,18.248; p<0.001) independently contributed to the risk factors for predicting mid-term outcome. The AUC values of the combined models in the training and validation groups were 0.846 and 0.724 respectively, outperformed the clinical model (0.714;0.556), PET based model (0.664; 0.589), NCCN-IPI model (0.523;0.406) and IPI model (0.510;0.412) in predicting mid-term treatment outcome. DCA showed that the combined model incorporating RadScore, clinical risk factors, and PET metabolic metrics has optimal net clinical benefit. COX indicated that the high RadScore group had worse prognosis and survival in progression-free survival (PFS) (HR, 2.1737,95%CI: 1.2983, 3.6392) and overall survival (OS) (HR,2.1356,95%CI: 1.2561, 3.6309) compared to the low RadScore group. KM survival analysis also showed the same prognosis prediction as Cox results. The combined model incorporating RadScore, sex, B symptoms and SUVmax demonstrates a significant enhancement in predicting medium-term efficacy and prognosis in high-risk DLBCL patients. RadScore using 7×7 machine learning cross-combinatorial methods for selection and classification holds promise as a potential method for evaluating medium-term treatment outcome and prognosis in high-risk DLBCL patients.

Specific mortality in patients with diffuse large B-cell lymphoma: a retrospective analysis based on the surveillance, epidemiology, and end results database.

The full potential of competing risk modeling approaches in the context of diffuse large B-cell lymphoma (DLBCL) patients has yet to be fully harnessed. This study aims to address this gap by developing a sophisticated competing risk model specifically designed to predict specific mortality in DLBCL patients. We extracted DLBCL patients' data from the SEER (Surveillance, Epidemiology, and End Results) database. To identify relevant variables, we conducted a two-step screening process using univariate and multivariate Fine and Gray regression analyses. Subsequently, a nomogram was constructed based on the results. The model's consistency index (C-index) was calculated to assess its performance. Additionally, calibration curves and receiver operator characteristic (ROC) curves were generated to validate the model's effectiveness. This study enrolled a total of 24,402 patients. The feature selection analysis identified 13 variables that were statistically significant and therefore included in the model. The model validation results demonstrated that the area under the receiver operating characteristic (ROC) curve (AUC) for predicting 6-month, 1-year, and 3-year DLBCL-specific mortality was 0.748, 0.718, and 0.698, respectively, in the training cohort. In the validation cohort, the AUC values were 0.747, 0.721, and 0.697. The calibration curves indicated good consistency between the training and validation cohorts. The most significant predictor of DLBCL-specific mortality is the age of the patient, followed by the Ann Arbor stage and the administration of chemotherapy. This predictive model has the potential to facilitate the identification of high-risk DLBCL patients by clinicians, ultimately leading to improved prognosis.

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.

The Efficacy and Safety of Decitabine Combined with Fludarabine and Cyclophosphamide As Conditioning Regimen in Second CD19 CAR-T Infusion

Background Chimeric antigen receptor (CAR) T therapy is widely accept for the first line treatment of refractory/relapsed diffuse large B cell lymphoma(DLBCL). However, insufficient remission and short duration of response are the barrier of current CART therapy. More and more patients meet the failure of CD19 CART therapy. Pre-CART condition treatment is an important factor in durable responses. Decitabine, the hypomethylating agent, has been found that it can reduce T cell exhaustion, enhance T cell cytotoxicity and overcome tumor immune escape in the tumor microenvironment. We initiated this study to evaluate the efficacy and safety of decitabine combined with fludarabine and cyclophosphamide as conditioning regimen in 2nd CD19 CART therapy. Methods Patients are enrolled in the clinical trail (NCT02537977), inclusion and exclusion criteria are described. Pre-CART conditioning regimen is decitabine 10mg/m2 D-7~-3, fludarabine 25mg/m2 D-5~-3 and cyclophosphamide 300mg/m2 D-5~-3. The dose of second CD19 CART infusion is 5*10^6/kg. Primary outcome is the safety and secondary outcome is the efficacy. Results 6 patients are enrolled in this trail and all patients are failure of CD19 CART. The media age is 57 year old and median line of treatment is 4. 4 patients(67%) are female. 5 patients(83%) have TP53 mutation/deletion and double expression, 4 patients(67%) have IPI ≥3.4 patients(67%) got complete remission(CR) after the first CD19 CART therapy and 2 patients(33%) got partial remission(PR). The median time of relapse/progression after first CD19 CART is 9 months. The objective response rate is 50%, 2 patients(33%) got CR and 1 patient(17%) got PR. With median 13 months followup, the 3 patients(50%) maintain remission more than 1 year and are still under follow up. 2 patients(33%) got stable disease for 3 months and undergo salvage therapy. 1 patient(17%) had no response in 1 month and died due to disease progression. Of all 6 patients, only 1 patient(17%) received 2nd CART infusion in 3 months after 1st infusion and had detecable CART cell in peripheral blood. 2 patients(33%) got grade 1-2 cytokines release syndrome(CRS). No sever CRS and Immune effector cell-associated neurotoxicity syndrome(ICANS) were observed. Grade 3-4 adverse events are hematologic toxicity. Conclusion Decitabine combined with fludarabine and cyclophosphamide maybe an alternative conditioning regimen in CD19 CART therapy in high risk DLBCL patients. More patients are needed for further study and the trail is undergoing.

