Inferior Progression-free Survival Research Articles

MRD-driven treatment with venetoclax-R2 in mantle cell lymphoma: the Nordic Lymphoma Group MCL7 VALERIA trial

AbstractDespite improvements in treatment of mantle cell lymphoma (MCL), most patients eventually relapse. In this multicenter phase 1b/2 trial, we evaluated safety and efficacy of minimal residual disease (MRD)–driven venetoclax, lenalidomide, and rituximab (venetoclax-R2) in relapsed/refractory (R/R) MCL and explored the feasibility of stopping treatment in molecular remission. The primary end point was overall response rate (ORR) at 6 months. After dose escalation, the recommended phase 2 dose was lenalidomide 20 mg daily, days 1 to 21; venetoclax 600 mg daily after ramp-up; and rituximab 375 mg/m2 weekly for 4 weeks, then every 8 weeks. MRD monitoring by RQ-PCR was performed every 3 months. When MRD-negativity in the blood was reached, treatment was continued for another 3 months; if MRD-negativity was then confirmed, treatment was stopped. In total, 59 patients were enrolled, with a median age of 73 years. At 6 months, the ORR was 63% (29 complete remission [CR], 8 partial remission [PR]), and 40% (4 CR, 2 PR) for patients previously failing a Bruton tyrosine kinase (BTK) inhibitor. Median progression-free survival (PFS) was 21 months, with median overall survival of 31 months. TP53 mutation was associated with inferior PFS (P < .01). Overall, 28 patients (48%) discontinued treatment in molecular remission, and 25 remain MRD negative after a median of 17.4 months. Hematological toxicity was frequent, with 52 of 59 (88%) patients with G3-4 neutropenia and 21 of 59 (36%) patients with G3-4 thrombocytopenia. To conclude, MRD-driven venetoclax-R2 is feasible and tolerable and shows efficacy in R/R MCL, also after BTK inhibitor failure. This trial was registered at www.ClinicalTrials.gov as #NCT03505944.

Minimal Residual Disease Status Predicts Outcome in Patients With Previously Untreated Follicular Lymphoma: A Prospective Analysis of the Phase III GALLIUM Study.

We report an analysis of minimal residual/detectable disease (MRD) as a predictor of outcome in previously untreated patients with follicular lymphoma (FL) from the randomized, multicenter GALLIUM (ClinicalTrials.gov identifier: NCT01332968) trial. Patients received induction with obinutuzumab (G) or rituximab (R) plus bendamustine, or cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) or cyclophosphamide, vincristine, prednisone (CVP) chemotherapy, followed by maintenance with the same antibody in responders. MRD status was assessed at predefined time points (mid-induction [MI], end of induction [EOI], and at 4-6 monthly intervals during maintenance and follow-up). Patients with evaluable biomarker data at diagnosis were included in the survival analysis. MRD positivity was associated with inferior progression-free survival (PFS) at MI (hazard ratio [HR], 3.03 [95% CI, 2.07 to 4.45]; P < .0001) and EOI (HR, 2.25 [95% CI, 1.53 to 3.32]; P < .0001). MRD response was higher after G- versus R-chemotherapy at MI (94.2% v 88.9%; P = .013) and at EOI (93.1% v 86.7%; P = .0077). Late responders (MI-positive/EOI-negative) had a significantly poorer PFS than early responders (MI-negative/EOI-negative; HR, 3.11 [95% CI, 1.75 to 5.52]; P = .00011). The smallest proportion of MRD positivity was observed in patients receiving bendamustine at MI (4.8% v 16.0% in those receiving CHOP; P < .0001). G appeared to compensate for less effective chemotherapy regimens, with similar MRD response rates observed across the G-chemo groups. During the maintenance period, more patients treated with R than with G were MRD-positive (R-CHOP, 20.7% v G-CHOP, 7.0%; R-CVP, 21.7% v G-CVP, 9.4%). Throughout maintenance, MRD positivity was associated with clinical relapse. MRD status can determine outcome after induction and during maintenance, and MRD negativity is a prerequisite for long-term disease control in FL. The higher MRD responses after G- versus R-based treatment confirm more effective tumor cell clearance.

Preoperative C-reactive protein to albumin ratio as a novel prognostic biomarker for the oncological outcomes of radical nephroureterectomy

PurposeThis study aimed to evaluate the prognostic impact of the preoperative C-reactive protein to albumin ratio (CAR) on progression-free survival (PFS) and cancer-specific survival (CSS) in patients with upper urinary tract urothelial carcinoma (UTUC) undergoing radical nephroureterectomy (RNU). MethodsA retrospective analysis was conducted using data from a single-center nephroureterectomy registry between January 2011 and December 2017. Participants were categorized into high and low CAR groups based on the optimal CAR cut-off value determined using the Youden index. The primary endpoint was PFS, the time from RNU to metastasis or disease recurrence. The secondary endpoint was CSS, the time from RNU to UTUC-related death. Median PFS and CSS were compared between the high and low CAR groups using Kaplan–Meier analysis and log-rank test. Multivariable Cox proportional hazard regression analysis was performed to assess the prognostic significance of CAR, adjusting for known prognostic factors. ResultsWe included 491 patients with UTUC in the analysis. The optimal CAR cut-off value was determined to be 0.036, which resulted in classifying 49.3% (242/491) of patients into the high CAR group. The high CAR group had older patients (69.8 vs. 67.4, p-value = 0.01), advanced T and N stages (p-value<0.001), high-grade tumor (p-value = 0.03), and a higher incidence of preoperative hydronephrosis (p-value < 0.01) than the low CAR group. The high CAR group demonstrated significantly inferior median PFS (78.3 vs. 100.3 months, p-value < 0.01) and CSS (73.2 vs. 96.1 months, p-value < 0.01) than the low CAR group. Moreover, high CAR independently increased the risk of disease progression (hazard ratio [HR]: 1.80, 95% confidence interval [CI]: 1.23–2.64, p < 0.01) and UTUC-related mortality (HR: 1.79, 95% CI: 1.15, p < 0.01). ConclusionPre-operative CAR is independently associated with poor PFS and CSS in patients with UTUC undergoing RNU. Moreover, CAR may be an independent UTUC prognostic factor, offering a cost-effective and minimally invasive marker. However, further validation through large-scale, multi-center studies is necessary to confirm these findings and determine the optimal CAR cut-off value.

HPV ctDNA Allows for the Detection of Residual Disease in Cervical Cancer

Abstract The presence of HPV ctDNA after chemoradiation is predictive of inferior progression-free survival.

Prognostic value of pretreatment lymphocyte-to-monocyte ratio in patients with glioma: a meta-analysis

BackgroundMany studies have explored the prognostic role of the lymphocyte-to-monocyte ratio (LMR) in patients with glioma, but the results have been inconsistent. We therefore conducted the current meta-analysis to identify the accurate prognostic effect of LMR in glioma.MethodsThe electronic databases of PubMed, Web of Science, Embase, and Cochrane Library were thoroughly searched from inception to July 25, 2023. The pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to estimate the prognostic role of LMR for glioma.ResultsA total of 16 studies comprising 3,407 patients were included in this meta-analysis. A low LMR was significantly associated with worse overall survival (OS) (HR = 1.35, 95% CI = 1.13–1.61, p = 0.001) in glioma. However, there was no significant correlation between LMR and progression-free survival (PFS) (HR = 1.20, 95% CI = 0.75–1.91, p = 0.442) in glioma patients. Subgroup analysis indicated that a low LMR was significantly associated with inferior OS and PFS in glioma when using a cutoff value of ≤ 3.7 or when patients received mixed treatment.ConclusionsThis meta-analysis demonstrated that a low LMR was significantly associated with poor OS in glioma. There was no significant correlation between LMR and PFS in glioma patients. The LMR could be a promising and cost-effective prognostic biomarker in patients with glioma in clinical practice.

