Minimal Residual Disease Negative Patients Research Articles

Recommendations on Minimal Residual Disease Monitoring in Acute Promyelocytic Leukemia Treated with Combination of All- Trans Retinoic Acid and Arsenic Trioxide

Background All- trans retinoic acid (ATRA)-arsenic trioxide (ATO) combination therapy significantly reduces acute promyelocytic leukemia (APL) relapse compared to ATRA combined with chemotherapy, leading to a cure rate of 80-90% in APL patients. The ideal frequency and duration of minimal residual disease (MRD) monitoring needs to be identified in the era of ATO. Methods A total of 1072 APL patients were enrolled in this study, comprising 855 from the APL2012 trial (NCT01987297) and 217 from our center between January 2018 to June 2022 (APL2018 study). The APL2018 protocol was adjusted for consolidation and maintenance therapy based on APL2012 (Chen et. al. PNAS 2021): In consolidation therapy, low-, intermediate-, and high-risk patients were performed following the ATO-group scheme of corresponding risk stratification in the APL2012 study. From December 2019, chemotherapy was no longer administered to high-risk group during consolidation phase. In maintenance treatment, ATO was replaced with oral arsenic realgar-indigo naturalis formula, and methotrexate was removed. RT-PCR or RQ-PCR was routinely used for MRD monitoring, detecting PML::RARA fusion genes in bone marrow before each consolidation and maintenance therapy. MRD testing was conducted every 3 months within the first year, every 6 months within the second year, and annually from 3 to 5 years after treatment. Results A total of 993 patients achieved complete remission (CR) after induction therapy. The negative MRD rates were observed in 61.7% of patients before 1 st consolidation , 95.4% before 2 nd consolidation, 99.3% before 3 rd consolidation, and 99.5% before maintenance therapy , respectively. The median time to achieve negative MRD was 57 (22-301) days with no significant difference between relapsed and non-relapsed patients (p=0.166). A total of 34 patients (30 in the APL2012 trial and 4 in the APL2018 study) relapsed, including 5 at low risk, 9 at intermediate risk, and 20 at high risk. The median recurrence time was 13.5 (3.5-49.0) months after achieving CR, with 94% (32/34) relapsing within 3 years of achieving CR. After induction remission, the risk of relapse decreased with time (44% occurred within the first year, 29% within the second year, 21% within the third year, and only 6% after three years, Figure 1). The median follow-up for the 179 evaluable patients after achieving CR was 39.7 (3.5 -64.6) months in the APL2018 group. The estimated 5-year disease-free survival (DFS) rates were 95.8% (95% CI 92.8-98.9). Prior to the 1 st, 2 nd, and 3 rd consolidation treatment, and before maintenance therapy, the estimated 5-year DFS rates for MRD positive and negative patients were 92.4% (95% CI 86.1-99.2) versus 97.8% (95% CI 94.8-100), p=0.100; 57.1% (95% CI 30.1-100) versus 97.3% (95% CI 94.7-100), p<0.001; 0 versus 96.5% (95% CI 93.1-100), p<0.001; 0 versus 96.8% (95% CI 94.0-99.6), p<0.001, respectively. The results of the APL2012 study were similar to those of APL2018. In the APL2018 study, the estimated 5-year cumulative incidence of relapse (CIR) was 2.51% (95% CI 2.48-2.54), which was 0%, 1.16% (95% CI 1.13-1.19), and 7.83% (95% CI 7.44-8.22) in the low-risk, intermediate-risk, and high-risk patients, respectively (p=0.057). The estimated 5-year CIR for the low to intermediate risk group was 0.74% (95% CI 0.73-0.75), which was statistically different from high-risk patients (p=0.019). The estimated 5-year CIR for MRD positive and negative patients prior to the 1 st and 2 nd consolidation treatments was 4.92% (95% CI 4.76-5.08) versus 1.10% (95% CI 1.08-1.12), p=0.194; 14.29% (95% CI 10.30-18.28) versus 2.08% (95% CI 2.05-2.11), p=0.010, respectively. Conclusion Our results show that MRD after one course of consolidation treatment, rather than before, is a crucial predictive factor for APL relapse. Patients with positive MRD after 1 st consolidation therapy have a high risk of relapse. ATRA-ATO based treatment has significantly reduced APL relapse, so the value of frequent MRD monitoring may be limited. It is recommended to implement an optimized MRD monitoring for risk stratification. For low to intermediate-risk patients and those with negative MRD after a consolidation treatment, the frequency and duration of MRD testing can be minimized. For high-risk patients with sustained MRD negativity for more than 3 years, MRD testing can be considered for discontinuation.

Prospective Evaluation of Minimal Residual Disease in Waldenström Macroglobulinemia across Different Tissues and Treatments: Results of the “BIO-WM” Trial of the Fondazione Italiana Linfomi (FIL)

Introduction. MYD88 L265P is the hallmark mutation in Waldenström Macroglobulinemia (WM) and is becoming increasingly important in the management of IgM-gammopathies, due to its role as prognostic and predictive biomarker. Recently, MYD88 L265P detection by allele-specific quantitative PCR was proposed as reliable minimal residual disease (MRD) marker in bone marrow (BM) samples (Varettoni, Hematol Oncol 2022). Novel, more sensitive, techniques as droplet digital PCR (ddPCR) might extend the feasibility of MRD detection in WM also in non-invasive tissues, as peripheral blood (PB) or plasmatic cell-free (cf) DNA. This was a secondary endpoint of the multicenter, observational “BIOWM” (NCT03521596) trial, sponsored by the Fondazione Italiana Linfomi (FIL) and the International WM Foundation/Leukemia and Lymphoma Society. From 2018 to 2020 this trial enrolled 300 consecutive patients with primary diagnosis of WM or IgM-MGUS and a systematic biobanking was performed. Here are presented the first results of the MRD study on the patient subset who received frontline treatment, with the aim of driving correlations with clinical response, type of therapy received and outcome prediction. Methods. Paired BM, PB and plasma samples were collected for each patient at baseline (T0) and follow-up and MYD88 L265P detection was performed by ddPCR in all specimens (Drandi, Haematologica 2018). As of today, 59/300 patients (58 WM and 1 IgM-MGUS) received frontline treatment and MRD levels after therapy (T2) were detected by ddPCR: actually, T0-T2 paired samples were available in 49 (BM), 56 (PB) and 48 (cfDNA) cases, respectively. Moreover, MRD was evaluated also by 8-color multiparametric flow cytometry (MFC) in 23 BM and 24 PB samples with a minimum of 10 million cells acquired. Results. Median age of the 59 treated patients was 68 years (24-85), 32% were female, median IgM value was 2420 mg/dL (101-8840), median Hb 10.5 g/dL (8 -17) and IPSS-WM was high in 35% and intermediate in 46%. MYD88 L265P mutation rate was 94% (46/49) in BM, 80% (45/56) in PB and 90% (43/48) in cfDNA samples, respectively. Treatment was started because of anemia in 32% cases, lymphadenopathy or splenomegaly in 34%, hyperviscosity in 20%, other reasons in 13%, and the only MGUS patient required treatment for anti-myelin-associated glycoprotein polyneuropathy. Thirty-one out of 59 patients received bendamustine-rituximab (BR), 23/59 dexamethasone, rituximab and cyclophosphamide (DRC) or DRC-like regimen and 5/59 a single agent therapy (namely, 3 rituximab, 1 cyclophosphamide, 1 ibrutinib). Overall response rate was 90% (87% for BR and 96% for DRC), with 19% CR, 29% VGPR and 35% PR in BR and 4% CR, 0% VGPR and 91% PR in DRC, respectively. Overall, MRD negativity after treatment was 30% (14/46) in BM, 89% (40/45) in PB and 54% (23/43) in cfDNA, respectively. Moreover, among patients still MRD positive after treatment, median MYD88 L265Ptumor burden shrinkage was about 2 Logs in BM (5.5E-03 vs 2.1E-01) and around 1 Log in cfDNA (5.9E-03 vs 3.7E-02) and in PB samples (3.6E-03 vs 2.5E-02), respectively (Figure 1). Interestingly, the MRD shrinkage in BM was deeper among patients receiving BR (48% MRD negative with a median MYD88 L265P decrease of more than 2 Logs among patients still MRD positive) vs patients receiving DRC (10% MRD negative with a median decrease of 1 Log), Figure 2. Similar trends in favor of BR in inducing MRD negativity were reported in PB (96% vs 88%) and cfDNA (70% vs 35%), respectively. MRD results by MFC were overall in line with ddPCR results: namely 27% of patients reached MRD negativity in BM and 69% in PB. The median follow-up for the whole series was 41 months, resulting in a 3-years PFS of 71% and 3-years OS of 89%, respectively. Interestingly, despite the current limited follow-up for such an indolent disease, MRD positivity by ddPCR in PB predicted a dismal clinical outcome if compared with MRD negative patients (3-years PFS 40% vs 73%, p=0.038). Conclusions. To the best of our knowledge, this is the first report of prospective MRD evaluation in a WM clinical trial. Our preliminary results suggest that MYD88 L265P monitoringby ddPCR is a suitable target for MRD analysis, in the contest of common immunochemotherapeutic schedules. Moreover, different levels of disease persistence were described across BM, PB and cfDNA, with MRD monitoring in non-invasive tissues showing promising predictive value for outcome discrimination.