Retrospective Analysis of Clinical Outcomes of Utilizing R-CHOP Plus Zanubrutinib for High-Risk Diffuse Large B Cell Lymphoma Patients with Extranodal Involvement or MYC/BCL2 Double Expression

Background: Diffuse large B-cell lymphoma (DLBCL) patients with extranodal involvement and double expression of MYC and BCL2 often have a poorer prognosis when treated with the standard first-line R-CHOP regimen. The 5-year progression-free survival (PFS) and overall survival (OS) rates are both below 40% for these high-risk DLBCL cases. In this study, we perform a retrospective analysis of the clinical outcomes and adverse events of the high-risk DLBCL patients with extranodal involvement and double expression, who were admitted to our center and used Bruton's tyrosine kinase inhibitor (BTKi) zanubrutinib on the basis of R-CHOP regimen. Methods: We conducted a retrospective analysis of 26 DLBCL patients with extranodal involvement or MYC/BCL2 double expression who were admitted to our center and used R-CHOP regimen in combination with zanubrutinib between 1 st January 2021 and 30 th April 2023 (Figure A). Of these patients, 19 had extranodal involvement, and 7 were identified as double expression cases. The patients had a median age of 70 years, ranging from 34 to 87. Among them, 20 cases (77%) were aged over 60, and 8 cases (30%) were aged over 75. Moreover, 20 cases (76.9%) exhibited an Eastern Cooperative Oncology Group (ECOG) performance status score higher than 2 points. All the patients received an induction regimen consisting of zanubrutinib in combination with R-CHOP for a total of 4 cycles. After the fourth cycle, treatment efficacy was assessed using 18FDG-PET/CT scans. Patients achieving partial response (PR) or better continued with rituximab in combination with zanubrutinib for consolidation maintenance therapy, lasting for 1 year. For patients at risk of central nervous system involvement, oral administration of zanubrutinib continued for 2 years. During the treatment period, imaging evaluations were performed every 3 months to assess treatment response (Figure B). Results: After the induction therapy, a mid-term PET/CT evaluation showed that all patients achieved PR or above, resulting in an overall response rate (ORR) of 100%. Among them, 19 patients (73.1%) achieved complete response (CR), while 7 patients (26.9%) achieved PR. During the consolidation and maintenance therapy, 7 patients who were initially in PR converted to CR. As of the latest follow-up, with a median follow-up duration of 11 months (ranging from 4.1 to 30.0 months), 2 patients (7.7%) were lost to follow-up, and 2 patients (7.7%) experienced disease progression. The median PFS and OS were not reached. The 2-year PFS was 69.7±13.4% (Figure C), and the 2-year OS was 77.4±12.2% (Figure D). Non-hematological adverse events included fatigue (38.5%), hypertension (15.38%), neurological symptoms (7.7%), and infections (100%). Among the infections, 3 cases (11.5%) were of &amp;gt;3-grade and led to treatment discontinuation. Additionally, one patient experienced immune encephalitis and went into a coma. Hematological adverse events included granulocytopenia (55%), anemia (26%), thrombocytopenia (42.3%), and bleeding (38.5%). Among them, &amp;gt;3-grade granulocytopenia occurred in 4.5% of the patients. Five patients who were on long-term anticoagulant therapy due to concurrent cardiovascular diseases experienced a reduction in zanubrutinib dosage during the follow-up period, resulting in an improvement in bleeding symptoms. Conclusions: The combination of 4 cycles of R-CHOP regimen with zanubrutinib in the induction treatment of DLBCL patients with high-risk factors, such as extranodal involvement or MYC/BCL2 double expression, has shown promising clinical outcomes especially in older or unfit/frail patients. This approach allows for a reduction in the number of cytotoxic drug cycles, thereby improving the patients' tolerance to the treatment. Moreover, the efficacy of this regimen is superior to the traditional 6-8 cycles of R-CHOP induction therapy. Furthermore, the consolidation and maintenance therapy with rituximab in combination with zanubrutinib has demonstrated good safety and manageable adverse effects. This approach exhibits hope for further enhancing the prognosis of high-risk DLBCL patients.

Identification of lactate regulation pattern on tumor immune infiltration, therapy response, and DNA methylation in diffuse large B-cell lymphoma.

Lactate, produced through glycolytic metabolism in the tumor microenvironment (TME), is implicated in tumorigenesis and progression in diverse cancers. However, the impact of lactate on the remodeling of the TME in diffuse large B-cell lymphoma (DLBCL) and its implications for therapy options remain unclear. A lactate-related (LAR) scoring model was constructed in DLBCL patients using bioinformatic methods. CIBERSORT, XCELL, and ssGSEA algorithms were used to determine the correlation between LAR score and immune cell infiltration. Tumor Immune Dysfunction and Exclusion (TIDE), rituximab, cyclophosphamide, adriamycin, vincristine, and prednisone (R-CHOP) cohorts, and Genomics of Drug Sensitivity in Cancer (GDSC) were utilized to predict the therapeutic response of DLBCL patients. The impact of the hub gene STAT4 on tumor biological behavior and DNA methylation was experimentally validated or accessed by the TSIDE database. The LAR scoring model was developed based on 20 prognosis-related lactate genes, which enabled the division of DLBCL patients into high- and low-risk groups based on the median LAR score. Patients with high-risk DLBCL exhibited significantly worse survival outcomes in both the training cohorts (GSE181063) and the validation cohorts (GSE10846, GSE32918, and GSE69053), as indicated by statistically significant differences (all P<0.05) and area under the curve (AUC) values exceeding 0.6. Immune analyses revealed that low-risk DLBCL patients had higher levels of immune cell infiltration and antitumor immune activation compared to high-risk DLBCL patients. Furthermore, DLBCL patients with high LAR scores were associated with a lower TIDE value and poor therapeutic efficacy of the R-CHOP regimen. GDSC analysis identified 18 drugs that exhibited significant response sensitivity in low-risk DLBCL patients. Moreover, in vitro experiments demonstrated that overexpression of the lactate key gene STAT4 could suppress proliferation and migration, induce cell cycle arrest, and promote cell apoptosis in DLBCL cells. Transcriptional expression and methylation of the STAT4 gene were found to be associated with immunomodulators and chemokines. The lactate-based gene signature effectively predicts the prognosis and regulates TME in DLBCL. Our study underscores the role of lactate gene, STAT4, as an important tumor suppressor in DLBCL. Modulating STAT4 could be a promising strategy for DLBCL in clinical practice.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: TP53 mutation (TP53-mut) correlates with inferior survivals in many cancers, whereas its prognostic role in diffuse large B-cell lymphoma (DLBCL) is still in controversy. TP53-mut is frequently enriched in A53 subtype and also can be found in other gene subtypes. However, A53 subtype which was associated with fatal prognosis cannot be identified using the next-generation sequencing (NGS) which used mostly in clinical practice. Therefore, more precise risk stratification is need to be further explored for TP53-mut DLBCL patients. Methods: The available clinical information and corresponding mutation data of DLBCL were retrieved and obtained from published articles. Ultimately, 2637 DLBCL patients in six cohorts were enrolled in the final analysis. Among the 109 DLBCL patients in the Jiangsu Province Hospital (JSPH) study cohort, all tumor tissue samples were collected to perform NGS while 104 samples were analyzed the gene expression levels using RNA-seq. Results: Among the 2637 DLBCL patients from the integrated cohort, 14.0% patients (370/2637) had TP53-mut. The distribution of mutation events was mainly located in the DNA binding domain (N = 333, 83.3%), containing 34 at Arg248, corresponding to the TP53 hotspots in non-Hodgkin lymphoma described in previous studies (Figure 1A). Compared with TP53 wild type (TP53-wt) patients, significant p value was generated by Breslow test (p = 0.0014), whereas Log Rank test uncovered border-line p value for overall survival (OS) (p = 0.0840). Such a result indicated that adverse survival of TP53-mut patients just occurred during early survival while the 10-year OS was even slightly better than the TP53-wrt patients (Figure 1B). Accordingly, we sought to construct a model to identify the truly high-risk patients. As shown in Figure 1C, three variables (age, international prognostic index score and MCD subtype) retained independent prognostic factors for progression free survival (PFS) and OS in TP53-mut patients. The TP53 prognostic index (TP53-PI) model could significantly distinguish the prognosis of TP53-mut DLBCL patients (p < 0.0001, Figure 1D). To calculate the weight of each selected factor, a nomogram was generated, which was used to predict the survival rates. Differential expression analysis by RNA-seq analysis in JSPH cohort offered insights into the underlying biological mechanisms of poor survivals for high TP53-PI risk DLBCL patients. The immune-associated biological processes occupied a large proportion in the result of Gene Ontology functional enrichment analysis between low and high TP53-PI risk DLBCL patients (Figure 1E). Of note, the TP53-mut group had a unique immune microenvironment. Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, microenvironment

