Minimal Residual Disease State Research Articles

Current Approaches and Novel Agents in the Treatment of Chronic Lymphocytic Leukemia.

The treatment of CLL has evolved from traditional chemoimmunotherapy (CIT) to an increasing number of targeted and biologic approaches. Randomized trials have demonstrated superiority of covalent bruton tyrosine kinase inhibitors (cBTKis) over CIT, and second-generation compounds such as acalabrutinib and zanubrutinib appear to have a more favorable efficacy/safety profile than ibrutinib. The noncovalent BTKi, pirtobrutinib, has shown impressive activity after failure of the cBTKis and is quite tolerable. The Bcl-2 inhibitor venetoclax plus a CD20, generally obinutuzumab, provides a high level of efficacy as initial treatment or after failure on a cBTKi, with many patients achieving a state of undetectable minimal residual disease. Promising novel approaches include BTK degraders, bispecific antibodies, and chimeric antigen receptor T-cell (CAR-T)-cell therapy. What is clear is that CIT is archaic, and current and future targeted approaches will continue to improve the outcome for patients with chronic lymphocytic leukemia.

EPCO-14. IDENTIFYING EPIGENETIC VULNERABILITIES OF MINIMAL RESIDUAL DISEASE IN GLIOBLASTOMA USING IN VIVO CRISPR SCREENS

Abstract Therapy for glioblastoma (GBM) includes surgical resection, radiation, and chemotherapy, which functionally reduces tumor burden to a state of minimal residual disease (MRD). However, recurrence over time is universal. Large-scale longitudinal studies of GBM patient samples did not identify selection pressure for specific DNA mutations in recurrent disease, highlighting the importance of epigenetic mechanisms during relapse. In this study, we aim to model MRD and the functional and transcriptional effects of epigenetic perturbations in GBM patient derived xenografts (PDXs). We developed an in vivo inducible genome editing system by engineering 827 GBM stem cells to express doxycycline inducible Cas9 and a pool of 14 CRISPR sgRNAs targeting 5 epigenetic factors (readers/erasers of H3K27me3 and H4 acetyltransferase KAT5) and controls. Tumor cells are implanted orthotopically and mice are followed by serial MRIs until tumor volumes reach 2mm3, after which in vivo editing is induced by administration of doxycycline. Cells are harvested for scRNA-sequencing and targeted amplification of sgRNA libraries, allowing for assignment of sgRNAs to individual cells. Genome editing efficiencies ranged from 50-90% in vivo. Knockout of core Polycomb (PcG) complex members led to dysregulated expression of genes involved in therapeutic resistance and altered cell state distributions. Current directions include optimization of sgRNA targeting and recovery of CRISPR-edited cells from tumors for scaling up screens. In addition, we have developed a “standard of care” treatment regimen in GBM PDX models whereby temozolomide and radiation therapy is dose optimized to achieve a latent state that mimics MRD followed by eventual tumor recurrence. Future experiments will combine MRD modeling in PDXs with in vivo CRISPR screening to characterize functional dependencies of MRD in GBM. This data provides proof of concept for utilizing these tools for studying genotype to phenotype connections in a complex GBM model system.

Efficacy of CLAE Chemotherapy Regimen Followed by Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Relapsed/Refractory Acute Leukemia