Impact of Cytogenetic Abnormalities, Induction and Maintenance Regimens on Outcomes After High-Dose Chemotherapy and Autologous Stem Cell Transplantation in Patients With Newly Diagnosed Multiple Myeloma: A Decade-Long Real-World Experience.

High-dose chemotherapy and autologous stem cell transplant (HDT-ASCT) has become a standard of care for transplant eligible newly diagnosed multiple myeloma (NDMM) patients. While cytogenetic abnormalities have been shown to affect outcomes after HDT-ASCT in clinical trials, these trials often exclude or underrepresent elderly patients with comorbidities and those belonging to ethnic minorities. We describe our institutional experience highlighting the impact of high-risk cytogenetic abnormalities (HRCAs) on outcomes after HDT-ASCT for NDMM patients. A total of 449 patients with NDMM who underwent HDT-ASCT between February 2012 and August 2022 were included in this retrospective analysis. HRCAs included the presence of one or more of: deletion 17p, t(14;16), t(4;14), and amplification 1q. Survival analyses, including progression-free survival (PFS) and overall survival (OS), were performed using Kaplan-Meier estimator. With a median follow-up of 29 (1 - 128) months for the entire patient population, the best overall response rate for the patients with HRCAs was lower compared to those with standard risk cytogenetics (90% vs. 96%; P = 0.01). Patients with HRCAs had an inferior PFS compared to patients with standard-risk cytogenetics (29 vs. 58 months; P < 0.001) without a difference in OS (70 months vs. not reached; P = 0.13). In a multivariable analysis adjusting for factors including age, race, and comorbidities, HRCAs, non-lenalidomide-based maintenance, non-proteasome inhibitor-based maintenance, and age greater than 65 were associated with inferior PFS. Amongst these factors, only non-lenalidomide-based maintenance was associated with inferior OS.

Unified framework for patient-derived, tumor-organoid-based predictive testing of standard-of-care therapies in metastatic colorectal cancer

Predictive drug testing of patient-derived tumor organoids (PDTOs) holds promise for personalizing treatment of metastatic colorectal cancer (mCRC), but prospective data are limited to chemotherapy regimens with conflicting results. We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options. In an Australian community cohort, PDTO predictions based on treatment-naive patients (n= 56) and response rates from first-line mCRC clinical trials achieve 83% accuracy for forecasting responses in patients receiving palliative treatments (18 patients, 29 treatments). Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment, AGITG FORECAST-1 (n= 30 patients). "Resistant" predictions are associated with inferior progression-free survival; misclassification rates are similar by regimen. Liver metastases are the optimal site for sampling, with testing achievable within 7weeks for 68.8% cases. Our findings indicate that PDTO drug panel testing can provide predictive information for multifarious standard-of-care therapies for mCRC.

Predictors of Outcomes with Venetoclax-Based Treatment in Patients with Progressive Disease or Intolerance after Covalent BTK Inhibitors

Background: Venetoclax (Ven) is a standard of care for the treatment of patients (pts) with CLL/SLL. In the relapsed setting, there is very limited data on the efficacy of Ven after covalent BTK inhibitors ([cBTKi], ibrutinib, acalabrutinib, or zanubrutinib), and there is no prospective trial data on the feasibility of a Ven-based fixed-duration therapy after cBTKi failure. Recently, novel treatment options like pirtobrutinib, a non-covalent BTKi, have shown high efficacy after cBTKi, prompting scrutiny of Ven-based therapies after cBTKi failure, including Ven monotherapy and combination treatment with anti-CD20 antibodies. In this study, we aim to evaluate: 1) The efficacy of Ven-based therapy after cBTKi, 2) Predictors of the efficacy of Ven in this setting, and 3) The feasibility of fixed-duration therapy with Ven after BTKi. Methods: In this single-institution, retrospective study, we used our institutional CLL registry to identify patients with a CLL/SLL diagnosis who received Ven (with or without anti-CD20 antibodies) for at least two months after prior treatment with a cBTKi. Patient-level clinical data were extracted from electronic medical records. Progressive disease (PD) was defined by the treating physician based on clinical, laboratory, and/or imaging data. Progression-free survival (PFS) and Overall survival (OS) were calculated using the Kaplan-Maier estimates. Results: We identified 80 pts who started Ven between 1/2016-12/2022 after prior treatment with a cBTKi. Median age was 62 (range: 40-80), and 53% were male. FISH abnormalities included: 58% del17p, 25% del11q, 48% del 13q, and 19% with trisomy 12. The median prior lines of treatment were 2 (range: 1-7). Prior first cBTKis were ibrutinib (92.5%), acalabrutinib (6.5%), and zanubrutinib (1.5%). A total of 7 pts (9%) received more than one prior cBTKi (1 ibrutinib, 6 acalabrutinib). Prior cBTKi was used as first-line treatment in 21 pts (26%), and 51 (64%) pts received prior treatment with chemotherapy. The median duration of cBTKi treatment was 25 months (range 1-92). Reasons for cBTKi discontinuation were PD (n=56, 70%) and intolerance (n=24, 30%). Most pts started Ven-based therapy immediately after cBTKi (n=53, 66%), although 21 (26%) received one interim line, and 6 (7.5%) received more than one interim line. Ven was used either as monotherapy (n=45, 56%) or with anti-CD20 antibodies (n=35, 44%). The median time on Ven was 11 months (2-55 months). At the time of analysis, 75% of pts had stopped Ven, with the most common reason for discontinuation being PD (31, 53%), followed by treatment completion (19, 32%). Median PFS after starting Ven was 29 months (95% CI: 17-44) for the entire cohort. Median PFS was significantly inferior in pts who experienced PD on prior cBTKi compared with those who were intolerant to prior cBTKi (17 months vs. 55 months, p=0.002). Inferior PFS was also observed in patients with del17p (7 months vs. 26 months without del17p, p=0.003) and in pts who received Ven monotherapy vs. combination therapy with anti-CD20 antibodies (21 months vs. not reached, p=0.01). There was no difference in PFS based on prior treatment with chemotherapy or for pts who received treatments between cBTKi and Ven. In multivariable analysis, having del17p (HR 3.04, 95%CI [1.38-6.65]), Ven monotherapy (HR 3.19, 95% CI [1.52-6.68]) and PD on prior cBTKi (HR 4.29, 95% [1.50-12.29]) were associated with inferior PFS. With a median of 28 months of follow-up, 26 pts (32%) stopped Ven and did not experience PD. This included 23% of pts with PD after prior cBTKi and 54% of pts intolerant to prior cBTKi (p=0.007). Conclusion: Venetoclax-based therapy after cBTKi provides disease control, but not to the extent reported by the only prospective trial in this setting (Jones, Lancet Oncol, 2018). Inferior PFS was observed in pts who had PD after prior cBTKi (vs. being intolerant), pts who received Ven monotherapy (vs. combined with anti-CD20 antibodies), and those with del17p. While this study may be limited in assessing the feasibility of fixed-duration Ven therapy after cBTKi, our results indicate that such an approach may only be feasible in a subset of pts who stopped cBTKi because of intolerance and not PD.