Diffuse large B-cell lymphoma at risk of secondary CNS involvement: The inefficacy of intravenous high-dose methotrexate CNS prophylaxis and the importance of baseline cerebrospinal fluid analysis.

High-dose intravenous methotrexate (HD-MTX) CNS prophylaxis in high-risk diffuse large B cell lymphoma (DLBCL) remains controversial. We describe real-world CNS relapse incidence following baseline cerebrospinal fluid (CSF) analysis to exclude asymptomatic leptomeningeal involvement in newly diagnosed high-risk DLBCL patients with versus without single-route HD-MTX CNS prophylaxis. Consecutively diagnosed high-risk systemic DLBCL patients without leptomeningeal involvement by CSF analysis (noCNS) were identified retrospectively. Five-year CNS relapse incidence and survival outcomes were examined, as stratified by receipt of HD-MTX prophylaxis. Secondary analysis of survival outcomes in patients with synchronous leptomeningeal involvement (CNSinv) by CSF analysis at diagnosis were compared with the noCNS group. No significant difference in 5-year CNS relapse incidence was observed following HD-MTX prophylaxis versus no prophylaxis (total n = 445) despite similar CNS-IPI risk; 6.2% versus 5.6%, adjusted HR 1.08 (95% CI 0.41-2.85), p = .88; nor in 5-year progression free survival (PFS) or overall survival (OS) risk. Of CNSinv patients, 93.3% had ≥1 extranodal site. Increased CNS relapse/progression risk (5-year risk; HR 10.7 [95% CI 5.35-21.37], p < .0001) and inferior PFS and OS were observed in CNSinv versus all noCNS patients. The CNSinv group had superior OS compared with noCNS patients who later experienced CNS relapse (HR 0.55, p = .052). HD-MTX prophylaxis does not reduce CNS relapse risk in high-risk systemic DLBCL without leptomeningeal involvement by CSF analysis at diagnosis. Asymptomatic patients with synchronous leptomeningeal involvement on baseline CSF examination are at increased risk of further CNS disease events and inferior survival compared to patients without CSF involvement.

Frontmind: A Phase III, Multicenter, Randomized, Double-Blind Study of Tafasitamab + Lenalidomide + R-CHOP Versus R-CHOP Alone for Newly Diagnosed High-Intermediate and High-Risk Diffuse Large B-Cell Lymphoma