To observe the efficacy and safety of CLAE intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with relapsed/refractory acute leukemia (R/R AL). CLAE regimen [cladribine 5 mg/(m2·d), d 1-5; cytarabine 1.5 g/(m2·d), d 1-5; etoposide 100 mg/(m2·d), d 3-5] followed by allo-HSCT was used to treat 3 R/R AL patients. The patients received CLAE chemotherapy in relapsed or refractory status and underwent bone marrow puncture to judge myelodysplastic state. After an interval of 3 to 5 days, followed by preconditioning regimen for allo-HSCT [fludarabine 30 mg/(m2·d), d -7 to d -3; busulfan 0.8 mg/kg q6h, d -6 to d -3 or d -5 to d -2. If the bone marrow hyperplasia was not active and the blasts were less than 10%, busulfan should be used for 3 days. If the bone marrow hyperplasia was active and the blasts were more than 10%, busulfan should be used for 4 days]. Cyclosporin A, mycophenolate mofetil and short-term methotrexate were used for graft-versus-host disease (GVHD) prevention. After transplantation, the status of minimal residual disease (MRD) and bone marrow chimerism were regularly monitored in all 3 patients, and demethylation drugs or dasatinib were used to prevent recurrence 3 months after transplantation. 2 patients with t(11;19) translocation and relapse/refractory acute myeloid leukemia recurred within 6 months after induction of remission, and received intensive chemotherapy with CLAE regimen followed by haploidentical allo-HSCT and unrelated donor allo-HSCT, respectively. The two patients both relapsed 6 months after transplantation, then achieved complete remission by donor lymphocyte infusion, interferon, interleukin-2 and other methods, and disease-free survival was 2 years after transplantation. The other patient was chronic myelogenous leukemia who developed acute lymphoblastic leukemia during oral administration of tyrosine kinase inhibitor, accompanied by T315I and E255K mutations in ABL1 kinase region and additional chromosomal abnormalities. After morphological remission by induction chemotherapy, central nervous system leukemia was complicated. Intensive chemotherapy with CLAE regimen followed by sibling allo-HSCT was performed in the positive state of MRD. The patient relapsed 3 months after transplantation, and achieved remission after chimeric antigen receptor T-cell (CAR-T) therapy, however, he died 5 months after transplantation because of severe cytokine release syndrome (CRS) and GVHD. CLAE regimen followed by allo-HSCT may be an effective salvage treatment option for R/R AL patients to prolong the overall survival.

Long-Term Host Immune Changes Following Treatment with Venetoclax Plus Rituximab in Relapsed/Refractory Chronic Lymphocytic Leukemia

Background: Immune dysregulation is a hallmark of chronic lymphocytic leukemia (CLL) and often leads to hypogammaglobulinemia and frequent infections. Treatment with anti-CD20 antibodies (Abs) such as rituximab (R) is a known risk factor for hypogammaglobulinemia. Despite the widespread use of anti-CD20 Abs in combination with venetoclax (Ven), a B cell lymphoma 2 inhibitor, little has been reported on the long-term impact of this therapy on B cell recovery. We report long-term immune changes post treatment in patients (pts) with relapsed/refractory CLL in the MURANO phase 3 trial (NCT02005471) who completed the full VenR course without progressive disease (PD). Methods: Overall, 389 pts were randomized to either VenR (2 years of Ven with R for the first 6 months) or 6 months of bendamustine-R. Minimal residual disease (MRD) status has been previously reported (Kater et al. J Clin Oncol 2020). In the VenR arm, immunoglobulin (Ig) G, A and M, and T, B, and natural killer (NK) cells were measured centrally at baseline (BL), cycle 4 day 1 (C4D1), end of combination treatment (EOCT), end of treatment (EOT), and every 3 months for up to 3 years, then every 6 months until PD. This is a post hoc analysis among evaluable pts; statistics are descriptive only. Results: In total, 130 pts completed 2 years of VenR without PD (27 pts were excluded from this analysis as they received Ig therapy during the study). BL characteristics amongst those included were consistent with the whole population. Median change from BL by visit (EOT, 12 months, and 24 months post EOT) was calculated among pts with paired evaluable BL and visit samples (Table 1). IgG was significantly decreased at EOT vs BL (n=91; median change from BL, -5.9%). At 12 months (n=77; median change from BL, -4.8%) and 24 months (n=58; median change from BL, 1.1%) post treatment, IgG was not significantly different to BL levels. While there was no reduction in IgA from BL at EOT, at 12 and 24 months post treatment, IgA levels were significantly increased (median change from BL, 12.5% and 35.1%, respectively). IgM levels were significantly reduced at EOT vs BL (median change from BL, -32.3%), but returned to BL levels at 12 months post treatment (median change from BL, 11.1%). At 24 months post treatment, IgM had significantly increased by 49.5% compared with BL levels. There was no significant difference in median IgG, IgA and IgM levels at any time point amongst pts who achieved undetectable (u)MRD at EOT vs pts with MRD at EOT. In pts who received VenR the overall infection rate was low; however, there was a non-statistically significant, but numerically higher rate of grade ≥3 infections occurring on treatment in those with uMRD at EOT compared with those who had MRD at EOT (15.2% [10/66] vs 7.1% [2/28]; p=0.5). T, B and NK cell data were available for all 130 pts. A depletion in CD19+ B cells was observed on treatment, which was more pronounced in pts with uMRD at EOT. After EOCT, B cell recovery started ~1 year later, and stabilized ~30 months after EOCT, regardless of EOT MRD status (Figure 1). Given the differences in disease burden at EOT, comparable CD19+ B cell recovery in pts with uMRD and MRD is likely to be representative of a larger relative proportion of normal B cells vs CLL cells. Additional analyses to test this are underway. At BL, total CD3+ T cells were increased (median [range], 1,991 [135-17,334]; reference values as previously reported, 700-2,100/µL [de Weerdt et al. Blood Adv 2019]). Though a reduction of CD3+ T cells was observed during combination treatment (C4D1), the number of CD3+ T cells were maintained within the reference range post treatment. A similar pattern was observed for NK cells. The median (range) CD4/CD8 ratio at BL was 1.10 (0.33-5.77) and was maintained throughout the study. Conclusions: Overall, pts demonstrated immune recovery following fixed-duration treatment with VenR in this post hoc exploratory analysis. This is the first time that post-treatment serial Ig levels have been reported following VenR therapy. Recovery of IgG, IgA and IgM levels post treatment was observed regardless of EOT MRD status. While the overall incidence of grade ≥3 infections was low, the rate was numerically higher in pts with uMRD at EOT compared with pts with MRD. Additional analyses are underway to assess the correlation between infection and MRD state. A normalization of CD3+ T cells during and after VenR is consistent with previous reports. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MODL-05. DISCOVERY OF DYNAMIC MINIMAL RESIDUAL DISEASE STATES IN ADULT GLIOBLASTOMA USING SINGLE CELL TECHNOLOGY