Co-Expression of C-MYC/BCL2 Is Associated with Inferior Survival Outcomes in Relapsed/Refractory Diffuse Large B-Cell Lymphoma after Autologous Stem Cell Transplantation - a Nationwide Retrospective Analysis in Singapore

Introduction Co-expression of C-MYC and BCL2 in diffuse large b-cell lymphomas (DLBCL), also termed double-expressor lymphomas (DEL), has been shown to be associated with poorer outcomes after standard R-CHOP induction therapy. Approximately 70% of DEL will relapse within 5 years. The subsequent traditional approach with potential for cure has been high dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT). Whilst factors such as remission status after front-line treatment and early relapse have been associated with poorer outcomes after ASCT, data are limited regarding significance of DEL status on survival outcomes post-ASCT. We retrospectively studied the prognostic impact of DEL status on outcomes in patients with relapsed or refractory (R/R) DLBCL who underwent ASCT. Methods This was a retrospective, nationwide study of adults patients, &amp;gt;18 years old, with R/R DLBCL who underwent autologous hematopoietic stem cell transplantation (ASCT) between 2010 and 2022 at the only 2 transplant centers in Singapore - Singapore General Hospital and National University Cancer Institute. Patients with double/triple hit lymphoma, defined as concurrent rearrangements of MYC and BCL2 and/or BCL6 by fluorescence in situ hybridization (FISH) analysis, were not included in this study. Patients with transformed indolent B-cell NHL (non hodgkin lymphoma), primary mediastinal B-cell lymphoma, primary CNS (central nervous system) lymphoma, or Richter transformation of chronic lymphocytic leukemia were excluded. Only patients that had available MYC and BCL-2 immunohistochemistry (IHC) were included in this study. Overall survival (OS) and progression free survival (PFS) were estimated using the Kaplan-Meier method. Cox proportional hazards regression models were used to assess associations between covariates of interest and OS or PFS. Results A total of 72 patients were included, 44 (61.1%) non-double expressor DLBCL (non-DEL) and 28 (38.9%) double expressor DLBCL (DEL). Patient demographics and disease characteristics at relapse were similar in both groups aside from a higher incidence of CNS involvement in the non-DEL group as compared to DEL group, 34.1% vs 10.7% (Table 1). Both groups achieved a &amp;gt;90% response (CR and PR) after second-line salvage chemotherapy as well as a &amp;gt;90% response post-transplant (CR and PR). Patients with DEL however were more likely to relapse post-transplant, 69.7% vs 40.9% (p = 0.032) and were also associated with increased odds of all-cause mortality, 60.7% vs 36.4% (p=0.043). This significance was maintained on multivariable analysis, HR 4.22 95% CI 1.20 - 14.84 (p=0.025) and HR 4.32 95% CI 1.10 - 17.15 (p=0.038) respectively. The median follow up for survivors were 42 months (range, 26 to 64 months) in the DEL patients and 60 months (range, 46 to 88 months) in the non-DEL patients. The 5-year PFS in patients with DEL was inferior to non-DEL patients, 23.3% vs 49.6% respectively, p=0.022. DEL patients were also associated with inferior 5-year OS, 40.2% vs 65.7%, p=0.041. In multivariable model, DEL status remained significantly associated with inferior PFS and OS. We also observed that patients with CNS involvement at relapse had inferior PFS and OS. Male gender and a less than complete metabolic response (CR) status prior to transplant, assessed on PET-CT, was also associated with inferior OS. Conclusion Co-expression of C-MYC/BCL2 was associated with inferior outcomes after ASCT in patients with R/R DLBCL. While ASCT has been the standard curative approach in the R/R setting, considerations for novel targeting therapies to achieve a deeper response prior to transplant or the upfront use of chimeric antigen receptor T-cell therapy at first relapse may be further explored in this high-risk subset of patients.

Resistance Mechanisms Impacting Bispecific Antibody (BsAbs) and Chimeric Antigen Receptor (CAR) T-Cell Therapy Outcomes in Large B Cell Lymphoma (LBCL) Patients

Introduction: BsAbs and CAR T-cell therapy have revolutionized the treatment landscape of patients with relapsed/refractory (R/R) LBCL. Regrettably, less than 40% of patients will attain durable responses, and determinants of response remain unclear. Our aim was to identify tumoral characteristics that can predict resistance to CAR T-cell therapy and BsAbs in LBCL patients. Methods: We retrospectively collected clinical data and obtained pre-treatment formalin-fixed and paraffin-embedded specimens prior CAR T-cell and BsAb treatment from 56 LBCL patients from 4 centers. Targeted NGS was performed to identify variants and copy number variations (CNVs) across a custom gene panel of 200 genes. These include lymphoma related genes, and genes involved in tumor antigen presentation, B and T lymphocyte interaction, and cell death pathway. Variables assessing response where Overall Response Rate (ORR), Complete Response Rate (CRR), Progression-Free Survival (PFS), and Overall Survival (OS). To ascertain each gene's influence on these endpoints, a logistic model was used for ORR and CRR comparisons, while Kaplan-Meier and Cox methods were employed to analyze PFS and OS. Results: A total of 56 patients were included in the study, 36 patients treated with CAR T-cell and 36 with BsAbs (16 patients received both). Samples were obtained for 31 patients prior to CAR T-cell therapy (CAR T-cell cohort) and 34 cases prior to BsAbs (BsAbs cohort). Baseline characteristics of the patient population are shown in Table 1. In summary, median age was 62 years, 57% were male, 42% had a previous indolent lymphoma and median previous lines were 2 (range 1-4). Best response included CRR in 59%, with a median PFS and OS of 13.6 (CI95% 5.98 - Not reached [NR]) and 29.5 (CI95% 15.7 - NR) months, respectively. Median follow-up for the full cohort was 18.7 months (CI95% 15.1 - 24). As per the CAR T-cell cohort, ORR and CRR were 81% and 68%, respectively. Median PFS and OS for CAR-T-cell cohort was 29.4 months (CI95% 5.98 - NR) and NR, respectively. Regarding the BsAb cohort, ORR and CRR were 66% and 50%, respectively. Median PFS and OS was 10 (CI95% 5.16 - NR) and 22.5 (CI95% 14.4 - NR) months, respectively. For the full cohort, the most frequently altered genes, considering both mutations and CNVs, were KMT2D (66%), TP53 (60%), CREBBP (58%), IGLL5 (51%), REL (42%), EZH2 (38%), BCL2 (38%), TNFRSF14 (37%), SGK1 (32%) and KLHL6 (32%) (Figure 1). The most frequent CNVs were gains of 2p16.1 (34%), 3q28 (15%), 6p(14%), 11q (12%), 21q (12%), 3q (10%); and losses of 17p (32%), 6q (26%), 1p36.32 (25%), 13q14.2 (18%), 19p13.3 (18%), 4q35.1(16%), (10q23.31 (12%), 9p21.3 (10%). Focusing on the CAR T-cell cohort (n=31), mutations and/or deletions in EP300 (7%, n=2)( p=0.01) and KLHL6 (n=2, 7%) ( p=0.04) were associated with an inferior PFS. In addition, mutations and/or deletions in KLHL6 ( p=0.02) and EP300 (n=3, 10%) ( p=0.05), and amplifications in MYC (n=3,10%) ( p=0.031) were associated with shorter OS. No differences in PFS and OS were oberved in patients with mutations and/or deletions in TP53 (n=19, 63%). Regarding RHOA patients with mutations or deletions (n=2, 7%) did not present an inferior PFS ( p=0.88) or OS ( p=0.52). The analyzed alterations did not show an impact on response rate. In terms of patients who received BsAbs (n=34), we observed that mutations and/or deletions in TP53 (n=19, 58%) ( p=0.04) , RHOA (n=5, 15%) ( p=0.05) and GNAI2 (n=5,15%) ( p=0.02)were associated with aninferior PFS. Mutations and/or deletions in TP53 (p=0.04) , RHOA (p=0.009) , GNAI2 ( p=0.01) and CD274(PDL1)-PDCD1LG2(PDL2) (12%, n=3) ( p=0.01) were associated with an inferior OS. Regarding MYC, 8 cases were mutated (n=2, 6%), amplified (n=5, 15%) or both (n=1, 3%), but these alterations were not associated with an inferior PFS (p=0.46) or OS ( p=0.27). Again, no impact on response rate was observed for patients carrying alterations. Conclusions: In our study, a pattern of genetic alterations predicting worse OS was observed in patients treated with CAR T-cells ( KLHL6 EP300 and MYC) and BsAbs (TP53, RHOA, PDL1, PDL2, GNAI2). This important information will aid in the development of targeted strategies to overcome resistance and improve treatment outcomes.