Background R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) is the standard of care for newly diagnosed and untreated diffuse large B-cell lymphoma (DLBCL); however, approximately 30-40% of patients relapse or become refractory. Furthermore, the 5-year overall survival (OS) is lower in high-risk DLBCL patients than in low-risk patients. Co-administration of lenalidomide (LEN) with R-CHOP improved both progression-free survival (PFS) and OS in the ECOG-ACRIN E1412 trial but not in the Phase III ROBUST study, in which a lower dose of LEN was used, despite a positive trend in 2-year PFS rate in a subgroup analysis of patients with high-risk disease. POLARIX (NCT03274492) evaluated a modified regimen substituting polatuzumab vedotin for vincristine (pola-R-CHP), showing a modest improvement in PFS without OS benefit between treatment arms, indicating an unmet clinical need remains. Tafasitamab, an Fc-enhanced, humanized, anti-CD19 monoclonal antibody, in combination with LEN was granted accelerated approval in the US (2020) and conditional/accelerated approval by the EMA (2021) and other regulatory authorities for the treatment of adult patients with relapsed/refractory DLBCL not otherwise specified, including DLBCL arising from low-grade lymphoma, and who are ineligible for ASCT. It is a preferred regimen in the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for this setting. CD19 and CD20 are broadly expressed on the surface of DLBCL cells, and a treatment strategy targeting both of these B cell surface molecules may limit target evasion in patients exhibiting low CD20 expression and reduce resistance to R-CHOP. Additionally, LEN is an immunomodulatory drug with antineoplastic activity and enhances antibody-dependent cellular cytotoxicity with tafasitamab and rituximab. The addition of tafasitamab + LEN to R-CHOP may provide further therapeutic benefit in patients with newly diagnosed and untreated DLBCL. The Phase Ib First-MIND (NCT04134936), randomized, safety study of R-CHOP + tafasitamab ± LEN in patients with newly diagnosed and untreated DLBCL showed that tafasitamab + LEN does not affect the dosing and scheduling of R-CHOP, with toxicities similar to those expected with R-CHOP alone (ASH 2021; #3556). frontMIND (NCT04824092) will investigate the efficacy and safety of R-CHOP + tafasitamab + LEN versus R-CHOP alone in previously untreated patients with high-intermediate and high-risk DLBCL. Methods frontMIND is a Phase III, multicenter, randomized, double-blind, placebo-controlled study. As of June 2, 2022, more than 350 study sites have been activated in over 25 countries globally (Figure 1). The study is currently enrolling, with a planned sample size of 880 patients; as of June 6, 2022, more than 50% of the planned sample size have been enrolled in the study. Eligible patients include adults aged 18‒80 years with previously untreated, local biopsy-proven CD20+ DLBCL and high-grade B cell lymphoma (double-hit/triple-hit lymphoma if deemed ineligible for a more aggressive treatment) with IPI score 3-5 (age-adjusted IPI 2-3 if ≤60 years), Eastern Cooperative Oncology Group performance status 0-2, and a time from diagnostic biopsy to start of treatment within 28 days (Figure 2). Patients will be randomized 1:1 to receive six 21-day cycles of either R-CHOP + tafasitamab (12 mg/kg IV, Days 1, 8, and 15) + LEN (25 mg orally, Days 1-10) in the experimental arm or R-CHOP + tafasitamab placebo (0.9% saline solution IV, Days 1, 8, and 15) + LEN placebo (orally, Days 1-10) in the control arm. Patients will be followed for safety and efficacy up to 60 months after the treatment ends. The primary endpoint is investigator-assessed PFS. Secondary endpoints include investigator-assessed event-free survival, OS, safety, and tafasitamab serum concentration (trough and Cmax levels). The sensitivity and specificity of minimal residual disease (MRD) for early detection of disease progression is an exploratory endpoint; further MRD parameters may also be investigated. The study is funded by MorphoSys AG and conducted with the scientific support of members of the Fondazione Italiana Linfomi and the German Lymphoma Alliance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combination of Phosphodiesterase 4 (PDE4) Inhibitor Roflumilast and R-CHOP As First-Line Therapy for High-Risk Diffuse Large B Cell Lymphoma Patients

BACKGROUND PDE4 inhibition is a biologically well-informed strategy with activity in B-cell malignancies including diffuse large B-cell lymphoma (DLBCL) (PMID: 15331441, PMID: 26503641). Roflumilast (R) is a PDE4 inhibitor, currently FDA-approved for COPD (PMID: 26338438 ). Building on preclinical and clinical activity of R in B-cell lymphomas, both alone and in combination with other agents (PMID: 27756749); a phase 1 evaluation was conducted, adding R to rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients with untreated high risk DLBCL. METHODS: This is a single center, phase 1b, open label, non-randomized, single arm, investigator initiated trial (IIT) of R + R-CHOP (RR-CHOP) for treatment naïve high-risk DLBCL patients. Key eligibility criteria included: non-GCB DLBCL (Han's algorithm), NCCN-IPI risk score of ≥ 2, Ann Arbor stage II-IV; key exclusion criteria include active CNS involvement and documented history of severe depression (a specific concern related to the use of PDE4 inhibitors). Patients received R-CHOP every 21 days for 6 cycles and R PO daily (500 mcg), throughout the 18-week treatment period. Paired germline-tumoral exome sequence was completed for 70% of patients enrolled in the trial, and DLBCL genetic subtype defined by the probabilistic classification tool LymphGen (PMID: 32289277). Primary end point was assessment and grading of adverse events (AE) according to the CTCAE version 4.03 criteria. Eligible patients were included in the intent-to-treat analysis. Secondary end points were estimation of complete response (CR) as per Lugano criteria, progression free survival (PFS), overall survival (OS) and their relationship to DLBCL genetic subtypes. RESULTS: Ten patients met eligibility criteria for enrollment. Sixty percent were male, 50 % were Hispanics (HI), median age at diagnosis was 59 years old (y.o) (33 y.o-79 y.o), 50% were stage 2, and 50% were stage 3-4; 50% received CNS prophylaxis given high risk and 70% had International Prognostic Index (IPI) scores of 3 to 5. Standard doses of R-CHOP were able to be delivered safely, with addition of 500 mcg of R on daily basis. Regarding hematologic adverse events (AE); 1 patient (10%) experienced neutropenia grade 3, and two patients (20%) had neutropenia grade 2. Anemia occurred in 2 patients (20%), all grade 1-2. Regarding non-hematologic AE; anorexia occurred in 40% of the cohort, all grade1; diarrhea occurred in 50%, all grade 1; headache occurred in 60%, with 40% being grade 2, and 20% being grade 3, with the latter judged to be unrelated to RR-CHOP by the investigators. Forty percent had grade 1 increase of BNP; 40% of patients had nausea, 30% being grade 1-2, and 10% being grade 3; and 40% experienced weight loss, all grade 1-2. Of 10 enrolled patients, 9 were found evaluable for assessment of efficacy, with 66% achieving CR, 22% partial response and 11 % stable disease. At 50 months of follow up, PFS was 60% at 1 year (y) and 50% at 2 y. Median PFS was not reached. OS was 100% at 1 y and 86% at 2 y, with a median OS of 38.9 months. (Figure 1 and Figure 2). The recommended R dose for phase 2 is 500 mcg PO daily. LymphGen assigned the DLBCLs in this cohort to the A53, MCD, EZB and B2N subtypes. Notably, 3 of the 3 DLBCLs in our cohort classified as MCD or A53 DLBCLs, which normally predict poor outcome, reached CR and are long term responders. Examination of 461 primary DLBCLs (NCBI dbGaP Genomic Variation in DLBLC cohort) uncovered significantly higher PDE4B expression in in the MCD subtype, suggesting that addition of R may be particularly beneficial in this DLBCL subset. CONCLUSION: Phosphodiesterase Inhibition in DLBCL is a well biologically informed strategy with the potential to enhance and perhaps optimize the duration of responses to R-CHOP. On this difficult to treat subgroup of non-GC DLBCL, with known subpar outcomes, RR-CHOP demonstrated to be safe, with manageable AEs that did not differ from what we have historically seen with R-CHOP alone. Early efficacy data from this combination shows 10% improvement compared to our own institutional historical controls of treatment naïve non-GC DLBCL patients treated with R-CHOP. Based on this data, we are pursuing a phase 2 multisite IIT of RR-CHOP in patients with newly diagnosed high risk DLBCL. Despite the small size cohort, our data suggest that tailoring this strategy to the poor outcome MCD/A53 genetic subgroups may increase the likelihood of success. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CYCLON and NPM1 Cooperate within an Oncogenic Network Predictive of R-CHOP Response in DLBCL.