Abstract Persister states are proposed oncogenic substrates of disease recurrence in cancer. Recent concepts suggest persisters may occur in dynamic states of the cell cycle (such as cycling and non-cycling states). We therefore investigated biological programs at the post-treatment minimal residual disease (MRD) state following standard chemoradiotherapy in patient-derived xenograft models of GBM. Our analysis of single cell RNA sequencing (scRNA-seq) data from 2704 tumor cells (929 cells post treatment, 1775 matched controls) yielded a cellular profile for non-cycling and cycling persister states. We validated these programs in 3 independent scRNA-seq datasets of human glioblastoma specimens consisting of over 16,000 cells from various genetic backgrounds, including an internal two patient-matched primary and recurrent GBM pairs with over 13,000 cells. Finally, we determined that clones identified based on large scale chromosomal rearrangements converge on previously identified persister states including the dynamic states discovered in our study. Our results provide new evidence towards dynamic persister states in glioblastoma, further analysis of these dynamic states is critical to targeting this incurable disease.

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma.

Recurrence of solid tumors renders patients vulnerable to advanced, treatment-refractory disease state with mutational and oncogenic landscape distinctive from initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profile barcoded tumor stem cell populations through therapy at tumor initiation, MRD, and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors show distinct patterns of recurrence in which clonal populations exhibit either a pre-existing fitness advantage or an equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing reveals a tumor-intrinsic immunomodulatory signature with prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from patients with GBM. Our results provide insight into the innate and therapy-driven dynamics of human GBM and the prognostic value of interrogating the MRD state in solid cancers.

TMOD-15. CHARACTERIZATION OF THE MINIMAL RESIDUAL DISEASE STATE REVEALS DISTINCT EVOLUTIONARY TRAJECTORIES OF HUMAN GLIOBLASTOMA

Abstract Recurrence of solid tumors renders patients vulnerable to a distinctly advanced, highly treatment-refractory disease state that has an increased mutational burden and novel oncogenic drivers not detected at initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cancer cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profiled barcoded tumor cell populations through therapy at tumor initiation/engraftment, MRD and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors showed distinct patterns of recurrence in which clonal populations exhibited either a priori, pre-existing fitness, or equipotent fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing revealed a tumor-intrinsic immunomodulatory signature with strong prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from GBM patients at all stages of disease. Our results provide insight into the innate and therapy-driven dynamics of human glioblastoma, and the prognostic value of the interrogating of the MRD state in solid cancers.

Abstract 513: Simultaneous monitoring of immune cell subsets and minimal residual disease in bone marrow aspirate using full-spectrum flow cytometry