A Single Center Real World Study of Outcome and Resistance of Bruton Tyrosine Kinase Inhibitors (BTKi) in Chinese Patients with Chronic Lymphocytic Leukeima/Small Lymphocytic Lymphoma

Introduction Acquired BTK/PLCG2 mutations remain to be the driver of Bruton Tyrosine Kinase inhibitor (BTKi) resistance. But the underlying mechanisms need to be further explored. In this study, we retrospectively analyzed clinical and biological characteristics of BTKi-treated CLL patients and report the outcome and resistance in our center (the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital). Method 262 patients treated with single BTKis continuously were collected and follow up the outcome in our center. Progression-free survival (PFS) was defined as from BTKi initiation to disease progression on BTKi. Next-generation sequencing (NGS) and High sensitivity droplet digital PCR (ddPCR) acquired at treatment initiation /or at the time of disease progression were used to describe mutational landscape. Kaplan-Meier method and the log-rank test were used for analysis. Results A total of 262 patients were enrolled from January 2014 to March 2023, and the median age was 63 (range 28-88) years. Ibrutinib, Zanubrutinib, Orelabrutinib were administered in 55.3%,18.3% and 17.9% of patients respectively, in addition to 6 patients with Acalabrutinib and one with Loxo-305, among them 47.7% (125/262) received BTKi as first line treatment. With a median follow up of 27.3 months, 208 patients were in durable remission while 54 patients relapsed, the median exposure of BTKi was 26.8 (range, 1-73.8) sand 27.7 (range, 3-69.8) months respectively. The median PFS was 5.8 years (5.0-NA, 95% CI) for the whole cohort. Clinical and biological characteristics of both cohorts at initiation of BTKi treatment were described and compared in Table 1. Higher proportion of bulky disease (50.0% vs 23.5%), IGHV unmutated status (84.3% vs 50.5%), del(17p) (44.7% vs 12.9%), TP53 mutation (37.8% vs 15.2%), TP53 aberrations (62.5% vs 24.3%) and complex karyotype (40.5% vs 21.1%) were shown in the BTKi relapsed cohort as compared to responsive cohort. Inferior PFS was observed in patients with TP53 aberration vs without TP53 aberration ( 45.6 mos vs not reached, p&amp;lt;0.0001), unmutated IGHV vs mutated IGHV (56.2 mos vs not reached, p=0.0005), with del(17p) vs without del(17p) (36.1 mos vs not reached, p&amp;lt;0.0001), with complex karyotype and without complex karyotype (69.8 mos vs not reached, p=0.0401), bulky disease vs no bulky disease (45.6 mos vs 69.8 mos, p=0.0006) and elevated LDH vs normal LDH (66.5 mos vs not reached, p=0.0083). Moreover, patients with del(11q) or BTKi given as second or greater line showed a tendency of inferior PFS ( p= 0.1963, p= 0.0735, respectively). Regarding the relapsed cohort, 43 patients displayed CLL progression while 11 patients displayed Richter transformation (RT, including 1 Hodgkin lymphoma [HL]) , the median time from initiation to progression were 33.7 months and 9.5 months, respectively（p&amp;lt;0.0001). BTK/PLCG2 mutations were the most common mutation at the timepoint of disease progression. By integrating NGS and ddPCR results, 54.9% (28/51) relapsed patients acquired BTK/PLCG2 mutation. BTKC481S mutation was the dominant hotspot mutation in all kinds of BTKi relapsed cases. Other mutations at the C481 site included C481R, C481Y, C481F. However, BTKT474 mutation was only detected in Orelabrutinib-resistant cases (Figure 1). 6 patients presented with multi-hit mutations. Treatment after disease progression on BTKis is still challenging. In our relapsed cohort, the median PFS was 5.7 months and poorer in RT as compared with CLL progression (3.0 months vs 6.7 months, p= 0.0874). Venetoclax-based regimen might be an effective salvage therapy which could showed a benefit trend in PFS as compared to other post BTKi therapy (10.1 months vs 3.1 months, p= 0.1818). Conclusion In our real world study, patients with IGHV unmutated status, TP53 aberrations, bulky disease and elevated LDH showed inferior PFS. Acquired BTK/PLCG2 mutations remained to be key drivers of BTKis resistance and BTKC481S mutation was the dominant mutation, while BTKT474 mutation was only detected in Orelabrutinib-resistant patients. The prognosis was rather poor for relapsed patients especially for RTs, treatment strategy after disease progression remains to be optimized.

Early Peripheral Blood Minimal Residual Disease Status By NGS in Patients with Newly Diagnosed Multiple Myeloma (MM) on a Phase 2 Trial Receiving Elotuzumab, Carfilzomib, Lenalidomide, and Dexamethasone (Elo-KRd)