Simple SummaryCYCLON is a nuclear protein, which has been associated with disease progression and treatment resistance in DLBCL, the most common form of aggressive B-cell lymphoma, but also represents a predictive factor of refractory disease and relapse for immuno-chemotherapy-treated DLBCL patients. The molecular mechanisms related to this unstructured protein remain largely uncharacterized. Here, we performed a mass-spectrometry-based identification of the CYCLON protein interactome that suggested it could exert nucleolar functions related to cell proliferation. Among the CYCLON oncogenic network, we performed an immunohistochemical evaluation of the multi-functional nucleolar protein NPM1 in a DLBCL cohort and showed that CYCLON/NPM1 concomitant expression delineates a poor prognosis subgroup of patients. Multivariate survival analyses demonstrated that specific sub-cellular localizations of CYCLON and NPM1 represent independent novel predictors specifically associated with refractory DLBCL.R-CHOP immuno-chemotherapy significantly improved clinical management of diffuse large B-cell lymphoma (DLBCL). However, 30–40% of DLBCL patients still present a refractory disease or relapse. Most of the prognostic markers identified to date fail to accurately stratify high-risk DLBCL patients. We have previously shown that the nuclear protein CYCLON is associated with DLBCL disease progression and resistance to anti-CD20 immunotherapy in preclinical models. We also recently reported that it also represents a potent predictor of refractory disease and relapse in a retrospective DLBCL cohort. However, only sparse data are available to predict the potential biological role of CYCLON and how it might exert its adverse effects on lymphoma cells. Here, we characterized the protein interaction network of CYCLON, connecting this protein to the nucleolus, RNA processing, MYC signaling and cell cycle progression. Among this network, NPM1, a nucleolar multi-functional protein frequently deregulated in cancer, emerged as another potential target related to treatment resistance in DLBCL. Immunohistochemistry evaluation of CYCLON and NPM1 revealed that their co-expression is strongly related to inferior prognosis in DLBCL. More specifically, alternative sub-cellular localizations of the proteins (extra-nucleolar CYCLON and pan-cellular NPM1) represent independent predictive factors specifically associated to R-CHOP refractory DLBCL patients, which could allow them to be orientated towards risk-adapted or novel targeted therapies.

Retrospective Review of the Safety and Efficacy of High-Dose Methotrexate for Prevention of CNS Relapse in Diffuse Large B-Cell Lymphoma at Kaiser Permanente- Northern California (Jan 2015 - June 2019)

Retrospective review of the safety and efficacy of high-dose methotrexate for prevention of CNS relapse in diffuse large B-cell lymphoma at Kaiser Permanente- Northern California (Jan 2015 - June 2019)Background: Central nervous system (CNS) relapse occurs in 10-12% of high-risk diffuse large B-cell lymphoma (DLBCL) patients. Prophylactic intravenous high-dose methotrexate (HD-MTX) is recommended by international guidelines to reduce this risk despite limited evidence to support such practice. Recent retrospective studies have cast doubt on the clinical benefit of such treatment. There is limited data on safety and efficacy of such treatment in a community oncology setting.Methods: We conducted a retrospective analysis of adults ≥ 18 years diagnosed with DLBCL treated with systemic therapy and HD-MTX as CNS prophylaxis at Kaiser Permanente Northern California from 1/2015 - 6/2019. We abstracted patient demographics, clinical information, treatment, toxicity, and health care utilization from the electronic health record. Descriptive statistics were used to evaluate patients' outcomes.Results: Of 33 patients (median age: 61; range: 23 - 81; age ≥ 60: 57.5%), most had stage IV disease (78.7%) and an ECOG performance status of 0 or 1 (66.5%). Patients with CNS-IPI score of 2-3 (intermediate-risk) was 30.3%, while higher CNS-IPI scores of 4-6 (high-risk) was 51.5%. Other patient characteristics include double hit lymphoma (12.1%), kidney/adrenal gland involvement (33%), and/or epidural involvement (24.2%). Most common therapies were R-CHOP (51.5%) and R-EPOCH (27.2%). The median number of HD-MTX doses was 3 (range 1-4). The median cumulative dose was 7 gm/m2 (range 3-10.5). With regards to the treatment schedule, 63.6% received HD-MTX intercalated with systemic chemotherapy and 36.4% received HD-MTX after completion of preplanned treatment. Overall, renal toxicity was the most common adverse side effect. The rates of grade 1, 2 and 3 renal toxicity were 12.1%, 9% and 6%, respectively. Other notable side effects experienced were neutropenic fever requiring hospitalization (27.2%) and grade 3 transaminitis (6%). No patients experienced grade 3 mucositis. The median duration of hospital stay was 12 days (range 4-37) and 12.1% required suspension of future HD-MTX. With a median follow up of 31.3 months (range: 0.79 - 58.4) 69.6% are alive and 15.1% had CNS relapse despite prophylactic HD-MTX.Conclusions:In this community oncology setting, patients with DLBCL who were deemed high risk for CNS relapse and received HD-MTX for prophylaxis experienced similar CNS relapse rate compared to those who did not in previous studies. Our findings are in line with recent retrospective reviews, which further support the lack of benefit of such prophylactic treatment. This study underscores the need for further research to prevent CNS disease and improved patient selection criteria for prophylactic treatment among high risk DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