Impact of Treatment with Inotuzumab Ozogamicin before or after Chimeric Antigen Receptor T-Cell Therapy in Children with Relapsed/Refractory Acute Lymphoblastic Leukemia

Background: Inotuzumab (InO), a monoclonal antibody targeting CD22 conjugated to calicheamicin, is widely used for relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) with good salvage rates (O'Brien, JCO 2022, Bhojwani, Leukemia 2019). Feasibility data on its use as a bridge to Chimeric Antigen Receptor T-cell therapy (CART) or as salvage therapy post CART is limited. Concern has been raised that InO given prior to leukapheresis might impact the quality of T-cell collection and, when used for bridging therapy prior to CART, could result in insufficient B-cell antigen load at the time of CART infusion, therefore reducing efficacy. Methods: Data of patients who received InO monotherapy on Children's Oncology Group study AALL1621 Cohort 1 and who received any form of CART prior to study enrollment or within 1 year of coming off protocol therapy, irrespective of other anti-leukemic treatments, were collected retrospectively using a standardized Case Report Form. Results: Forty-eight patients received InO on AALL1621; 3-year overall survival (OS) rate was 35.4±6.9%. Thirteen received InO prior to CART and thirteen received InO post CART including 1 patient who received CD19 CART prior to InO and CD22 CART post InO (Table 1). InO prior to CART (11 CD19 CART, 1 CD22 CART, 1 unknown): Seven patients went directly to CART post InO, 5 received intervening chemotherapy, and 1 had an intervening transplant with relapse post. Nine patients received tisagenlecleucel and 3 received an investigational CART. Patients were infused CART 1-150 days (median 45 days) after coming off protocol therapy. At CART infusion, 10 patients had detectable ALL, only 2 had no CD19 expressing cells at infusion (ALL or normal B-cells) and 1 unknown. At post CART bone marrow evaluation on day 28, 7/12 (58.3%) were in a minimal residual disease (MRD) negative (<0.01%) remission, 1 had MRD=0.021%, 1 had unknown response. Three had residual disease including the single patient who received CD22-targeted CART. Four MRD negative patients who received CART post-InO are alive without relapse at a median of 3.8 years post CART and 1 is alive with a subsequent relapse. At 3 years post CART infusion OS is 53.9±13.8%. T-cells were collected immediately following 3-9 doses of InO in 8/13 (61.5%) patients (1-6 months from date of enrollment on the study), 2 were collected prior to InO and three had unknown collection dates. All had a CART product manufactured successfully. Post CART 7/13 patients did not develop thrombocytopenia (< 50,000/uL); 5 who developed thrombocytopenia had measurable ALL at day 28 post CART or soon after. One other patient went to planned transplant post CART and remained with low platelets and ANC. Almost all patients (11/13) had ANC < 500/uL post CART, of whom 3 did not recover including two with persistent ALL. InO Post CART (11 CD19-targeted CART, 2 CD22-targeted CART): Of the 13 patients who received InO post CART therapy, 7/13 (53.8%) achieved an MRD-negative complete remission (CR) to CART but then developed detectable disease 2-36 months post CART. Five did not have a CR post-CART, 4 of whom enrolled on AALL1621 within one month; 1 received intervening chemotherapy. One had an unknown response. On univariate analysis CR rate with InO therapy was no different for those who received CART prior to InO than those who did not. As well, risk of a dose limiting toxicity event (DLT) or developing sinusoidal obstructive syndrome (SOS) during InO therapy or during subsequent transplant after InO was not different in patients with CART exposure prior to AALL1621 enrollment. Conclusion: The ability to collect and manufacture CART cells was feasible within weeks of receiving InO. Response to CART post InO was lower than retrospective cohorts (Ceolin, Leukemia 2022) but 3-year OS post CART was consistent with published CD19-targeted CART trials at 53.9% (Maude, NEJM 2018; Pasquini, Blood Adv 2020). InO did not appear to impact durability of remission in those who attained an MRD negative remission with CART. Given most patients had a CD19 target due to disease presence at the time of CART infusion, differential response to CART based upon antigen could not be analyzed. Prior InO exposure did not appear to worsen cytopenias post CART when compared to published CART trials and was associated with a comparable 3-year OS. As well, CART exposure prior to InO did not appear to effect response or increase the risk of DLT or SOS during subsequent InO therapy.

Crebbp Mutations Are Associated with Slow Minimal Residual Response to Upfront Induction Chemotherapy in Pediatric High-Hyperdiploid B-Cell Precursor Acute Lymphoblastic Leukemia Treated in the AIEOP-BFM ALL 2009 Protocol

Introduction High-hyperdiploidy (HHD) represents the most common cytogenetic abnormality found in B-cell precursor acute lymphoblastic leukemia (B-ALL). Despite the overall good outcome of this entity, up to 15% of patients suffer from disease relapse. Genome-wide ALL studies with next-generation sequencing (NGS) approaches identified a distinct pattern of somatic mutations in HHD ALL, enriched in mutations occurring in genes activating the RAS pathway and in CREBBP gene, especially in relapsed patients. Although several studies have attempted to explore the prognostic significance of these mutations that are potentially susceptible of targeted therapies, their impact in uniformly treated patients with newly diagnosed B-ALL is not well characterized, and there is no consensus on their clinical significance. In this study, we aimed to investigate the frequency of recurrent mutations and to define their prognostic role in the setting of newly diagnosed HHD B-ALL, by using end of induction minimal residual disease (MRD) and event-free survival (EFS). Methods We included 101 consecutive patients, aged between 1-17 years, with newly diagnosed HHD B-ALL and enrolled in the AIEOP-BFM ALL 2009 protocol (NCT01117441). HHD was defined by the presence of a DNA index ≥1.16 and lack of ETV6::RUNX1, BCR::ABL1, KMT2A::AFF1 translocations. Diagnostic leukemia samples of 89 patients were sequenced using a validated clinical NGS panel profiling 39 genes involved in leukemia pathogenesis. Mutations affecting RAS pathway ( NRAS, KRAS, PTPN11, NF1, BRAF) and mutations affecting CREBBP gene were included. In addition, digital multiplex ligation-dependent probe amplification (digital MLPA) analysis for the identification of IKZF1 deletion and IKZF1plus phenotype was evaluable for 67 patients. A Fisher exact test and Wilcoxon rank sum test were used for categorical and continuous variables. MRD was assessed by an IG/TR PCR assay at day +33 of induction (timepoint 1, TP1) and at day +78 (timepoint 2, TP2). EFS was estimated by using Kaplan and Meier method and differences between groups were tested using the log-rank test. Resistance to treatment, relapse, death or second malignant neoplasm were considered as events, whichever occurred first. Results Patients' characteristics according to the presence of studied mutations are shown in Table 1. Mutations affecting RAS pathway and CREBBP occurred in 57.3% and 12.3% of HHD B-ALL patients, respectively. Clonal RAS pathway mutations, defined by variant allele frequency (VAF) ≥25%, were found in 16.8% of cases. Among the 11 patients with mutations of CREBBP, 7 had also a mutation affecting KRAS and the association was significant (p=0.03). Seven patients (10.4%) had deletion of IKZF1, of whom 5 (7.4%) were IKZF1plus. There was no association between any mutations or IKZF1 status and patient presenting characteristics, as including sex, age and WBC counts. MRD was evaluable in 84 patients at the end of induction (TP1). MRD level ≥5x10 -4 was found in 36.4% of patients with CREBBP mutations and in 9.6% of those without, and this association was statistically significant (p=0.02). Additionally, MRD negativity was found in 9.1% and 38.4%, respectively. At TP2, MRD was still detectable in 45.5% of patients with CREBBP mutations compared to 20.6% of those with CREBBP wildtype (p=0.08). Similarly, we observed an association between RAS pathway mutations and MRD at TP1 (p=0.01) with a marked difference in MRD negative patients (21.3% vs 51.4% in patients with and without mutations). No association was observed between MRD level and clonal RAS pathway mutations or IKZF1 status. Despite the association between CREBBP mutations and high MRD at TP1, no significant difference in final high-risk group was found (27.3% vs 11.5%, p=0.17). A total of 8 events were observed, of which 6 were relapses. No impact of any mutations or IKZF1 status in term of 5-years EFS was found in our cohort. Conclusion Our study shows that CREBBP mutations in HHD B-ALL are associated with inferior early response to treatment in terms of MRD level at the end of induction (TP1) but not with worse EFS with our whole current chemotherapy backbone. Therefore, the inclusion of CREBBP mutated patients in very low risk protocols should be carefully evaluated. The validation of these findings is ongoing through the analysis of patients treated by other international study groups adopting different chemotherapy backbones.