Abstract Background: Treatment options for multiple myeloma (MM) patients have expanded and improved outcomes. Autologous stem cell transplantation, immunomodulatory drugs (IMiDs), monoclonal antibodies, and CAR-T cell therapy have direct and indirect immune modulating effects that favor patient responses. However, the majority of patients still relapse due to minimal residual disease (MRD). Current flow cytometry methods that monitor MRD and bone marrow immune cell populations require large cell numbers to achieve assay sensitivity. New approaches are needed to better capture high-resolution data given the limited recovery of bone marrow specimens. Here, we describe the simultaneous monitoring of immune cell subsets in the bone marrow and MRD using a single antibody panel and the Cytek Aurora full-spectrum flow cytometry platform. In a Phase 1/2 study to evaluate an off-the-shelf, allogeneic human placental-derived NK cell product in newly diagnosed MM, this method enables correlative analysis of MRD state with immune profiling. Methods: Bone marrow aspirate (BMA) from 5 patients with newly diagnosed MM following induction therapy were collected for MRD assessment in parallel using next-generation sequencing (NGS) and MRD evaluation by flow cytometry. BMA was lysed of red blood cells and 2x107 cells were stained using TCRγδ, CD16, CD8, CD33, CD117, CD11c, CD3, CD19, CD1c, CD45, CD38, CD14, HLA-DR, CD138, CD141, CD34, CD25, CRTH2, CD56, CD303, CD4, and CD127. Cells were acquired on a Cytek Aurora 4 laser system followed by data analysis using FlowJo software. MRD threshold of 10-5 was used for positivity for both modalities of testing. Results: The 22-parameter panel resolved the majority of immune cell subpopulations present in the bone marrow and peripheral blood including granulocytes, B cells, plasma cells, monocytes, MDSCs, plasmacytoid and classical dendritic cells, NK cells, and T cells (CD4, CD8, NKT, γδT, Treg). Total plasma cells were identified based upon CD38+CD138+ co-staining. Myeloma cells could be identified based on aberrant expression of CD45, CD19, CD56, and CD117. Of the five patients evaluated, one patient was MRD negative by NGS and flow cytometry with no detectable cells. One patient was MRD negative by NGS (0.7x10-5) but positive by flow cytometry (1.53x10-5). The three remaining patients were positive by NGS (6.32x10-4, 7.51x10-4, and 4.78x10-4) and flow cytometry (2.46x10-4, 5.37x10-4, and 9.74x10-4). Conclusion: Utilizing full-spectrum flow cytometry provides streamlined monitoring of MRD while capturing BMA-associated immune cell populations. Compared to validated measures of MRD testing by NGS, our flow cytometry approach provides comparable results while profiling myeloid and lymphoid cell subpopulations, which enables correlative analyses of underlying pathophysiological activities. Citation Format: William van der Touw, Bhavani Stout, James McCauley, Tanel Mahlakõiv, Robert Hariri, Xiaokui Zhang. Simultaneous monitoring of immune cell subsets and minimal residual disease in bone marrow aspirate using full-spectrum flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 513.

Clonotypic Features of Rearranged Immunoglobulin Genes Yield Personalized Biomarkers for Minimal Residual Disease Monitoring in Multiple Myeloma.

Due to improved treatment, more patients with multiple myeloma (MM) reach a state of minimal residual disease (MRD). Different strategies for MM MRD monitoring include flow cytometry, allele-specific oligonucleotide-quantitative PCR, next-generation sequencing, and mass spectrometry (MS). The last 3 methods rely on the presence and the stability of a unique immunoglobulin fingerprint derived from the clonal plasma cell population. For MS-MRD monitoring it is imperative that MS-compatible clonotypic M-protein peptides are identified. To support implementation of molecular MRD techniques, we studied the presence and stability of these clonotypic features in the CoMMpass database. An analysis pipeline based on MiXCR and HIGH-VQUEST was constructed to identify clonal molecular fingerprints and their clonotypic peptides based on transcriptomic datasets. To determine the stability of the clonal fingerprints, we compared the clonal fingerprints during disease progression for each patient. The analysis pipeline to establish the clonal fingerprint and MS-suitable clonotypic peptides was successfully validated in MM cell lines. In a cohort of 609 patients with MM, we demonstrated that the most abundant clone harbored a unique clonal molecular fingerprint and that multiple unique clonotypic peptides compatible with MS measurements could be identified for all patients. Furthermore, the clonal immunoglobulin gene fingerprints of both the light and heavy chain remained stable during MM disease progression. Our data support the use of the clonal immunoglobulin gene fingerprints in patients with MM as a suitable MRD target for MS-MRD analyses.