Comparative assessment of efficacy and toxicity of R-DA-EPOCH and R-mNHL-BFM-90 induction courses in the treatment of patients with diffuse large B-cell lymphoma with poor prognostic factors in a randomized multicenter clinical trial “DLBCL-2015”

Background. Diffuse large B-cell lymphoma (DLBCL) is one of the most common and aggressive tumors of the lymphatic system. Despite the frequency of occurrence, there is no single algorithm for treating DLBCL patients with poor prognostic factors. R-CHOP therapy does not allow achieving long-term complete remissions. Therefore, there is a need for second and subsequent lines of therapy. At the same time, the effectiveness of each subsequent therapy is low, while the toxicity increases. There are many randomized trials of the DLBCL treatment; however, there are only a few studies on the comparative efficacy of high-dose chemotherapy at the induction stage.The objective of the study: the evaluation of the effectiveness and toxicity of R-DA-EPOCH and R-mNHL-BFM-90 induction courses in DLBCL patients with poor prognostic factors in a randomized multicenter clinical trial “DLBCL-2015”.Materials and methods. As of April 2021, 140 patients from 13 medical institutions in Russia were included in the randomized multicenter clinical trial DLBCL-2015. As part of this study, the analysis of pharmacoeconomic factors and effectiveness of combined immunochemotherapy R-DA-EPOCH and R-mNHL-BFM-90 in patients with prognostically unfavorable DLBCL had been performed. From January 2018 to April 2021, this study included 41 patients (21 men, 20 women) with a newly diagnosed DLBCL, with 2 or more factors of an unfavorable prognosis, who were treated at the National Research Center for Hematology of the Ministry of Health of the Russian Federation. Of these, 21 patients received R-DA-EPOCH, and 20, R-mNHL-BFM-90 therapy. Median age for R-DA-EPOCH patients was 52 years (range 30–64); for R-mNHL-BFM-90 patients, 40 years (range 18–60). All patients had high-intermediate and high risk according to the international (IPI) and age-adjusted (aaIPI) prognostic index. The primary protocol endpoints were rates of complete remission, partial remission, disease progression, and hematologic and non-hematologic toxicity. Side effects were assessed in accordance with the Common Terminology Criteria for Adverse Events (CTCAE) criteria.Results. By the end of 6 induction courses, the frequency of achieving complete remission on R-mNHL-BFM-90 therapy was 100 % (20/20) compared to R-DA-EPOCH, where the complete remission rate was 71.4 % (15/21) (p = 0.0097), partial remission and progression were 14.3 % (n = 3) and 14.3 % (n = 3), respectively. Hematological toxicity on therapy according to the R-mNHL-BFM-90 scheme exceeded that on R-DA-EPOCH in terms of myelotoxic agranulocytosis (p = 0.0536), anemia (p = 0.0464) and thrombocytopenia grade III–IV (p = 0.0206). When assessing non-hematological toxicity at the compared courses, no statistically significant differences were noted, all complications occurred with the same frequency.Conclusion. Treatment according to the R-mNHL-BFM-90 protocol is highly effective as first line therapy in high-intermediate and high-risk DLBCL patients. The hematologic toxicity is higher on the R-mNHL-BFM-90 than on the R-DA-EPOCH therapy, but it is acceptable. Non-hematological toxicity in both programs is comparable.

The Efficacy of Systemic High Dose Methotrexate As Central Nervous System Prophylaxis in Patients with High Risk Diffuse Large B-Cell Lymphoma: A Propensity Score-Matched Analysis

Background: While the outcome of patients with diffuse large B-cell lymphoma (DLBCL) has been improved with the introduction of rituximab, central nervous system (CNS) relapse is still associated with poor prognosis. Although intrathecal methotrexate has been widely used for prophylaxis, its role has been questioned. Another way of utilizing the agent, systemic high dose methotrexate (HD-MTX) has been advocated from a few retrospective studies. However, they had no comparison group or they were compared with imbalanced, unmatched group even if existed. With prospectively collected cohort, we performed a propensity score-matched analysis to evaluate the efficacy of IV HD-HTX as CNS prophylaxis in high risk DLBCL patients.Methods: The registry data set of DLBCL patients, collected from January 2010 through March 2015 at a single institute, Asan Medical Center, was retrospectively reviewed. From July 2013, all consecutive DLBCL patients who were considered at high risk for CNS recurrence received systemic HD-MTX with standard R-CHOP therapy. High risk CNS relapse was defined by the involvement of ≥2 extranodal sites and elevated lactic dehydrogenase (LDH); or high-risk CNS international prognostic index (CNS-IPI ≥4); or involvement of specific high-risk extranodal sites including bone marrow, breasts, testes, paranasal sinuses. HD-MTX was administered at a dose of 3-3.5 g/m2 on day 15 of alternating cycles of R-CHOP or delivered 2 to 5 weeks after the completion of the primary therapy. We analyzed the progression-free survival (PFS), CNS relapse-free survival (CNS-RFS) and overall survival (OS) of the patients receiving CNS prophylaxis and compared with the patients who received R-CHOP only. Multivariate Cox regression and propensity score analysis were used to evaluate the treatment effect of systemic HD-MTX on survival with multiple covariates including age, sex, LDH, ECOG score, stage, CNS-IPI, extranodal involvement and involved sites.Results: A total of 197 patients with DLBCL and CNS risk factors who were treated with standard R-CHOP therapy were identified between January 2010 and March 2015. Among them, 47 patients received systemic HD-MTX as CNS prophylaxis. In the R-CHOP only group (n=150), with a median follow-up of 58.88 (1.34-84.3) months, 14 CNS relapses occurred within 2 years. The estimated 2-year PFS, CNS-RFS and OS rates were 58.6%, 89.5%, 71.3%, respectively. In the HD-MTX prophylaxis group (n=47), with a median follow-up of 27.24 (3.38-42.02) months, 3 CNS relapses occurred within 2 years. The estimated 2-year PFS, CNS-RFS and OS rates were 62.3%, 93.1% and 74.5%, respectively. Systemic HD-MTX did not significantly increase the PFS (HR 0.619, 95% CI 0.363-1.057, P = .079), CNS-RFS (HR 0.547, 95% CI 0.156-1.924, P=.347), and OS (HR 0.628, 95% CI 0.331-1.19, P = .154) in the multivariate survival analysis. The results seems to consistently show no survival benefit of systemic HD-MTX regardless of the different propensity score analysis methods except for PFS by weighting HR: PFS (matching HR 0.652, 95% CI 0.36-1.18, P=.157; weighting HR 0.689, 95% CI 0.503-0.945, P=.02),CNS-RFS (matching HR 0.635, 95% CI 0.161-2.499, P=.516; weighting HR 0.587, 95% CI 0.287-1.203, P=.146), OS (matching HR 0.625, 95% CI 0.319-1.227, P=.172; weighting HR 0.726, 95% CI 0.505-1.044, P=.083).Conclusion: Systemic HD-MTX prophylaxis for CNS relapse did not show significant survival improvement in high risk DLBCL patients in this study although there was a trend for better outcomes in patients given HD-MTX, as different statistical methods incorporating stringent propensity score-matched analysis were applied. However, these results are still limited by small cohort size its retrospective nature. Multi-center, prospective randomized studies are necessary to appraise the efficacy of systemic HD-MTX as CNS prophylaxis in high risk DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