Outcome of Infants Treated on Total Therapy for Infants with Acute Lymphoblastic Leukemia I: Results from a Non-Randomized Multi-Center Study

Infants with KMT2A rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival (EFS) of 33.6-36.9% on recent Children's Oncology Group and Interfant trials. In preclinical studies, we identified bortezomib and vorinostat as novel active agents in primary KMT2Ar infant ALL specimens that have not been included in infant regimens to date. Total therapy for infants with acute lymphoblastic leukemia I (TINI) evaluated this novel combination in a multi-institutional pilot trial for infants with de novo ALL. In this study, bortezomib and vorinostat were incorporated into an ALL chemotherapy backbone containing dexamethasone, mitoxantrone, and pegasparaginase during induction and reinduction chemotherapy cycles. A run-in dose escalation evaluated 3 dose levels of vorinostat which was then followed by a dose expansion phase. The primary endpoint of the study was tolerability; secondary endpoints included minimal residual disease (MRD), 3-year EFS and overall survival (OS). Fifty patients enrolled between 2017-2021. Median follow up was 3.4 years (range 1.6-6.8 years). The six patients in the dose escalation phase had no dose-limiting toxicities. Forty-four patients received vorinostat at the third dose level (DL3) of 180 mg/m 2/dose. 68% of patients treated at DL3 had CNS involvement at diagnosis. 68% of DL3 patients carried a KMT2Ar. The most frequent KMT2A partner genes were AFF1 (40%), MLLT1 (20%), and MLLT3 (13%). The most frequent grade 3-4 non-hematologic adverse events during induction include neutropenic fever (45%), hypertension (43%), infection (40%), anorexia (32%), diarrhea (18%) and perineal skin ulceration (16%). There were 3 episodes of sepsis and 1 induction death due to parainfluenza pneumonia. Fewer events and no deaths or proven sepsis occurred during reinduction, with only grade 3 hypertriglyceridemia exceeding 10% of patients affected (18%). 79% of all patients and 69% of KMT2Ar patients became MRD negative by flow cytometry on day 22 of induction following our investigational block. Following induction intensification with cyclophosphamide, cytarabine, and mercaptopurine, 77% and 66% of all patients and KMT2Ar patients respectively were MRD negative at the time of count recovery. Eight of the forty-four patients, all KMT2Ar, underwent stem cell transplant in first remission due to persistent MRD positivity at the end of consolidation. The 3-year EFS and OS probabilities (SE) of all forty-four DL3 patients were 56.5% (8.0) and 70.5% (7.3), respectively. The 3-year cumulative incidence of relapse (CIR) with death considered a competing event was 29.3% (6.9) and the cumulative incidence of death (CID) as a first event was 8.9% (4.5). Patients lacking a KMT2Ar had superior outcomes with 85.7% (9.4) EFS and 92.9% (6.9) OS. The EFS and OS of KMT2Ar patients were 44.8% (9.5) and 62% (9.1) respectively. CIR and CID for KMT2Ar patients was 43.5% (9.6) and 7.2% (5) respectively. KMT2Ar patients ≥90 days at diagnosis had superior outcomes compared to their younger counterparts with 59.1% (10.5) EFS and 77.3% (8.9) OS. End of induction MRD was predictive of an event in this subgroup, with 85.6% (9.4) EFS for MRD negative patients and 14.3% (13.2) EFS for MRD positive patients. In contrast, KMT2Ar patients < 90 days of age at diagnosis had poor outcomes irrespective of MRD with 12.5% (11.7) EFS and 25% (15.3) OS. Notably KMT2Ar patients < 90 days who were MRD negative at the end of induction had a median time to relapse of 600 days (range 282-969 days), a late time point for this malignancy that typically relapses within a year from initial diagnosis. This suggests the regimen effectively reduced tumor burden leading to an extended duration of remission but failed to sufficiently eradicate disease to prevent relapse. We conclude that the incorporation of bortezomib and vorinostat for treatment of infants with ALL was well tolerated. MRD responses in KMT2Ar patients were robust. Although the study was not powered to evaluate outcomes, EFS and OS in KMT2Ar patients suggest a clinical signal worth pursuing given the low proportion of patients transplanted in first remission. The successor study, TINI 2 (NCT05848687), will build upon the bortezomib/vorinostat backbone by incorporating 2 cycles of blinatumomab and the menin inhibitor ziftomenib in combination with chemotherapy during reinduction.

Urinary comprehensive genomic profiling predicts urothelial carcinoma recurrence and identifies responders to intravesical therapy.

Intravesical therapy (IVT) is the standard of care to decrease risk of recurrence and progression for high-grade nonmuscle-invasive bladder cancer. However, post-IVT recurrence remains common and the ability to risk-stratify patients before or after IVT is limited. In this prospectively designed and accrued cohort study, we examine the utility of urinary comprehensive genomic profiling (uCGP) for predicting recurrence risk following transurethral resection of bladder tumor (TURBT) and evaluating longitudinal IVT response. Urine was collected before and after IVT instillation and uCGP testing was done using the UroAmp™ platform. Baseline uCGP following TURBT identified patients with high (61%) and low (39%) recurrence risk. At 24 months, recurrence-free survival (RFS) was 100% for low-risk and 45% for high-risk patients with a hazard ratio (HR) of 9.3. Longitudinal uCGP classified patients as minimal residual disease (MRD) Negative, IVT Responder, or IVT Refractory with 24-month RFS of 100%, 50%, and 32%, respectively. Compared with MRD Negative patients, IVT Refractory patients had a HR of 10.5. Collectively, uCGP enables noninvasive risk assessment of patients following TURBT and induction IVT. uCGP could inform surveillance cystoscopy schedules and identify high-risk patients in need of additional therapy.

Maintenance Therapy Cessation for Sustained MRD Negative Multiple Myeloma Patients

Introduction Maintenance therapy with lenalidomide (len) and other drugs is a cornerstone therapeutic strategy for sustaining deep durable remissions in multiple myeloma (MM). However, indefinite treatment, as currently recommended, may lead to financial, emotional, and physical toxicity. Current guidelines lack data on optimal duration of maintenance in MM patients (pts) who have achieved sustained negative minimal residual disease (MRD) status. Here, we present the first pre-planned interim results from a phase II study evaluating maintenance cessation in patients with sustained MRD negative status for at least 3 years at the time of enrollment on study. Methods The current two-stage designed study of fifty MM pts, having already demonstrated >3 years of durable MRD negative responses, will be evaluated for MRD negative sustainability after ceasing maintenance therapy. Enrolled pts stop maintenance therapy and undergo active surveillance for three years, including serum protein testing every 3 months, bone marrow MRD testing by multiparametric flow cytometry (single 10-color tube with limit of detection of at least 6x106 (6 cells in 1 million) every 6 months, and annual PET-CTs. Primary endpoint includes sustained MRD negative rate after 1 year. Secondary endpoints include sustained MRD negative rate 3 years of cessation, re-treatment responses, microbiome and immune studies, health-related quality of life outcomes (HRQoL), and progression-free survival (PFS). The first stage includes 15 pts, and if 8 or more remain MRD negative at the end of 1 year, study enrollment continues to stage two (n=35). Results At data cutoff, on July 31, 2022, twenty-three pts (15 - stage 1; 8 - stage 2) have been enrolled on study. Median follow-up was 14.8 months (95% LL-UL: 11.1 - 25.1) after study enrollment, included 17(74%) male and 6(26%) female; 19(83%) ISS-stage 1, 2(9%) ISS-stage 2, and 2(9%) ISS-stage 3; and 4(17.3%) pts with high-risk cytogenetics [1 - t(4,14) and t(14,16); 1 - t(4,14) and +1q21gain; 2 - +1q21gain]. All pts enrolled onto study were receiving lenalidomide maintenance at the time. For stage 1, 13/15 (87%) remained MRD negative at 12 months of stopping maintenance therapy, and study accrual is ongoing with enrollment of 8 additional pts onto second stage. Overall, MRD negative sustained rates at 6 months and 12 months are 17/18 (94%) (95% CI: 73%-99%) and 14/16 (88%) (95% CI: 62%-98%), respectively. Progression free survival at 12 months was 94% (95% CI: 83%-100%). Two pts converted from MRD negative to MRD positive disease states while meeting clinical progression criteria with new FDG avid bone lesions. These pts have transitioned to new regimens: Pt 13 initiated carfilzomib, pomalidomide, and dexamethasone with VGPR; Pt 2 with a plan to initiate daratumamab-based therapy locally. Pt 10 converted to MRD positivity without clinical progression and opted to restart maintenance therapy with repeat MRD testing pending. Among the three MRD converters, one pt demonstrated standard risk cytogenetics and two pts had unknown risk cytogenetics. Additional HRQoL outcome data will be presented at the time of the meeting. Conclusions Initial first stage results of stopping maintenance in sustained MRD negative patients (>3 years) warrants further investigation and study continuation. This study and similar ones, such as MRD2STOP (NCT04108624), set a benchmark for future trials investigating maintenance cessation. One such trial includes SWOG 1803, which randomizes maintenance continuation vs. discontinuation in two-year MRD negative sustained patients. While maintenance therapy demonstrates clear benefit for MM patients, feasibility of treatment-free periods remain an unanswered critical question in the field. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MRD Status By Clonoseq ® Is a Poor Predictor of Long-Term Outcomes after Bispecific LV20.19 CAR T-Cell Therapy for Relapsed, Refractory B-Cell NHL