Background: Assaying for minimal residual disease (MRD) in the peripheral blood (PB) of patients with multiple myeloma (MM) could solve issues of patchy and extramedullary disease leading to false-negative MRD tests based on bone marrow (BM) only assessments. However, PB testing is challenging due to limited circulating myeloma cells and/or paraprotein. Further, early PB assessments for MRD by paraprotein are unreliable due to the extended half-lives of immunoglobulins. In this analysis, we investigated the concordance and prognostic significance of early MRD status after 4 cycles of treatment with Elo-KRd by next generation sequencing (NGS) in both the BM and PB and by International Myeloma Working Group (IMWG) response criteria. Methods: All patients received 12 cycles of Elo-KRd in 28-day cycles without intent for stem cell transplant: Elo per standard dosing; K 56/70 mg/m 2 (highest tolerated) days 1, 8 and 15 (20 mg/m 2 on day 1 of cycle 1) ; R 25 mg days 1-21; and dexamethasone 40 mg days 1, 8, 15, 22. An MRD-adapted design was used to determine the duration of carfilzomib as previously reported (Derman et al. JAMA Oncology 2022). MRD by NGS in BM (sensitivity threshold 10 -6,clonoSEQ, Adaptive Biotechnologies) was performed at multiple timepoints per protocol. PB mononuclear cells were evaluated for MRD using the same assay; any quantifiable signal was considered positive in PB. Conventional response was assessed per IMWG response criteria. Paired IMWG response and MRD status in the PB and BM were assessed for concordance and prognostic significance at the end of cycle 4 (C4). The Kaplan-Meier method was used to assess progression free survival (PFS) based on a landmark assessment at C4. Results: A total of 46 patients initiated treatment with Elo-KRd; patient baseline characteristics were reported previously (Derman et al. JAMA Oncology 2022). With a median follow-up of 39 months, estimated 4-year PFS was 75%. Of the 42 patients in response at C4, 39 (93%) achieved &amp;gt;VGPR, and 13 (31%) reached &amp;gt;CR. When stratified by &amp;gt;CR vs &amp;lt;CR at C4, there was no difference in PFS (HR 1.4, 95% CI 0.3-6.9, p=0.7). BM MRD status at C4 was available for 34 patients: 11 (32%) patients were BM MRD(-)and 23 (68%) were MRD(+). Matched PB MRD samples at C4 were available for 31 patients; 22 (69%) patients were PB MRD(-) using the best available threshold based on sample input (16 at 10 -6, 6 at 10 -5). Of the 9 (31%) patients with PB MRD(+), 6 (67%) had at least one high-risk cytogenetic abnormality (del17p in n=4, +1q in n=2, t(4;14) in n=1). Extramedullary disease relapse was a manifestation of progression in 4 (44%) patients. BM MRD(-) at C4 was associated with superior PFS (HR not estimable, p=0.04; Figure 1A). No patient with MRD(-) BM after C4 had disease progression. PB MRD(+) at C4 was also associated with inferior PFS (HR 21, 95% CI 2.3-184, p=0.002; Figure 1B). In comparing BM and PB MRD status, 8/9 (89%) patients with PB MRD(+) at C4 had BM MRD(+), and 14/22 (61%) patients with BM MRD(+) at C4 had PB MRD(-). 3/9 (33%) patients with PB MRD(+) at C4 were in &amp;gt;CR; 16/22 (73%) patients with PB MRD(-) at C4 were in a PR/VGPR. With progression serving as the reference standard, PB MRD(+) carried a sensitivity of 71%, a specificity of 83%, a positive predictive value of 56%, and a negative predictive value of 91%. There were 19 matched BM MRD(+)/PB MRD(+) samples across all timepoints; Pearson's correlation coefficient of detected cells per million between BM and PB MRD was r=0.93. BM MRD detected aberrant cells at higher concentrations, with a median log difference of 1.38 compared to PB MRD (range 0.07-3). Conclusions: MRD by NGS in the PB was less sensitive compared to the same assessment in the BM by 1-2 logs; however, PB MRD status following 4 cycles of induction therapy was strongly prognostic given its association with PFS. PB MRD status early in treatment may represent a leading indicator of early response and/or a marker of high-risk disease features, and validation of these findings may help to eventually guide intensification of therapy.

Real-World Outcomes of Zanubrutinib Monotherapy in Chronic Lymphocytic Leukemia: A Multicenter Retrospective Study

Background The second-generation Bruton's Tyrosine Kinase inhibitor (BTKi) zanubrutinib had better BTK specificity and less off-target inhibition than first-generation BTKi ibrutinib. SEQUOIA study demonstrated superior efficacy of frontline zanubrutinib than bendamustine-rituximab combination, and ALPINE study showed improved overall response rate (ORR) and progression free survival (PFS) of zanubrutinib than ibrutinib in relapsed/refractory chronic lymphocytic leukemia (R/R CLL). Meanwhile, adverse event (AE) remained one of the main reasons of zanubrutinib dose reduction and discontinuation. We previously reported the first real-world data of zanubrutinib monotherapy in Chinese CLL patients in our single center, demonstrating zanubrutinib discontinuation significantly deteriorated PFS and overall survival (OS). Herein, we reported the updated results to a multicenter scale, with a special focus on the impact of zanubrutinib dose reduction. Methods From Aug. 2020 to Mar. 2023, CLL pts received zanubrutinib monotherapy for at least 3 months in 7 medical centers in Shanghai were enrolled. Data cut-off time was Jul. 2023. AEs were evaluated and graded according to CTCAE 5.0 and IWCLL criteria. ORR included partial response with lymphocytosis or above. Results In total, 124 pts were enrolled and analyzed. 83 (66.9%) were treatment-naïve (TN) pts and 41 (33.1%) were R/R pts. Zanubrutinib was given 160mg, twice a day. The median age of zanubrutinib initiation was 68 years old (range, 37-87) with a M/F ratio of 2.1:1. Among evaluable pts, 25.0% had TP53 aberration (deletion and/or mutation) and 45.2% had unmutated IGHV. With a median follow-up time of 21.8 months, ORR of the entire cohort, TN and R/R pts were 93.5%, 96.4% and 87.8%, respectively. The estimated PFS and OS rates at 24 months in the entire cohort were 79.6% (95%CI 75.0-84.2%) and 94.2% (95%CI 91.9-96.5%). When stratified by line of therapy, the estimated 24-month PFS rates of TN and R/R pts were 82.7% (95%CI 77.5-87.9%) and 71.8% (95%CI 62.1-81.5%), and 24-month OS rates were 96.1% (95%CI 93.9-98.3%) and 89.1% (95%CI 83.1-95.1%), respectively. Pts with TP53 aberration had a trend of inferior PFS ( P=0.102), and pts received 1 st or 2 nd line zanubrutinib monotherapy had significantly longer PFS compared with 3 rd line or above ( P&amp;lt;0.001) (Fig.1A). No significant impact of gender, age, Binet or Rai stages, 11q deletion or IGHV mutation status on PFS or OS was observed. One hundred and two (82.3%) pts experienced AEs of any grade, and 30 (24.2%) pts experienced AEs of grade 3 or above. The most common grade 3 or above AEs were neutropenia (10.5%) and pneumonia (8.1%). Two pts (1.6%) experienced grade 2 atrial fibrillation. AEs of interest (AEI) are shown in Table 1. Overall, 21 (16.9%) pts had zanubrutinib dose reduction, and the most common reason was AE (61.9%). Zanubrutinib reduction was associated with both inferior PFS (Fig.1B) and OS. Nineteen (15.3%) pts had zanubrutinib discontinuation, and the most common reason was progressive disease (57.9%). Conclusion In our real-world study, the efficacy of zanubrutinib monotherapy in both TN and R/R Chinese CLL pts were slightly inferior to that of clinical trials. Pts receiving ≤ 2 nd line zanubrutinib monotherapy had significantly improved PFS. The toxicity spectrum was similar to data previously reported. Despite an improved safety profile, AE remained the main reason of zanubrutinib dose reduction, and dose reduction significantly associated with shorter PFS and OS. Therefore, better treatment strategies such as combined targeted therapy are required to improve the outcomes of these pts.