Targeting DNA Repair Mechanisms to Overcome Drug Resistance in Diffuse Large B Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of adult non-Hodgkin lymphomas. Most DLBCL patients achieve long-term remission after treatment, but a third relapse after conventional Rituximab (R)-based chemotherapy regimens, such as CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) (Siegel et al, 2012).Cancer cells are exposed to chronic replication stress, which impedes the duplication of their genome and induces mitotic catastrophe (Shaheen et al, 2011). Functional DNA repair pathways are therefore important for the survival of cancer cells. This dependence can be exploited therapeutically to hamper repair of the intrinsic DNA damage occurring during replication or to exacerbate DNA damage induced by chemotherapy (Shaheen et al, Blood 2011). Furthermore, high-risk DLBCL patients overexpress genes potentially involved in resistance to CHOP-based regimens, such as genes of the nucleotide excision repair (NER) pathway (Bret et al, Oncotarget 2012, Cell Cycle 2013).We recently developed GEP-based DNA repair scores that allow to identify high-risk DLBCL patients that could benefit from treatment with DNA repair inhibitors (Bret at al, BJH 2015).DLBCL treatments include cyclophosphamide, a nitrogen mustard derivate that induces interstrand crosslinks (ICLs), and doxorubicin, a DNA topoisomerase inhibitor that induces DNA double-strand breaks, DNA adducts and formaldehyde-dependent ICL formation. Inhibiting DNA repair is a promising strategy to improve the efficacy of genotoxic drugs and overcome drug resistance. Our data support the view that inhibitors of DNA damage signaling and DNA repair have potential therapeutic interest in DLBCL.We characterized the drug-response of 16 DLBCL cell lines to 9 DNA repair inhibitors including PJ34 (PARP inhibitor), NU7441 (DNAPK inhibitor), KU55933 (ATM inhibitor), PF477736 (CHK1/2 inhibitor), AZD6738 (ATR inhibitor), MK8776 (CHK1 inhibitor), AZD1775 (Wee1 inhibitor), MP-470 (Rad51 inhibitor), Lomeguatrib (MGMT) and genotoxic agents used in DLBCL treatment (Cyclophosphamide, Gemcitabine, Doxorubicin and Etoposide) (Table 1). All drugs induced significant apoptosis (Annexin V staining and PARP cleavage) and significant inhibition of proliferation (BrdU incorporation) in the different DLBCL cell lines tested (P <0.05). CHK1/2 inhibitor, Wee1 inhibitor, Cyclophosphamide, Gemcitabine and Doxorubicin induced DNA damages monitored by H2AX phosphorylation. Correlating drug response of each compounds with our GEP-based DNA repair scores (Bret et al, BJH 2015), we identified a significant correlation between FANC score and response to ATR inhibitor (AZD6738) and HRR score/BER score and response to Etoposide (P<0.05). High-risk DLBCL patients identified with GEP-based FANC, HRR and BER scores may benefit from treatment by ATR inhibitors or Etoposide respectively.Since DNA repair pathways play a role in drug resistance, we sought to identify new synthetic lethal and synergistic combinations associating IC20 of DNA repair targeted treatments with conventional genotoxic agents in DLBCL. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone, a total of 3 synthetic lethal combinations have been identified including cyclophosphamide with CHK1/2 inhibitor (PF477736) (IC50 reduced from 2.63 µM to 1.20 µM), cyclophosphamide and ATR inhibitor (AZD6738) (IC50 reduced from 2.63 µM to 0.74 µM) and doxorubicin with DNAPK inhibitor (NU7441) (IC50 reduced from 57 nM to 14 nM).Furthermore we identified new potent synergistic combinations (combination index < 1) including CHK1/2 inhibitor (PF477736) with etoposide (IC50 reduced from 560 nM to 437 nM), ATR inhibitor (AZD6738) (IC50 reduced from 560 nM to 364 nM) with etoposide and ATM inhibitor (KU55933) with etoposide (IC50 reduced from 560 nM to 269 nM).Despite overall improvements in the treatment of DLBCL, including the use of rituximab, approximately one-third of patients fail to achieve complete remission or experience relapse. This remains a major cause of morbidity and mortality. The DNA repair scores could be useful to identify high-risk DLBCL patients and define the best synthetic lethal approach combining DNA repair inhibitors with conventional chemotherapy. These results open new perspectives to improve the treatment of DLBCL patients and provide new strategies to overcome drug resistance. [Display omitted] DisclosuresCartron:Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Celgene: Consultancy, Employment; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding.