INTRODUCTION: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) uses next-generation sequencing (NGS) based testing to identify disease-specific immunoglobulin sequences and detect evidence of minimal residual disease (MRD). MRD assessments have been shown to predict progression-free survival and overall survival in non-Hodgkin's lymphoma (NHL) patients. We report MRD results via the commercially available clonoSEQ® assay and its association with durability of response in patients treated with bispecific LV20.19 CAR in our phase 1/2 trial (NCT04186520). METHODS: We conducted a phase 1/2 single-center prospective trial (NCT04186520) to evaluate the efficacy and safety of LV20.19 CAR T-cells in patients with NHL at 2.5x106 cells/kg dose. LV20.19 CAR T-cells were manufactured onsite at varying lengths of manufacturing (8 vs 12 days) in the CliniMACS Prodigy device with IL7+15 for cell expansion, with the goal of fresh infusion. The commercial clonoSEQ® assay (by Adaptive Biotechnologies) was used to identify and track tumor clonotypes in responding patients at first assessment performed between Day 28-60. We evaluated associations between MRD status and long-term response post LV20.19 CAR-T cell therapy. Descriptive statistics were utilized for baseline characteristics. Survival analyses were calculated utilizing the Kaplan-Meier method and differences between groups were evaluated via the log-rank test. RESULTS: Among the 32 patients enrolled in the trial, there were 26 responders on Day 28. Of these responders, 23 patients had clonoSEQ® B-cell MRD assay done between Day 28-60 post CAR infusion (Table 1). MRD was performed in 11 patients with Diffuse Large B-cell Lymphoma (DLBCL), 3 patients with Follicular lymphoma (FL), and 9 patients with Mantle Cell Lymphoma (MCL). The median age was 64 years (45-74) and the median lines of prior therapy were 4 (2-11). Of the 23 patients with early MRD status available, the day 28 objective response rate (ORR) was 100% (complete response=57%, n=13 and partial response=43%, n=10). Eighteen patients (78%) were MRD negative at first assessment between days 28-60 and 5 (22%) were MRD positive. Of the 5 patients who were MRD positive, 3 became MRD negative at day 90, 1 patient was non-evaluable due to a non-relapse mortality event, and the remaining patient remains positive but with decreasing MRD status at Day 90. None of the patients with MRD positivity at day 28 have relapsed to date. Among patients who achieved early MRD negativity (n=18), 7 (39%) have relapsed, and all were patients with either DLBCL or FL. No MRD-negative MCL patient has relapsed to date. The duration of response in MRD-negative patients was significantly improved in MCL patients compared to DLBCL/FL patients (Figure 1), p=0.02. Among the 7 MRD negative patients who relapsed, only 1 patient was detected to have disease relapse by MRD positivity, 2 were detected simultaneously on MRD testing and imaging, while 3 were detected by physical exam or imaging findings and did not have MRD testing. Interestingly, 1 patient had a central nervous system relapse while being MRD negative. CONCLUSION: Relapse after CAR T-cell therapy remains a significant clinical challenge. New predictors are indicated to determine which responding patients after CAR-T cell therapy are more likely to relapse. Utilizing the commercially available Adaptive clonoSEQ® assay, early MRD assessment was not predictive of long-term clinical outcomes in DLBCL and FL. MRD positive patients maintained durable remission and converted to MRD negativity, while early MRD negative patients continued to relapse. In contrast, MRD negativity post-CAR-T therapy did appear to predict durable clinical remission in MCL patients. Newer assays, including cell-free technologies, may be more informative for non-MCL histologies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax and Hypomethylating Agents Followed By Allogeneic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia

Background So far, it was a treatment paradigm to induce remission in patients with either newly diagnosed (ND) or relapsed/refractory (R/R) acute myeloid leukemia (AML) prior to allogeneic stem cell transplantation (allo-SCT) using intensive therapies with anthracyclins in combination with low- or intermediate to high-dose cytarabine. For AML patients ineligible for intensive therapy, the combination of hypomethylating agents (HMA) and the BCL2 inhibitor Venetoclax (VEN) has become the new treatment standard due to high and rapidly achieved remission rates and a favorable toxicity profile. These properties make this combination also attractive for frontline induction of ND-AML or as salvage therapy in R/R-AML patients with borderline fitness to tolerate and/or limited chance to achieve remission by intensive therapy. Considering the limited evidence from the literature so far, we here report our single-center experience of allo-SCT after HMA/VEN-based therapy. Design/Methods: We retrospectively analyzed all consecutive ND and R/R AML patients, who had received HMA/VEN therapy followed by allo-SCT at our center from 2021 to 2022, regarding outcome after transplant. The study was approved by the ethics committee of the University Duisburg/Essen (approval number: 22-10708-BO) and all patients gave written informed consent for scientific use of their data. Results: Overall, 26 patients (median age 64 years, range 26 to 75) with ND (n=14, 54%) or R/R (n=12, 46%) AML were treated with a median of 2 cycles HMA/VEN (range, 1 to 5; Azacitidine n=10, Decitabine n=16). Overall response rate (CR/CRi, MLFS) in the entire cohort was 58% with no difference between patients with ND and R/R AML (57% vs. 58%, p>0.99). Minimal residual disease (MRD) determined by molecular genetics as part of the clinical routine was not detectable prior transplant in 43% of the responding patients. Allo-SCT using PBSC (n=25, 96%) or BM (n=1, 4%) from a matched sibling (n=4, 15%), unrelated (n=19, 73%) or haploidentical donor (n=3, 12%) was performed in median 98 days (range, 31 to 220) after start of HMA/VEN treatment. Neither the rate of MRD negativity nor time to allo-SCT differed between ND and R/R patients. A reduced-intensity conditioning (RIC) was used in all patients including 11 patients (42%) receiving melphalan- or FLAMSA-based sequential conditioning. Median follow-up from start of HMA/VEN therapy was 10 months (range, 4 to 19 months) and from allo-SCT 6 months (range, 1 to 15 months), respectively. Median overall survival (OS) calculated from start of HMA/VEN therapy and from allo-SCT is both not reached, while the estimated 1-year OS rates were 81% and 80% respectively. OS after transplant was numerically higher in patients with CR at transplant (90% vs. 69%), but did not differ between patients with ND and those with R/R AML (75% vs. 80%) nor between MRD negative and positive CR patients (75% vs. 100%). All patients achieved CR following allo-SCT, but 1 patient had a persistence of his cytogenetic marker and two patients had mixed chimerism. Incidence of acute GvHD was 31% (including 8% with acute GvHD grade III/IV) and of chronic GvHD 4%. A total of 6 patients relapsed after allo-SCT and a total of 4 patients have died due to relapse (n=3) or treatment-related toxicity (n=1). Median relapse-free survival (RFS) after allo-SCT was not reached and the estimated 1-year RFS rate was 67%. Again, RFS numerically differed between in patients in remission and with active disease at transplant (84% vs. 48%), but did not differ between patients with ND and those with R/R AML (70% vs. 64%) nor between MRD negative and positive CR patients (83% vs. 86%). Conclusion: Treatment with HMA/VEN either as induction or salvage therapy in ND or R/R AML leads to reasonable remission rates facilitating subsequent allo-SCT with promising survival rates. This warrants confirmatory analyses in larger patient cohorts with longer follow-up, ideally in a prospective clinical trial.