Atypical Stem Cell, Pre-B-Cell, T-Cell and Myeloid Gene Expression Characterizes Early Waldenstrom's Macroglobulinemia Clones Which Diminishes with Advancing Disease and Has Therapeutic Implications

Background: In previous studies using multi-omic data for 253 treatment-naive patients with Waldenstrom's Macroglobulinemia (WM), we identified three subtypes of WM: B-cell like (BCL), Plasma cell like (PCL), and an intermediate clone enriched for early/smoldering WM from which the other two evolved (Hunter et al, ASH 2022). The BCL and PCL subtypes show distinct mutation and transcriptional signatures, as well as clinical characteristics. Diffusion pseudo-time (DPT) analysis suggests all patient tumors, regardless of subtype have a shared evolutionary path and can be divided into Early and Late DPT determined stages that mirror disease progression. Additional analysis of DPT suggests it represents a continuous process akin to clonal evolution. We therefore sought to further characterize the DPT signal in MYD88 mutated WM. Methods: RNASeq was performed on CD19-selected bone marrow (BM) samples from 249 of the 253 treatment-naive WM patients who were MYD88 mutated, as well as 13 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood samples. Whole exome sequencing of CD19-selected BM cells along with CD19-depleted peripheral blood mononuclear controls was also performed for 215 of the WM samples. To assess BM histology, slides from 66 BM paraffin blocks from these WM patients were analyzed. Results: WM patients had median age of 66 (range 31-95 years), BM involvement of 50% (5-95%) and serum IgM level of 3,162 (104-10,321 mg/dL). The median follow-up for the study cohort was 8.8 (range 0.2 - 33.8 years). Patients were stratified into 5 cohorts by DPT values. Analysis of BM involvement by DPT cohorts revealed a stepwise increase with the median involvement rising from 20% in the lowest to 80% in the highest cohort (p&amp;lt;0.0001). Increasing DPT predicted for shorter time from biopsy to first therapy (p=0.0003) but was not predictive of time from diagnosis to first therapy consistent with DPT alignment by stage of WM development. RNASeq analysis revealed high expression of genes atypical of mature B-cells that diminished with increasing DPT ( Figure 1). Among these were the myeloid chemo-attractants CXCL1, CXCL8 and CXCL12 which fell from a median of 4.4, 67.2 and 41.1 to 0.1, 3.6 and 0.8 transcripts per million (TpM) in the lowest and highest DPT cohorts, respectively (p&amp;lt;0.0001). Accordingly, all the top 20 gene ontology (GO) terms associated with DPT were related to activation, degranulation, and migration of myeloid cells and leukocytes. Pathological review of the 66 BM slides ranked by DPT, showed increased early myeloid cells and eosinophils clustering near small lymphoid aggregates, and as DPT increased, increased lymphocytes and mast cells with decreased myeloid cell involvement was observed. By RNASeq, pre-B-cell genes including MYB, the VDJ recombination genes RAG1, RAG2 and DNTT as well as the surrogate light chain genes IGLL1 and VPREB1 fell from 22.5, 10, 13.5, 61.1, 144 and 38.3 to 2.2, 1, 1.1, 9.1, 12.2 and 4.3 TpM, respectively (p&amp;lt;0.0001). T-cell and myeloid genes including CD4, CD8A, FCGR3A (CD16), CD14 and CD33 also decreased with increased DPT from 67.6, 7.1, 80.5, 200, and 23.2 to 2.4, 0.9, 1.7, 3.6 and 0.7 TpM, respectively (p&amp;lt;0.0005). A subclonal stem cell signature was also detected, including CD34 and KIT (CD117), which fell from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively (0&amp;lt;0.0001). CD34, KIT, preBCR, CD4, CD8A, FCGR3A, CEACAM8 (CD66b), and CD33 expression was confirmed by CD19+ light chain restricted flow cytometry in WM but not in HD samples. In addition, the MB and plasma blast genes PRDM1 and XBP1 were elevated in all samples regardless of DPT, consistent with a putative memory B-cell origin. Progression free survival (PFS) in response to proteasome inhibitors revealed strong stratification by subtype (p=0.001), as well as inferior PFS with BTK-inhibitors in low DPT patients (p= 0.044; Figure 2), suggesting less BTK dependence in the early DPT clones. Consistent with this finding, gene set enrichment identified increased BCR signaling with increased DPT. Conclusions: This analysis suggests that stem cell program reactivation and atypical marker expression characterize early WM clones that are ultimately replaced by clones exhibiting more typical B-cell markers with disease progression. The findings may be relevant to treatment selection and provide a framework for investigating subtype and early/late evolutionary staging in the treatment approach to WM.

Novel Drug to Target HK2 Overcomes Therapeutic Resistance in Preclinical B-Cell Lymphoma Models

Background: The Warburg's effect is a hallmark in cancer. Hexokinase II (HK2) is a glycolic kinase to phosphorylate glucose to glucose-6-phosphate, which can be utilized as the energy source for cells. HK2 is predominantly overexpressed in cancer cells and has been associated with poor clinical outcomes in cancer patients. Previously, we demonstrated that HK2 mRNA and protein levels were increased in diffuse large B-cell lymphoma patients, and were significantly associated with inferior progression free survival (PFS) and overall survival (OS). In our rituximab and chemotherapy-resistant cell lines (TRCL), we demonstrated an upregulation of HK2 expression level is association with voltage-dependent anion channel (VDAC) resulting in a) repress mitochondrial potential and b) global therapy resistance. Knocking out HK2 overcame the chemo-resistance phenotype observed in TRCL. Here, we designed a novel specific inhibitor of HK2 and tested its activity in lymphoma pre-clinical models. Material and Methods: A HK2 specific inhibitor (HK2-001) was designed and generated according to antisense peptide technology, CABS-docking system and ROSWTTA analyzing. A panel of B-cell lymphoma cell lines including rituximab/chemo-sensitive and resistant cell lines (Raji, Raji 4RH, RL and RL 4RH), diffuse large B-cell lymphoma (DLBCL) (DHL4, DOHH2, TMD8 and U2932) and mantle cell lymphoma (MCL) (HBL2, Granta, Mino and Z138) were used. In addition, primary tumor cells isolated from chronic lymphocytic lymphoma (CLL) or other B-cell lymphoma patients were used. Briefly, cell lines or primary tumor cells were exposed to escalating doses of HK2-001 alone or in combination with different chemotherapy agents (doxorubicin, dexamethasone), targeted agents (i.e. burton tyrosine kinase inhibitors, BH3 mimetics) or monoclonal anti-CD20 antibodies. Cell viability was evaluated using the Presto Blue assay in experiments testing pharmacological interactions between HK2-001 and chemotherapy or targeted agents. All experiments were performed in triplicates in three separate experiments. The coefficient of synergy was calculated using CalcuSyn. Results: In vitro exposure of lymphoma cell lines to HK2-001 induced cell death in a panel of B-cell lymphoma cell lines representing rituximab/chemo-sensitive and resistant lymphoma. In addition, synergistic activity was observed between HK2-001 and doxorubicin, ibrutinib, and Venetoclax. These effects were also observed in primary tumor cells isolated from B-cell lymphoma patients. In addition, HK2-001 potentiated the anti-tumor activity of Rituximab and Obinutuzumab ex vivo. Conclusion: We developed a novel peptide targeting HK2. Pre-clinical studies suggested anti-tumor activity as a single agent. More importantly, HK2-001 enhanced the anti-tumor activity of chemotherapy agents, BTK inhibitors, BH3 mimetics and monoclonal antibodies targeting CD20. HK2-001 is a promising novel therapeutic strategy for against B-cell lymphoma, especially in refractory or relapsed patients. Future studies aimed to evaluate the effects of HKII-001 drug combinations in lymphoma animal models are planned and to evaluate its effects on Chimeric antigen receptor T-cells (CART) or bi-specific antibodies.