Th1/2 Immune Response Signature Predicts Outcome after Dose-Dense Immunochemotherapy in Patients with High Risk Diffuse Large B-Cell Lymphoma - Results from Nordic Lymphoma Group Trials

Introduction: Despite better therapeutic options and improved survival of diffuse large B-cell lymphoma (DLBCL), 30-40% of the patients still relapse and have dismal prognosis. Recently, the impact of genomic aberrations, allowing lymphoma cells to escape immune recognition on DLBCL pathogenesis has been recognized. However, whether immune related signatures could be used as determinants for treatment outcome has not been rigorously evaluated. Here, our aim was to elucidate the immunologic characteristics of the tumor microenvironment, and associate the findings with outcome in patients with high-risk DLBCL.Patients and methods: We specifically examined gene expression in the tumor microenvironment, using RNA isolated from formalin fixed paraffin-embedded (FFPE) tumor samples from 81 patients with DLBCL. Gene expression analysis was conducted on the NanoString nCounter gene expression platform using a 770-gene PanCancer Immune panel. The predictive impact of the gene expression signatures was assessed using a cohort of 65 high risk DLBCL patients enrolled in the Nordic CHIC trial (Leppä et al., 2016). The findings were validated using exon array data from an independent cohort of 38 high risk DLBCL patients treated in the Nordic CRY-04 trial (Holte et al., 2013; Leivonen et al., 2017a). In both trials, the patients received dose-dense immunochemotherapy (R-CHOEP-14) and systemic central nervous system prophylaxis (HD-Mtx and HD-Ara-C).Results: Unsupervised hierarchical clustering revealed three major clusters of genes with differential expression between the patients. The clusters were enriched for the genes related to focal adhesion and extracellular matrix (ECM) receptor interactions (eg. COL3A1, ITGA, ITGB, VEGFC, VEGFA, PDGFRB, PDGFC, FN1 ), T/NK-cell signaling (eg. ICAM1, CD3, CD4, CD8 GZMB, ITGB2 ), and type 1/2 immune responses (eg. IL4, IFNL1, IFNL2, IL13, IL24 ). Interestingly, type 1/2 immune response signature correlated with survival. In the CHIC cohort, the group of patients (n=10) with high expression of type 1/2 immune response signature genes had a 4-year PFS of 60% in comparison to 91% for those (n=55) with low expression (p = 0.021). The corresponding 4 y OS rates were 64% vs 90% (p=ns). The risk of progression was 4.0 fold higher for the patients with high expression of type 1/2 immune response signature genes (95% CI 1.120-14.280, p=0.033). No differences were observed in sex, age, stage, IPI scores, or molecular subgroups between the low and high type 1/2 immune response subgroups. The prognostic impact of the signature was confirmed in the CRY-04 exon array cohort (4-y PFS; 46% vs 80%, p = 0.021, 4-y OS 62% vs 88%; p = 0.014). In the CRY-04 cohort, the risk of progression was 3.6 fold (95% CI 1.13-11.20, p=0.030) and death 4.8 fold (95% CI 1.20-19.26, p=0.027) higher for the group of patients with high expression of the type 1/2 immune response signature genes. In contrast to our data on primary testicular lymphomas (PTLs) (Leivonen et al., 2017b), T/NK cell signature did not correlate with outcome in either of the trial cohorts. Correspondingly, type 1/2 immune signature was not associated with outcome in the cohorts of PTL or DLBCL patients treated with conventional R-CHOP regimen.Conclusion: Our data suggest that immune related signatures exhibit treatment-specific roles in diffuse large B-cell lymphoma. For the high risk DLBCL patients treated with dose-dense immunochemotherapy, high expression of type 1/2 immune response signature genes predicts a poor outcome. A detailed characterization of immune cell composition in the tumor microenvironment and its impact on survival is ongoing. DisclosuresJørgensen:Roche: Research Funding. Holte:Novartis: Consultancy, Honoraria; Celgene: Consultancy; Janssen Cilag: Consultancy; Nordic Nanovector: Consultancy; Mundipharma: Consultancy. Leppa:Merck: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Janssen Cilag: Consultancy, Research Funding; Bayer: Research Funding.

Efficacy and safety of prophylactic high-dose MTX in high-risk DLBCL: a treatment intent–based analysis

AbstractDespite central nervous system (CNS) relapse occurring in >10% of high-risk diffuse large B-cell lymphoma (DLBCL) patients, the role of CNS-directed prophylaxis is controversial in the absence of randomized controlled trials. In this retrospective study, we aimed to evaluate the safety and efficacy of prophylactic high-dose methotrexate (HD-MTX) on CNS relapse and survival outcomes in 258 newly diagnosed R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone)–treated high-risk DLBCL patients, based on the initial treatment intent (ITT) of the physician on the use of prophylactic HD-MTX. Patients were classified into an ITT HD-MTX group (n = 128) and a non-ITT HD-MTX group (n = 130). The CNS relapse rate was not significantly different between these groups, with 2-year CNS relapse rates of 12.4% and 13.9%, respectively (P = 0.96). Three-year progression-free survival and overall survival rates in the ITT HD-MTX and non-ITT HD-MTX groups were 62.4% vs 64.5% (P = 0.94) and 71.7% vs 71.4% (P = 0.7), respectively. Also, propensity score–matched analyses showed no significant differences in the time-to-CNS-relapse, progression-free survival, or overall survival. The ITT HD-MTX group showed a higher incidence of grade ≥ 3 oral mucositis and elevated alanine aminotransferase. Prophylactic HD-MTX does not improve CNS relapse rate or survival outcomes in high-risk DLBCL patients, and it is accompanied by increased toxicities.