Detection of Minimal Residual Disease in Acute Myeloid Leukemia by Multi-color Flow Cytometry

To establish 10-color fluorescent antibody combination panels for the detection of minimal residual disease (MRD) of acute myeloid leukemia (AML) in our laboratory and discuss the value of clinical application. According to the antigen expression characteristics of leukemia cells of incipient AML patients, MRD in bone marrow were detected by multiparameter flow cytometry, and the test results were compared with both bone marrow cell morphology and PCR results, then 10-color fluorescent antibody combination panels in our lab for MRD detection was determined. The immunophenotypic characteristics of 392 incipient patients with AML in the First Affiliated Hospital of Zhengzhou University were analyzed, among them 357 (91.07%) cases showed abnormal immunophenotypes, which mainly included cross-lineage expression, cross-stage expression, deficiency of antigen expression or abnormal antigen intensity and other abnormal expression. The 10-color fluorescent antibody combination panels established according to abnormal immunophenotypic characteristics of leukemia cells were applied for detecting MRD in 156 patients with AML, the positive rate (43.6%) was higher than 26.8% of morphology, and the results were highly consistent with PCR detection results (96.49%), moreover, the recurrence rate of MRD positive patients (86.96%) was significantly higher than 5.75% of MRD negative patients. Therefore, this method could truly reflect the load of leukemia cells and prompt change of disease condition. Multiparameter flow cytometry can detect various abnormal immunophenotypes of AML. The 10-color fluorescent antibody combination panels in our lab based on the characteristics of antigens expression in leukemia cells can well detect MRD of leukemia cells, so as to predict relapse and provide basis for clinical treatment.

Residual Monoclonal Free Light Chain Positivity By Mass Spectrometry Identifies Patients at Increased Risk of Early Relapse Following First-Line Anti-Myeloma Treatment

Residual Monoclonal Free Light Chain Positivity By Mass Spectrometry Identifies Patients at Increased Risk of Early Relapse Following First-Line Anti-Myeloma Treatment

Venetoclax, Actinomycin D and Low Dose Cytarabine for Relapsed or Refractory Acute Myeloid Leukemia in Clinical Practice Setting

BackgroundVenetoclax based therapies have produced varying results in the relapsed or refractory acute myeloid leukemia (R/R AML) setting. Highest response rates were demonstrated after Venetoclax in combination with high dose chemotherapy. However, this approach is feasible mainly in fit, younger patients. Herein, we present a lower intensity combination therapy consisting of Venetoclax, low dose Cytarabine and Actinomycin D (ACTIVE) in patients with R/R AML administered in real-life clinical practice setting.MethodsWe performed an observational, retrospective, single centre study. The patients were at least 18 years of age and had R/R AML. All patients provided informed consent for treatment as well as data collection. Venetoclax ramp-up was administered over either 3 or 5 days until the daily dose of 600mg/d was reached. The ACTIVE regimen consisted of Venetoclax 600mg/d p/o on days 1-28, Cytarabine 20mg/m 2 s/c on days 1-10, Actinomycin D 12.5 µg/kg i/v on days 1, 2 and 3 (on days 1 and 2 for patients ≥65 years). Strong/moderate CYP3A inhibitors/inducers or Venetoclax dose reductions were not allowed. Indications for stopping Venetoclax before Day 28 were life threatening infectious complications or faster hematological recovery with the addition of G-CSF in responders. A second ACTIVE cycle was administered in non-responders without evidence of progressive disease after Cycle 1 or in responders with positive minimal residual disease (MRD).We evaluated baseline characteristics, composite CR (CRc = CR + CRi + CRp), overall response (ORR = CRc + MLFS), MRD negativity rates, overall survival (OS), relapse-free survival (RFS), event-free survival (EFS), Grade 3-5 non-hematological toxicities and Day 30 and Day 60 mortality rates.ResultsFifty R/R AML patients were treated with ACTIVE and 56% (28/50) were male. The median age was 65 years (20-84). The median Eastern Cooperative Oncology Group (ECOG) status was 1 (0-3) and 40% (20/50) had ECOG status of 2 or 3. Secondary AML was confirmed in 48% (24/50) of cases. Adverse cytogenetics were identified in 28% (14/50) of patients whereas 60% (30/50) were stratified to adverse risk group in accordance with ELN 2017 guidelines. The most common gene mutations were IDH1/2 30% (15/50), FLT3 28% (14/50), ASXL1 22% (11/50), NPM1 14% (7/50), N/KRAS 12% (6/50) and RUNX1 12% (6/50).The median number of prior therapies was 2 (1-5). Intensive chemotherapy was administered in 80% (40/50) of whom 38% (15/40) had primary refractory disease and 48% (19/40) had previously received Fludarabine + Cytarabine + Idarubicin (FLAG-Ida) or Cytarabine + Mitoxantrone (HAM). Eight percent (4/50) had had prior Venetoclax exposure. Thirty-six percent (18/50) had relapsed after allogeneic stem cell transplantation (alloSCT) with a median time of 7.4 months (1.6-37.3) from transplant to relapse.One cycle of ACTIVE therapy was administered in 76% (38/50) of cases, whereas 24% (12/50) received 2 cycles. The median number of Venetoclax 600mg/d days per cycle was 18 (8-28). Additional FLT3/RAS/BCR-ABL1 inhibitors Gilteritinib, Trametinib or Dasatinib were administered in 12% (6/50).Forty-nine patients were evaluable for response and one patient died of sepsis before response evaluation (Table 1). The ORR was 73% (36/49) and the CRc rate was 67% (31/46). MRD negativity was confirmed in 61% (19/31) of CRc patients. Sixteen patients had undergone additional early bone marrow evaluations. Blast count reduction to <5% was observed in 50% (8/16) after Venetoclax ramp-up and in 88% (14/16) on Day 4.Half of the ACTIVE responders (18/36) continued maintenance therapy with Venetoclax + low dose Cytarabine and optional DLI, whereas 36% (13/36) proceeded to either first or second alloSCT. After 16.4 months of median follow-up, the median OS, RFS and EFS were 13.1, 7.2 and 4.5 months, respectively (Figure 1A). Median OS was not reached in MRD negative patients (Figure 1B). In multivariable Cox regression analysis, adverse cytogenetics (HR 3.48, 95% CI 1.39 -8.55) and primary refractory disease (HR 2.54, 95% CI 1.05-6.12) were associated with worse OS.The most common grade 3-5 non-hematological adverse events were febrile neutropenia (54%, 27/50) and bacteremia/sepsis (34%, 17/50). Day 30 and Day 60 mortality rates were 8% (4/50) and 16% (8/50), respectively.ConclusionsACTIVE was effective and well tolerated in this unselected prognostically unfavourable older R/R AML patient population. [Display omitted] DisclosuresZucenka: Jannsen: Honoraria, Other: Travel-expenses; Takeda: Other: Travel Expenses; Novartis: Honoraria, Other: Travel Expenses; Pfizer: Honoraria, Other: Travel Expenses; Astellas: Honoraria; Abbvie: Honoraria, Other: Travel Expenses. Maneikis: Abbvie: Honoraria. Pileckytė: Abbvie: Honoraria, Other: Travel Expenses. Griškevičius: Abbvie: Other: Travel Expenses. OffLabel Disclosure:Venetoclax has been used off-label for the treatment of R/R AML

Post-Transplant MRD Negativity on Day +100 Predicts Outcomes for Pre-Transplant Relapsed/Refractory AML Patients