Venetoclax as a therapeutic option for the treatment of chronic lymphocytic leukemia: the evidence so far

ABSTRACT Introduction Venetoclax, an oral, BCL-2 inhibitor, is approved by the FDA for treatment of CLL in all lines of therapy. Data from landmark studies, including the CLL14 and MURANO trials, demonstrated marked improvement in clinical outcomes compared to chemoimmunotherapy when venetoclax was used in combination with CD20 monoclonal antibodies for fixed treatment duration. Areas covered This article reviews the mechanism of action of venetoclax and discusses how curtailing the BCL signaling pathway undermines CLL pathophysiology. The authors also give their clinical experience with the drug, with emphasis on assessing and managing the risk of venetoclax-associated tumor lysis syndrome (TLS). Expert opinion Venetoclax has positioned itself as one of the primary treatment options for CLL, given the consistent efficacy and deep remissions it has elicited across multiple settings of the disease with a time-limited schedule. Accurate TLS risk evaluation and stringent adherence to the dose-escalation protocols will help optimize patient outcomes. Finally, we expect that current and future studies will (1) ascertain the ideal treatment duration using the minimal residual disease state as a guide and (2) help us understand the optimal role of venetoclax in combination or in sequence with other novel targeted therapies in the treatment of CLL.

Clonal Dynamics after Venetoclax-Obinutuzumab Therapy: Novel Insights from the Randomized, Phase 3 CLL14 Trial

Clonal Dynamics after Venetoclax-Obinutuzumab Therapy: Novel Insights from the Randomized, Phase 3 CLL14 Trial

Prognosis Impact of Positive Minimal Residual Disease By Flow Cytometry Prior to Transplant According to the Cut-Off Threshold in Patients with Acute Myeloid Leukemia

Introduction: Patients with Acute Myeloid Leukemia (AML) and positive Minimal Residual Disease (MRD) prior to allogeneic transplant are currently considered to be a group at high risk of relapse. Multiparameter flow cytometry is a standard technique to measure MRD, and generally we use a 0.1% threshold for positivity. The clinical significance of those patients with an MRD levels >0% but <0.1% is uncertain and it is recommended to define the prognosis of this subgroup. Material and methods: We performed a single-center retrospective analysis of 88 patients transplanted between 2012 and 2020. All patients achieved complete remission (CR) with or without hemoperipheral recovery prior to allogeneic transplant. We have divided our cohort into three groups according to MRD state by flow cytometry: Group 1 patients with negative MRD, Group 2 patients with MRD level >0% but <0.1% and Group 3 patients with MRD ≥ 0.1%. The baseline characteristics of each group were compared using the Chi2 test. The survival analysis was performed through Kaplan-Meier method and the risk was calculated with Cox regression. The Overall Survival (OS) was defined as the time from transplantation to death and the Relapse-Free Survival (RFS) as the time from transplantation to either relapse or death. P<0.05 was defined as statistically significant difference. Results: The baseline characteristics of our cohort are reflected in Table 1. We did not find statistical significant differences except for the response to induction. The median follow-up of the entire cohort was 13.5 months (range 6-43.5). The 4-year RFS (4y-RFS) was 47% and the 4-year OS (4y-OS) 50%. The 4y-RFS was 52.5% in Group 1 vs 59% in Group 2 vs 30% in Group 3. The 4y-OS was 60% in Group 1 vs 60% in Group 2 vs 31% in Group 3 (Image 1). The Hazard Ratio (HR) for RFS and OS comparing Group 1 vs Group 2 was 0.9 [95% CI ((0.3-2.5)] and 1.1 [95% CI (0.4-3)] respectively. The HR for the RFS and OS comparing Group 1 vs 3 was 1.2 [95% CI (0.9-1.7)] and 1.2 [95% CI (0.8-1.6)]. We have stratified patients according to the European LeukemiaNet risk classification. In Group 1, the 4y-RFS was 79% in patients with Favorable Risk (FR) vs 55% in those with Intermediate Risk (IR) and 53% in patients with Adverse Risk (AR) [HR 1.2, 95% CI (0.6-2.3)] and the 4y-OS was 79% vs 54% vs 53% respectively [HR 1.3, 95% CI (0.6-2.5)]. In Group 2, the 4y-RFS was 100% in those with FR vs 83% in IR vs 33% in AR [HR 3.9, 95% CI (0.4-30)] and the 4y-OS was 100% vs 82% vs 36% respectively [HR 4, 95% CI (0.5-32%)]. In Group 3, the 4y-RFS in patients with FR was 82% vs 0% in IR vs 0% in AR [HR 2.1, 95% CI (1.1-4.1)] and the 4y-OS was 82% vs 0% vs 0% respectively [HR 1.6, 95% CI (0.8-3.3)] (Image 2). Conclusions: In our cohort, positive MRD >0.1% prior to transplant identified a group with worse RFS and OS compared to those with negative MRD or positive MRD level >0% but <0.1%. Positive MRD >0.1% is especially relevant in the IR and AR groups of the European LeukemiaNet risk classification. In the AR subgroup even any detectable level of positive MRD could identify patients with unfavorable post-transplant OS and RFS outcomes. We must establish post-transplant strategies in these patients to improve survival. Disclosures Garcia-Gutiérrez: Pfizer: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Incyte: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Bristol-Myers Squibb: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Novartis: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding.