Early Subclones Showing Aberrant Co-Expression of Stem Cell, T Cell and Myeloid Genes Can be Detected By Flow Cytometry in MYD88 Mutated Waldenström's Macroglobulinemia Patients

Background: Our RNA-Seq analysis of 249 untreated MYD88 mutated WM patients showed aberrant expression of stem cell, pre-B-cell, T-cell and myeloid genes in early WM B clones. This expression pattern was associated with inferior progression free survival with BTK inhibitor therapy and diminished with advancing disease. We therefore sought to validate this early signature at the protein level using flow cytometry and evaluate its potential as a clinical assay to monitor WM disease evolution. Methods: RNA-Seq predictions were validated on an independent cohort of treatment naïve MYD88 mutated WM patients and healthy (HD) samples. We designed a 13-color panel for flow cytometry detection of surface markers CD19, CD138, k and l immunoglobulin (Ig) light chains, CD34, CD117 (KIT), CD4, CD8A, CD33, FCGR3A (CD16), CEACAM8 (CD66b), pre-BCR (using an antibody that recognizes a unique conformational epitope of the BCR complex formed by the association of IGLL1 and VPREB1); Zombie Yellow was used to label dead cells. Peripheral B (PBMC) and/or BM mononuclear cells (BMMC) were isolated from WM and HD samples by density gradient centrifugation using Ficoll-Paque medium. Data acquisition was performed for at least 10^6 nucleated cells per tube in BDLSR Fortessa flow cytometer using the FACSDiva software. Results: RNA-Seq analysis showed high expression of genes atypical of mature B-cells that significantly diminished with increasing disease burden. For validation purposes, we selected genes reaching at least a 10-fold drop in their expression levels between the earliest and the most advanced disease cohort. In particular, the T-cell and myeloid genes including CD4, CD8A, FCGR3A, CD33, CEACAM8 as well as the prePCR surrogate light chain genes IGLL1 and VREPB1 respectively decreased from 67.6, 7.1, 80.5, 23.2, 8.3, 114, 38.3 to 2.4, 0.9, 1.7, 0.7, 0.4, 12.2 and 4.3 transcripts per million (TpM). A subclonal stem cell signature was detected, including CD34 and KIT, which dropped from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively. It is well established that WM represents a mature, post germinal center clone. Consistent with this notion, expression of memory B-cell and plasma blast genes in our RNASeq data set, including PRDM1, XBP1 and SPIB, was high in all patients regardless of disease progression and expression of the studied atypical genes. Our independent patient validation cohort had a median age of 68 (range 50-88 years), BM involvement by immunohistochemistry of 40% (range 10-95%), hemoglobin of 13.2 (range 8.1-13.4 g/dL) and serum IgM of 686 (range 536-1,151 mg/dL). An IgMk monoclonal spike was detected in all cases. All patients were mutated for MYD88. Within the CD19+k+-selected cells, we detected a subpopulation of cells co-expressing CD34, KIT, CD4, CD8A, FCGR3A, CD33, CEACAM8 and preBCR in all the patients, including in PBMC. As predicted, these markers were relatively enriched in samples with lower BM disease burden ( Figure 1, 2). Even in those with the highest BM involvement, the subclone was still detectable in both cellular compartments. Conversely, HD controls showed normal B cell populations and no evidence of the aberrant subclone. Conclusions: In WM primary cells, we observed the existence of a clonal B cell population that appears to re-activate stem cell programming early on and progressively is replaced by clones bearing more typical B cell markers with disease expansion. This study lays the foundation to develop a widely accessible methodology to accurately diagnose IgM MGUS/early-stage WM and monitor disease evolution. Flow-based sorting of these subclones will allow targeted multi-omic studies with insights into disease pathogenesis and targeted therapeutic applications.

Outcomes of B-Cell Acute Lymphoblastic Leukemia Patients Who Relapse after Blinatumomab Salvage Therapy - a Tertiary Centre Experience

Introduction In recent years, the treatment of B-cell acute lymphoblastic leukemia (B-ALL) has evolved tremendously resulting in improved outcomes in patients with relapse/refractory (r/r) B-ALL and patients with measurable residual disease (MRD+). One such novel therapy is the CD3/CD19 bispecific antibody blinatumomab. There is however limited data on the outcomes and treatment options for B-ALL patients who relapse after blinatumomab was given as salvage therapy. Methods This IRB-approved study retrospectively reviewed the outcomes of all adult patients (≥18 years old) with B-ALL treated with blinatumomab at our center, Singapore General Hospital. The primary aim was to describe the incidence, risk factors and survival outcomes of B-ALL patients who relapse after blinatumomab salvage therapy. Results Between January 2017 to December 2022, a total of 27 patients were treated at our center with blinatumomab. Blinatumomab was used as salvage therapy in 13 patients and for MRD+ disease after front-line therapy in 7 patients. Characteristics of the 13 patients who received blinatumomab salvage therapy are shown in Table 1. Eight patients had received an allogenic hematopoietic stem cell transplant (alloHSCT) and 6 patients had received inotuzumab, a CD22 antibody-drug conjugate, prior to blinatumomab salvage. The median number of lines of therapy before blinatumomab salvage was 3 and the median number of cycles of blinatumomab administered was 4. All but 1 patient achieved MRD negativity after the prescribed number of blinatumomab cycles. Eight (61.5%) of 13 patients relapsed after blinatumomab salvage therapy. Median progression free survival (PFS) in this group of patients was 7 months (IQR 2-11) and overall survival (OS) of 19 months (IQR 8-26). Six (75%) of these 8 patients proceeded to receive salvage therapy with inotuzumab. Majority (5 of 6) achieved MRD negativity and 2 patients subsequently underwent alloHSCT and CAR-T therapy respectively. Unfortunately, all 6 patients eventually relapsed. Of note, 3 patients had received inotuzumab prior to blinatumomab salvage. We observed that patients (n=3) with extramedullary disease (EMD) (including central nervous system (CNS) involvement) at initiation of blinatumomab salvage therapy were associated with higher odds of both systemic and extramedullary relapse (p=0.014). These patients had inferior progression free survival (PFS) as compared to patients without EMD/CNS involvement, median PFS 2 months (95% CI 2 - 11) vs 21 months (95% CI 4 - 21), p = 0.041 (Figure 1). A trend towards inferior overall survival was also observed though statistical significance was not demonstrated, median OS 19 months (95% CI 8 - 19) vs 26 months (95% CI 14 - 26). Conclusion EMD/CNS involvement prior to blinatumomab salvage therapy was associated with higher risk for systemic and EMD/CNS relapse. Patients with B-ALL who relapsed after blinatumomab salvage could be successfully rescued by inotuzumab regardless of prior inotuzumab use. These groups of patients however represent very-high risk groups with inferior survival outcomes. Novel treatments and better sequencing of treatments may improve outcomes of these patients.