IntroductionPatients with relapsed/refractory (r/r) AML have in general a dismal prognosis. Allogeneic stem cell transplantation (allo-SCT) provides a curative approach for these patients, however the overall survival (OS) remains still low achieving 20-40%. Though post-transplant minimal residual disease (MRD) monitoring has been shown to be predictive for development of post-transplant relapses and lower survival, data focusing on pre-transplant relapsed/refractory AML patients is scarce. In this study, we investigated the impact of achieving MRD negativity on day +100 for relapses and survival for this high risk patients.Patients and MethodsWe analyzed post-transplant outcomes for pre-transplant r/r AML patients depending on their post-transplant MRD status at day +100. The day +100 was chosen concerning the possibility of early post-transplant interventions (e.g. tapering of immunosuppression or administration of donor lymphocytes).Fifty six consecutive adult patients (≥18 years old) with r/r AML (median age 58, range 20-76; male, n=34, 61%) who underwent allo-SCT (first, n=44, 79%; second, n=12, 21%) between 2015-2020 at the Department for Stem Cell Transplantation at the University Medical Center Hamburg (Germany) were included. The MRD was assessed on day +100 using multiparameter flow cytometry according to “different from normal” strategy.The patients experienced rather primary refractory disease (64%), secondary/therapy-related AML (55%) and abnormal cytogenetics (59%) at diagnosis. The median pre-transplant blast count was 25% (6-91%). A number of 29 patients (52%) showed blasts in peripheral blood. Myeloablative conditioning was used in 31 (55%) patients, whereas 25 (45%) patients received reduced intensity regimens. A number of 29 patients (52%) received a FLAMSA-based conditioning. Post-transplant donor lymphocyte infusions as well as other treatment were given to 13 (23%) and 17 (30%) patients, respectively.ResultsThe median follow up was 20 months (range 4-66). Forty patients (71%) achieved MRD negativity on day +100 and 16 (29%) remained MRD positive. The 2-year OS, LFS, relapses and NRM at 2 years for day +100 MRD negative patients were: 76% (95% CI: 60-87%), 59% (95% CI: 41-75%), 31% (95% CI: 17-50%) and 8% (95% CI: 3-19%), respectively. The 2-year OS, LFS, relapses and NRM at 2 years for day +100 MRD positive patients were: 35% (95% CI: 17-59%, p=p=0.001), 23% (95% CI: 9-46%, p<0.0001), 70% (95% CI: 45-87%, p=0.0002) and 6% (95% CI: 1-28%, p=0.88), respectively.Several factors were evaluated for possible association with day +100 MRD negativity (Table 1). There were no significant associations. Further, the incidence of acute (grade II-IV) GvHD at 100 days was not significantly different between the day 100 MRD positive und negative patients.Following factors had impact on post-transplant outcomes in multivariate analysis: presence (no vs yes) of peripheral blasts prior to allograft (OS: HR 0.3, 95% CI: 0.1-0.9, p=0.03; LFS: HR 0.4, 95% CI: 0.1-0.9, p=0.03; relapses: HR 0.4, 95% CI: 0.1-0.99, p=0.048; NRM: HR 0.5, 95% CI: 0.2-1.3, p=0.17), FLAMSA vs other preparative regimens (OS: HR 0.4, 95% CI: 0.1-0.93, p=0.03; LFS: HR 0.4, 95% CI: 0.2-0.98, p=0.04; relapses: HR 0.4, 95% CI: 0.2-1.0, p=0.05; NRM: HR 0.4, 95% CI: 0.2-1.0, p=0.06), and day +100 MRD (negative vs positive) (OS: HR 0.3, 95% CI: 0.1-0.7, p=0.009; LFS: HR 0.2, 95% CI: 0.1-0.6, p=0.001; relapses: HR 0.2, 95% CI: 0.1-0.4, p=0.0001; NRM: HR 0.2, 95% CI: 0.1-0.5, p=0.0006).ConclusionsPost-transplant MRD detection plays predictive role in pre-transplant r/r AML patients and may help to define possible candidates for early post-transplant interventions. DisclosuresNo relevant conflicts of interest to declare.

An Update on Outcomes Using the USC ALL Frontline Regimen (based on CCG-1882) after Further Modification of Protocol in the Era of Novel Agents in Ph-Negative ALL Patients; A Retrospective Study

▪INTRODUCTION: The use of pediatric inspired regimens in adolescent young adults and adults has improved outcomes in acute lymphoblastic leukemia (ALL). CALGB 10403 was prospectively tested in patients 17-39 years, showing a 3-year event free survival of 59%.In 2007, our institution reported outcomes after incorporating peg-asparaginase (PEG) dosed at 2000mg/m 2 in induction for patients aged 17- 55 years. This regimen was well tolerated and resulted in a complete remission (CR) rate of 96%. In 2014, we reported experience with the use of multiple doses of PEG (CCG-1882) for newly diagnosed ALL adult patients, with a 7-year overall survival (OS) of 51%. Since our last report, the USC ALL regimen consisting of 2 induction phases, has been further modified with a goal of maintaining good outcomes, and improving compliance and toxicities. Fractionated doses of cytarabine were changed to a single dose, the methotrexate dose of 1g/m 2 (2.5 g/m 2 if T-cell) given D1,15 of intensification phases was changed to 3g/m 2 (B and T cell) given in single doses, and consolidation was increased to six cycles allowing for PEG holidays (Table 1).Moreover, the approval and incorporation of novel agents such as blinatumomab and inotuzumab also changed outcomes in ALL. Therefore, this study reports an update of outcomes since further modification of the USC ALL pediatric-based regimen in the era of novel agents. METHODSThis is a retrospective review which included adults aged >18 with newly diagnosed Philadelphia negative ALL between 2016 and 2020 treated at USC/Norris Cancer Center and Los Angeles County Hospital (LAC). Primary objectives were over-all survival (OS), event-free survival (EFS), disease-free survival (DFS) at 3 years, and secondary outcomes were complete remission/complete remission with incomplete recovery (CR/CRi) rates and minimal residual disease (MRD) by flow cytometry. OS, DFS, EFS were reported through Kaplan Meier curves and Log-rank tests. Two-sided p value ≤0.05 was significant.RESULTS: 121 patients with Ph-negative ALL were identified (49.6% Ph-negative B-ALL, 24% Ph-like B-ALL, 0.8% B cell lymphoblastic leukemia (LBL), 9.1% T cell ALL, 4.1% T cell LBL, 4.1% early T-cell, 5.8% mixed phenotype acute leukemia (MPAL). Median age at diagnosis was 38.5 years and maximum age of patient to receive pegasparagase during induction 1 was 63 years. 71.9% Hispanic, 15.7% white, 9.9% Asian, 2.5% African American. 57.9% males, 42.1% females. 3.4% were favorable, 4.2% intermediate, 54.6% unfavorable and 37.8% unknown by karyotypic risk stratification. Median BMI was 28.9 kg/m 2.54.6% had hepatic steatosis either on history or imaging and 5.1% had CNS disease pre-induction.Post-induction 1, 81.4% of patients achieved CR/CRi and 50% MRD negative. Post induction 2, 82.2% achieved CR/CRi, 67.7% MRD flow negative. Post-consolidation 1, 90.9% were MRD flow negative. 33.1% subsequently received blinatumomab for MRD positive disease, 5% given inotuzumab for relapsed disease, 32.2% underwent allogenic hematopoietic stem cell transplant (allo-HSCT). Median number of pegasparagase doses received during treatment was 2, rate of relapse and mortality was 27.3% and 13% respectively.Median OS and DFS were not reached but median EFS was 35 months. 3-year OS was 85.3%, when stratified according to MRD post induction 2; 3-year OS was 91.7% for MRD positive patients and 91.2% for MRD negative patients (p=0.55). 3-year DFS was 83.2%; 88.2% for MRD positive patients and 97.4% for MRD negative patients (p=0.22). 3-year EFS was 47.3%; 51.3% for MRD positive patients and 50.6% for MRD negative patients (p=0.49) (Tables 2-3).Use of pegasparagase resulted in grade 3/4 toxicities including hypersensitivity (4.1%), transaminitis (21.5%), pancreatitis (5.4%), hypertriglyceridemia (49.5%), hypofibrinogenemia (45.5%), hyperbilirubinemia (21.5%) and thrombosis (16.5%). CONCLUSIONSPediatric-based modified USC ALL induction regimen led to a high 3-year OS (85.3%), DFS (83.2%) and EFS (47.3%) with predictable toxicity and compares favorably with original USC ALL regimen, CALGB 10407, GRAALL 2005 and USC/MSKCC regimen.Interestingly, MRD positivity after induction 2 did not adversely affect OS, DFS or EFS, which is likely due to incorporation of blinatumomab and inotuzumab. These agents could have allowed for deeper remissions allowing Ph-negative patients with residual disease to receive allo-HSCT. [Display omitted] DisclosuresChaudhary: Oncotartis: Consultancy; Pancella: Consultancy; Moderna: Current equity holder in publicly-traded company; Celldex: Current equity holder in publicly-traded company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company; Athelas: Consultancy, Current holder of stock options in a privately-held company; Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy . Douer: Servier: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Adaptive: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Speakers Bureau; Jazz: Consultancy. Yaghmour: Novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Alexion: Speakers Bureau; Astellas: Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Jazz: Speakers Bureau; Agios: Consultancy, Speakers Bureau.

Urine tumor DNA detection of minimal residual disease in muscle-invasive bladder cancer treated with curative-intent radical cystectomy: A cohort study.

BackgroundThe standard of care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy, which is typically preceded by neoadjuvant chemotherapy. However, the inability to assess minimal residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. Here, we sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis.Methods and findingsWe applied urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq), a targeted next-generation sequencing (NGS) method for detecting utDNA, to urine cell-free DNA (cfDNA) samples acquired between April 2019 and November 2020 on the day of curative-intent radical cystectomy from 42 patients with localized bladder cancer. The average age of patients was 69 years (range: 50 to 86), of whom 76% (32/42) were male, 64% (27/42) were smokers, and 76% (32/42) had a confirmed diagnosis of MIBC. Among MIBC patients, 59% (19/32) received neoadjuvant chemotherapy. utDNA variant calling was performed noninvasively without prior sequencing of tumor tissue. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. utDNA analysis revealed a median utDNA level of 0% in healthy adults and 2.4% in bladder cancer patients. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (pCR; n = 26), median utDNA levels were 4.3% vs. 0%, respectively (p = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD detection was highly correlated with the absence of pCR (p < 0.001) with a sensitivity of 81% and specificity of 81%. Leave-one-out cross-validation applied to the prediction of pathologic response based on utDNA MRD detection in our cohort yielded a highly significant accuracy of 81% (p = 0.007). Moreover, utDNA MRD–positive patients exhibited significantly worse progression-free survival (PFS; HR = 7.4; 95% CI: 1.4–38.9; p = 0.02) compared to utDNA MRD–negative patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 85%. Tumor mutational burden (TMB) in utDNA MRD–positive patients was inferred from the number of non-silent mutations detected in urine cfDNA by applying a linear relationship derived from The Cancer Genome Atlas (TCGA) whole exome sequencing of 409 MIBC tumors. We suggest that about 58% of these patients with high inferred TMB might have been candidates for treatment with early immune checkpoint blockade. Study limitations included an analysis restricted only to single-nucleotide variants (SNVs), survival differences diminished by surgery, and a low number of DNA damage response (DRR) mutations detected after neoadjuvant chemotherapy at the MRD time point.ConclusionsutDNA MRD detection prior to curative-intent radical cystectomy for bladder cancer correlated significantly with pathologic response, which may help select patients for bladder-sparing treatment. utDNA MRD detection also correlated significantly with PFS. Furthermore, utDNA can be used to noninvasively infer TMB, which could facilitate personalized immunotherapy for bladder cancer in the future.

Pre-Transplant Exhausted Marrow Infiltrating T Lymphocytes in MRD Negative Patients with Acute Leukemia Predict Early Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplant (alloSCT) is curative for patients with high-risk acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite this, relapse remains the leading cause of treatment failure. The use of flow cytometry to identify aberrant leukemia-associated immunophenotypes is an established method of detecting minimal residual disease (MRD). MRD status immediately prior to transplant remains one of the single best predictors of post-transplant outcome. Unfortunately, not all MRD(-) patients maintain remission post-transplant. Therefore, we sought to find additional predictors of post-transplant relapse. T cell exhaustion occurs following continuous exposure to antigen and leads to sustained up-regulation of multiple inhibitory receptors, progressive loss of function, and expression of a distinct transcriptional state. Bachireddy et al recently reported that exhausted CD8+ marrow infiltrating lymphocytes (MILs) were a novel predictor of response to donor lymphocyte infusion (DLI) for patients with relapsed chronic myelogenous leukemia (CML). Based on this principle, we hypothesized that exhausted CD8+MILs may also be predictive of outcome for MRD(-) patients prior to alloSCT.We matched pre-transplant samples from 17 MRD(-) patients who maintained remission for greater than 2 years post-transplant to 11 MRD(-) patients who suffered relapse within 2 years of transplant. Patients had either ALL or AML and did not differ by demographic details. MILs were evaluated for exhaustion using 2 multicolor flow cytometry panels which collectively included a viability dye, T cell markers (CD3 and CD8) surface inhibitory receptors (PD-1, Tim-3, and TIGIT), transcription factors (T-bet and Eomes), and intracellular cytokine staining (IL-2, TNF-α, and IFN-γ) in response to CD3/CD28 bead stimulation.There was no difference in the percentage of total T cells (CD3), the CD8+ T cells (CD3+CD8+), or CD8+ T cell subsets between the 2 groups. While there was also no difference in the mean fluorescent intensity (MFI) of Tim-3, both PD-1 and TIGIT were significantly elevated on the CD8+ MILs of MRD(-) patients that eventually relapsed (p=0.01 and p=0.03, respectively). Among CD8+ MILs, cells with the highest PD-1 expression (PD-1High) were significantly elevated in the relapse group (p=0.006), as was co-expression of multiple inhibitory receptors on single cells (p=0.01) (Figure A,B). In contrast, the proportion of CD8+ MILs lacking all 3 of these inhibitory receptors was significantly higher among the MRD(-) group that maintained remission (p=0.01) (Figure C). Interestingly, CD8+ MILs in the remission group with dual PD-1+TIGIT+ expression or expressing PD-1High were more likely to be central memory cells (both p=0.05), while these same immunophenotypes were significantly more often effector memory cells in patients who went on to relapse (p=0.03 and p=0.02, respectively).As noted above, inhibitory receptor expression alone does not define exhaustion. We further assessed the CD8+ inhibitory receptor expressing MILs in the relapse group for other phenotypic evidence of exhaustion by evaluating their function and transcription factor expression. Cells with either PD-1+TIGIT+ dual expression or PD-1High were directly compared to cells that lacked inhibitory receptors. The dual PD-1+TIGIT+ cells had impaired ability to produce TNF-α and IFN-γ (both with p<0.0001), while PD-1High cells produced significantly less TNF-α (p<0.0001), IFN-γ (P<0.0001) and IL-2 (p=0.0001). Moreover, both sets of MILs had significantly decreased polyfunctional cytokine production compared to cells without these surface receptors (p < 0.0001) (Figure D). Furthermore, transcription factor expression was also altered among inhibitory receptor expressing cells. They exhibited both increased early exhaustion as evidenced by high T-bet expression in PD-1 expressing cells as well as terminal exhaustion indicated by significantly elevated Eomes expression on PD-1High expressing cells (Figure E,F).Collectively, this study suggests that the presence of exhaustion in CD8+ MILs in acute leukemia patients who are MRD(-) prior to transplant may be at higher risk for post-transplant relapse. If validated, this data may be used to further risk-stratify patients and/or direct pre- or post-transplant therapy, including the potential use of checkpoint blockade. [Display omitted] DisclosuresBachanova:Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Wagner:Novartis: Research Funding; Magenta Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Maintenance Treatment with Lenalidomide for Multiple Myeloma Increases the Proportion of MRD Negative (Flow-/PET-CT-) Patients

IntroductionLenalidomide maintenance therapy in Multiple Myeloma has been used in order to prolong the Progression-Free Survival (PFS) obtained with any induction therapy through the depletion of residual tumor burden. In this way, it is well established the value of Minimal Residual Disease (MRD) assessed by flow cytometry (FCM) as an independent prognosis factor in Multiple Myeloma. Similarly, patients who become PET-CT negative (when previously they have proved lesions) have better survival outcomes; in fact, this has recently been accepted as a new response criterion.However, the available data about the complementary use of both techniques in terms of prognosis are limited. The aim of this study is to analyze the prognostic impact of achieving negative residual disease by FCM and PET-CT during Lenalidomide maintenance therapy.MethodsWe retrospectively selected 87 Multiple Myeloma patients from 3 Spanish hospitals (Hospital 12 de Octubre, Hospital Virgen de las Nieves and Hospital Ramón y Cajal). Main selection criterion was to start Lenalidomide maintenance therapy between 2008-2017; the previous induction therapy was heterogeneous. 75% of included patients had previously received an autologous stem cell transplant. We collected data about baseline variables, clinical and biological parameters, therapy administered and FCM and PET-CT assessment at diagnosis, post-induction and during maintenance. Statistical analysis was realized with SPSS 21.0 version (SPSS, Inc., Armonk, NY).ResultsResponse at the beginning of maintenance was VGPR or CR in 78% of patients and 48,3% were MRD-. Initial PET-CT was positive in 31%. After a median of 21 Lenalidomide cycles, 32% of patients with initial MRD+ achieve MRD-; meanwhile, 48,1% of patients with initial PET-CT+ become negative.When we jointly assessed FCM and PET-CT results during maintenance therapy (49 patients had been studied by both techniques), we detected a significant difference in terms of PFS between patients that achieve combined PET-/FCM- and the other groups (median PFS not reached vs 32 months, respectively; p<0,0001) (Fig.1). We also found a trend to longer OS in patients PET-/FCM- (4-year OS 100% vs 83%; p=0,053). Patients with standard-risk cytogenetics were more frequently PET-/FCM- than in the high-risk cytogenetics group (31,8% vs 5,6%; p=0,002); nevertheless high-risk cytogenetics patients that achieve PET-/FCM- (n=3) also had prolonged PFS (4-year PFS 100%).ConclusionLenalidomide maintenance therapy increases the proportion of patients that achieve combined PET-/FCM-. Both techniques are complementary and their combination allows us to identify a subset of PET-/FCM- patients with very good prognosis and PFS outcomes significantly superior than the other patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Significance of Minimal Residual Disease in Patients with Secondary Acute Myeloid Leukemia