Unraveling Diverse Mechanisms of Complex Structural Variant Interactions through Multiomic Data in Multiple Myeloma

Introduction: Structural variants (SVs) play a role in proliferation of plasma cells that characterizes multiple myeloma (MM). Several hallmark SVs have been identified in MM, including translocations involving the immunoglobulin heavy chain locus and several partner genes, as well as deletions and duplications affecting key genes such as TP53, RB1, and MYC. Complex SVs such as templated insertions, chromothripsis, and chromoplexy are associated with disease progression but have not yet systematically been studied. Methods: Paired short-read (Illumina, average depth 75x), long-read HiFi (PacBio, 16x), optical genome mapping (OGM; Bionano Genomics, 341x) and Micro-C (Cantata Bio, 45x) data were sequenced from 8 relapsed MM patient-derived xenograft samples and 2 cell lines, covering t(11;14), t(4;14), and hyperdiploid subgroups. Reads were aligned to the hg38 reference genome while complex SVs (chromothripsis, chromoplexy and templated insertion) were systematically identified and accurately reconstructed by combining short-read (Manta), HiFi (pbsv), OGM, and Micro-C data (EagleC). Chromosome organization including loops, topologically associated domains (TADs) and A/B compartments were called from Micro-C data (Mustache and Juicer). Haplotype phasing was conducted using small variants detected from HiFi data. De novo assembly of the genome was conducted using HiFi and Micro-C data and compared to the reference genome. Reconstructed derivative chromosome interactions due to complex SVs were further identified by aligning Micro-C reads to the newly assembled genomes. Previously identified super-enhancers from H3K27Ac ChIP-seq from MM cell lines were mapped onto these SVs and expression of target genes assessed by RNA-seq. Results: 1,340 SVs were found in 10 samples using OGM of which 828 were identified in short-read WGS data. 16 complex SVs including chromothripsis, chromoplexy and templated insertions were detected in 10 samples, and we describe two (Fig.1) in detail here. A complex templated insertion consisting of a t(2;6;8) was found in a t(11;14) PDX sample with copy number gains at breakpoints which could not be resolved from short-read data alone. OGM data revealed a single contig consisting of DNA from chromosomes 2, 6, and 8. Micro-C data revealed interactions between these chromosomes, specifically the gained regions on chromosomes 2 and 8 showed more interactions with chromosome 6 than with other chromosomes, indicating insertions into chromosome 6. As a result, both MYC at chromosome 8 and GALM at chromosome 2 were inserted into the TXNDC5 locus, containing a known MM super-enhancer, leading to the over-expression of both MYC and GALM (log 2FC=1.7 and 1.0) in this sample. A de novo genome assembly indicated neo-TAD formation encompassing the super-enhancer and both genes. In coMMpass dataset, high expression of GALM and MYC are significantly associated with inferior overall (p=0.002 and 0.05, logrank test) and progression-free survival (p=0.01 and 0.09). Another complex templated insertion consisting of a t(3;4;16) was identified in a hyperdiploid PDX sample, again with copy number gains at breakpoints. Although the short-read data indicated an assembly similar to the previous templated insertion SV, the OGM and Micro-C data indicated a 3-way chromosome swap where the short arm of chromosome 3 was translocated to the short arm of chromosome 4, the short arm of chromosome 4 was translocated to the long arm of chromosome 16, and the long arm of chromosome 16 was translocated to the short arm of chromosome 3. Separate contigs for the t(4;16), t(3;16) and t(3;4) were confirmed by OGM data and only 2-way chromosomal interactions were seen in the Micro-C data. This complex event led to numerous oncogenes juxtaposed to super-enhancers, which caused over-expression of target genes such as ACD and RANBP10 (log 2FC=1.0 and 0.4), both of which were on chromosome 16 and are significantly associated with inferior overall (p=0.02 and p=0.004) and progression-free survival (p=0.003 and p=0.005). Conclusion: By utilizing multiomic techniques we were able to reconstruct complex SV events, showing different mechanisms of generating templated insertion events and identifying their biological consequences. We demonstrated how these complex events could potentially facilitate tumor proliferation via engaging the super-enhancers and regulating the expression of key oncogenes.

Outcomes of Patients with Relapsed/Refractory Lymphoplasmacytic Lymphoma Treated with Venetoclax: A Multicenter Retrospective Analysis

Outcomes of Patients with Relapsed/Refractory Lymphoplasmacytic Lymphoma Treated with Venetoclax: A Multicenter Retrospective Analysis

Heterogeneity of TP53 Mutations in Mantle Cell Lymphoma- Challenges in Risk Stratification and Subclassification

Introduction: Somatic mutations in TP53 occur in approximately 10-20% of mantle cell lymphoma (MCL) patients (pts) at diagnosis. Eskelund and colleagues (Blood 2017) demonstrated poor survival among 20 pts with TP53-mutated MCL following intensive induction and transplant, and reported sequence-level variant data. However, TP53 mutations are heterogeneous, and few studies in MCL provide detailed sequencing data or variant allele frequency (VAF). Furthermore, the clinical impact of TP53 mutations may vary, and there is no algorithm shown to predict outcome for a given variant. We hypothesized that high VAF, or presence of a “disruptive” TP53 mutation as reported in squamous cell carcinoma (SCC; Poeta NEJM 2007), may adversely impact prognosis in MCL. To address this, we analyzed all MCL pts with available TP53 sequencing data at our center, including exact TP53 variant data and VAF, sMIPI score, and classification as disruptive vs not, and impact on survival from time of testing. Methods: Institutional records were analyzed with IRB approval, identifying 65 MCL pts who underwent sequencing for TP53 mutation from any tumor sample, as a single-gene assay or part of a panel. We analyzed VAF, type of mutation, and sMIPI score at time of TP53 testing. We classified TP53 mutations as “disruptive” or not according to method of Poeta and colleagues; these mutations result in a STOP codon, or an amino acid polarity change in the L2-L3 DNA binding domain of the TP53 protein (Poeta NEJM 2017). The Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS) from time of TP53 testing to death or last follow up, and the log-rank test was used to compare outcomes based on TP53 mutational status. Among TP53 mutated cases, Cox proportional analyses were performed to explore the impact of the following covariates on survival: disruptive status, VAF, sMIPI score at time of TP53 testing, and presence of complex cytogenetics. Results: Sixty-five pts underwent TP53 sequencing, and 23 (35%) had a TP53 mutation (mTP53+). mTP53+ pts had inferior PFS (p=0.03) compared to TP53 wild-type, and a trend toward inferior OS (p=0.07); median OS was 26 months (95% CI: 8.1, NA) from time of TP53 testing. Ki-67 index, blastoid histology, and age were similar between mTP53+ and wild-type status, but mTP53+ pts had a higher rate of complex cytogenetics (&amp;gt; 3 lesions; 48% vs 26%). SMIPI was also higher in mTP53+ pts (median score 6, interquartile range (IQR) range 4-7) vs TP53 wild-type pts (median 4, IQR range 3-5.2). MTP53+ pts were more likely to have received a BTK-inhibitor (BTK-I) (91 % vs 64%) and CAR-T therapy (39% vs 18%). Details of the mTP53+ subset are shown in Table 1; 4 pts had &amp;gt;1 TP53 mutation, median VAF was 40%, and 30% of pts had a disruptive mutation. Among mTP53+ pts, on univariate analysis, higher sMIPI at time of TP53 testing (p=0.003) was associated with worse OS (Table 2). In a multivariable Cox model including VAF &amp;gt;= 40%, presence of disruptive mutation, sMIPI, and complex cytogenetics, only sMIPI at time of TP53 testing was associated with inferior OS (p=0.02). Conclusion: TP53 variants in MCL are diverse, and in our cohort were largely unique from those reported by Eskelund and colleagues (Blood 2017). Despite now-routine application of sequencing and improvements in therapy, both of which may attenuate the negative prognostic impact of TP53 mutation, we confirmed poor outcomes in mTP53+ MCL. Among mTP53+ pts, only sMIPI predicted inferior OS on both univariate and multivariate analysis. Higher VAF and adoption of a clinical algorithm from SCC failed to discriminate among TP53 variants in terms of prognosis. Larger datasets of uniformly tested MCL pts are needed to elucidate prognosis among TP53 mutation variants, and improve understanding and management of mTP53+ MCL.