BACKGROUNDSecondary acute myeloid leukemia (AML) is defined as AML that develops from a prior myelodysplastic or myeloproliferative disorder, or from exposure to chemotherapy, radiation, or environmental toxins. Patients with secondary AML have inferior prognosis compared to those with de novo AML,1 and are more likely to be elderly with increased comorbidities and worse performance status.2 The detection of minimal residual disease (MRD) also has independent prognostic value in patients with AML.3-6 In elderly AML patients, MRD predicts worse outcomes independent of the poor treatment risk factors associated with increased age.7Due to the similar disease pathogenesis between elderly AML and secondary AML patients, it is likely that MRD testing will have similar implications. However, there is limited data on the prognostic role of MRD testing in secondary AML. METHODSWe retrospectively analyzed de novo and secondary AML patients at Washington University Medical Center who received post-induction, post-consolidation, pre-transplant, or post-transplant MRD testing during their treatment. MRD testing was performed with multiparameter flow cytometry testing using the “different from normal” method at one reference laboratory,8,9 with positive MRD defined as detection of an abnormal myeloblast population above 0.1%. A total of 180 patients were included. Any patient with at least one positive MRD was considered “MRD positive.” Baseline pretreatment characteristics were compared between groups with Mann-Whitney, Chi-square, or Fisher's exact test, when appropriate. We evaluated overall survival (OS) and relapse rates in de novo and secondary AML patients stratified by MRD status using Kaplan-Meier curves and the log rank test, or cumulative incidence function, as appropriate. Multivariate Cox proportional hazards model was also fitted to determine if MRD testing has similar prognostic value across de novo and secondary AML patients. RESULTSOur analysis included 133 patients with de novo and 47 patients with secondary AML. Secondary AML patients were significantly older at age of diagnosis (mean age 59.85 versus 51.17, p<0.001), however had similar rates of MRD positivity (28% versus 29%, p=0.830). There was a trend toward poorer cytogenetic risk profiles and worse Karnofsky Performance Status in secondary AML (Table 1). In all patients, we confirmed that MRD positivity confers significantly worse OS (45.5% versus 70.8%, p=0.020) and relapse rates at 2 years (53% versus 25.5%, p=0.029) (Figure 1). In de novo AML patients, MRD positivity continued to demonstrate significantly worse 2-year OS (44.1% versus 76.3%, p=0.006) and relapse rates (57.6% versus 20.7%, p=0.008). However, in secondary AML patients, there were no differences in OS (p=0.69) or relapse rates (p=0.98) between MRD positive and negative patients. Multivariate analysis demonstrated significant interaction (p=0.042) between MRD status and AML subtypes. MRD positivity emerged as an independent prognostic indicator of worse OS in de novo patients (HR 2.8, 95% CI 1.524-5.405) but not in secondary patients (HR 1.5, 95% CI 0.188-2.336). DISCUSSIONWe confirm that MRD status is a poor prognostic indicator in all AML patients, and this also holds true in patients with de novo AML. However, we found that MRD status has less prognostic value in secondary AML patients. This may be due to the overall poor prognosis seen in secondary AML patients, suggesting that MRD monitoring in this group of patients may be less useful for risk stratification. However, our study is limited by the small sample size, particularly in the number of secondary AML patients who displayed MRD positivity (N=13). In addition, we defined MRD positivity across all time points of testing, which may decrease patient homogeneity. A larger study with increased follow-up is necessary to confirm our findings, as these results have important implications in risk-stratification and treatment of patients with secondary AML. [Display omitted] DisclosuresDiPersio:BMS, Asterias, Amphivena, Bluebird: Other: Travel, Accomodations, Expenses; Hemedicus, DAVA Oncology: Speakers Bureau; Celgene, Bioline, Vasculox, Cellworks, Rivervest: Consultancy; Magenta Therapeutics: Equity Ownership.

Minimal Residual Disease (MRD) Assessment after Induction Chemotherapy in Children and Adults with Acute Lymphoblastic Leukemia (ALL): A Valuable Strategy in Resource Constraint Settings in the Indian Subcontinent

Background: In a developing country with limited resources and insurance coverage, it is very important to risk stratify patients early in the treatment of Acute Lymphoblastic Leukemia (ALL). This strategy will enable better utilization of resources and also identify a subset of patients who will benefit from early haematopoeitic stem cell transplant (HSCT). Historically, clinical and laboratory features such as age, gender, white blood cell counts at diagnosis, organomegaly, mediastinal mass, morphology, immunophenotyping and cytogenetics have been used to assess the outcome of patients with ALL. However recent studies have shown that assessment of minimal residual disease (MRD) was the most important prognostic determinants of both childhood and adult ALL. Currently multiparameter flow cytometry and quantitative polymerase chain reaction (PCR) of the immunoglobulin or TCR genes are most commonly used for the assessment of MRD. However availability of this technology is very limited. In this present study, we assessed the MRD at day 33 post induction chemotherapy by flowcytometry at our center, and risk stratified patients to determine outcomes.Methods: We analyzed a cohort of 70 de novo ALL patients who presented to our Hospital (Mazumdar Shaw Medical Center, Narayana Health City, Bangalore, India) between April 2014 and April 2017. Diagnosis was confirmed by bone marrow aspiration based on the morphology, cytochemistry, immunophenotyping and cytogenetics. Clinico-pathological features, immunophenotyping, cytogenetics were recorded at the time of presentation. Day 8 prednisolone response was recorded and MRD assessment was done at day 33, using 8 Color multiparameter Flowcytometry. The cost of MRD analysis is 220 US$.Results: Out of 70 patients, 47 (67%) were male. The median age was 9 years (range: 8 months, 69 years). Among the subtypes, B-ALL was found in 83% (n=58) and T-ALL in 17% (n=12). The most common presenting symptom was fever (66%). Hepatosplenomegaly was seen in 35% (n=23), lymphadenopathy in 35% (n=24). CNS involvement at presentation was seen in 8.6% (n=6). The median WBC count at presentation was 11x103/µl (0.8-437). Majority of adults (39%) and pediatric patients (44%) had normal cytogenetics. This was followed by hyperdiploidy (27%, n=11) in children and t(9;22) in adults (26%, n=6). Intermediate risk cytogenetics were seen in most of the patients (44%, n=26). 57 patients (81%) showed good prednisolone response at day 8. Twenty-seven (40%) patients were MRD positive at day 33. Among the MRD positive cases, nearly 50% patients were aged <1 yr or >10 yr. Seventy-five percent of patients in the age group between 1-9 yrs were MRD negative. 72% patients with WBC >50x103/µl, 40% of patients with CNS involvement and 50% of patients with PPR group had MRD positivity. The incidence of MRD positivity was similar in both B-ALL and T-ALL patients (40% vs 42%). The MRD status and clinical details are summarized in table 1. The disease free survival was significantly higher in MRD negative patients compared to MRD positive patients at 24 months (97% vs 58%, p<0.01) (Figure 1).CONCLUSION: MRD assessment at day 33 post chemotherapy is the most important factor in determining risk of relapse in patients with ALL, surpassing the prognostic information obtained from analysis of pretreatment characteristics alone. This determinant helps to take the decision on offering stem cell transplant to MRD positive patients, on the background of resource constraints, and poor outcomes of CR2 transplants. [Display omitted] Figure 1: Overall Survival at 24 months: MRD positive vs. MRD negative DisclosuresNo relevant conflicts of interest to declare.

Risk-adapted, ofatumumab-based chemoimmunotherapy and consolidation in treatment-naïve chronic lymphocytic leukemia: a phase 2 study

High-risk cytogenetics and minimal residual disease (MRD) after chemoimmunotherapy (CIT) predict unfavorable outcome in chronic lymphocytic leukemia (CLL). This phase 2 study investigated risk-adapted CIT in treatment-naïve CLL (NCT01145209). Patients with high-risk cytogenetics received induction with fludarabine, cyclophosphamide, and ofatumumab. Those without high-risk cytogenetics received fludarabine and ofatumumab. After induction, MRD positive (MRD+) patients received 4 doses of ofatumumab consolidation. MRD negative (MRD-) patients had no intervention. Of 28 evaluable for response, all responded to induction and 10 (36%) achieved MRD-. Two-year progression-free survival (PFS) was 71.4% (CI95, 56.5–90.3%). There was no significant difference in median PFS between the high-risk and the standard-risk groups. Ofatumumab consolidation didn’t convert MRD + to MRD-. In the MRD + group, we saw selective loss of CD20 antigens during therapy. In conclusion, risk-adapted CIT is feasible in treatment-naïve CLL. Ofatumumab consolidation didn’t improve depth of response in MRD + patients. Loss of targetable CD20 likely reduces efficacy of consolidation therapy.