Abstract IA10: Liquid biopsies as biomarkers of disease progression in multiple myeloma

Abstract Multiple myeloma (MM) is a plasma cell dyscrasia that is almost always preceded by the precursor states of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Some patients rapidly progress from MGUS/SMM to overt MM while others remain indolent for many years. Disease evolution also depends on the surrounding microenvironment, which can be either permissive or inhibitory of clonal expansion. The use of liquid biopsies for cancer detection and monitoring is rapidly gaining prominence. Current methods for the detection of circulating tumor cell-free DNA (cfDNA) include low-pass genome sequencing for copy number profiling and deep targeted sequencing for detection of recurrent somatic mutations. However, the sensitivity of these methods is limited among patients with minimal disease, such as MGUS or the minimal residual disease (MRD) state, on account of low tumor burden and a highly variable genomic landscape, whereby most patients do not carry any of the recurrent alterations. I will discuss our current data on detecting cfDNA and circulating tumor cells in the peripheral blood of patients with MGUS, SMM, and overt MM as well as novel personalized, sensitive methods to detect cfDNA with deep UMI-based sequencing for potential use to detect MRD. There is an urgent need for a liquid biopsy assay that can be integrated in the clinical diagnosis of patients for more sensitive tumor detection along with better characterization of their genomic and epigenetic alterations leading to much-needed solutions for integrative staging of MM precursor and MRD patients, with potential implications for patient monitoring and treatment.

IA17 Druggable Vulnerabilities in Therapy-Resistant Lung Cancers

Oncogene-targeted therapies often drive therapeutic responses in patients with advanced lung cancers, but frequently these responses eventually give way to acquired resistance. Blocking acquired resistance is a substantial and open-ended challenge whose difficultly is underscored by the fact that resistance within individual patients is often polyclonal in nature, driven by diverse and co-occurring mechanisms. Here, I will discuss two broad approaches we are employing in the search for therapeutic strategies that delay or circumvent resistance evolution. In the first area, we have identified vulnerabilities present in tumors at minimal residual disease states. For example, we have identified a molecular pathway through which targeted therapies such as EGFR, ALK, and BRAF inhibitors trigger double-strand DNA breaks in the cancer cells that comprise the minimal residual disease state. These cells rely upon an ATM-dependent DNA repair process for their survival and are thus hypersensitive to ATM inhibition. As such, combining oncogene-targeted therapies with an ATM inhibitor leads to more penetrant and durable responses to these agents in vivo. In the second area, we have identified vulnerabilities that arise specifically in tumor cells that develop acquired resistance to oncogene-targeted therapies. Importantly, we have identified scenarios in which these “collateral sensitivities” are conserved across heterogeneous resistant clones with distinct resistance mechanisms, implying that targeting these mechanisms may simultaneously eradicate diverse clones. I will describe examples of mechanism-based collateral sensitivities we have uncovered in lung cancers, melanomas, and leukemias, then demonstrate that by targeting these mechanisms in the upfront setting, it is possible to construct combination therapies that select against resistance.

Regulation of drug metabolizing enzymes in the leukaemic bone marrow microenvironment

The bone marrow (BM) microenvironment contributes to drug resistance in acute myeloid leukaemia (AML) and multiple myeloma (MM). We have shown that the critical drug metabolizing enzymes cytochrome P450 (CYP) 3A4 and cytidine deaminase (CDA) are highly expressed by BM stroma, and play an important role in this resistance to chemotherapy. However, what factors influence the chemoprotective capacity of the BM microenvironment, specifically related to CYP3A4 and CDA expression, are unknown. In this study, we found that the presence of AML cells decreases BM stromal expression of CYP3A4 and CDA, and this effect appears to be at least partially the result of cytokines secreted by AML cells. We also observed that stromal CYP3A4 expression is up‐regulated by drugs commonly used in AML induction therapy, cytarabine, etoposide and daunorubicin, resulting in cross‐resistance. Cytarabine also up‐regulated CDA expression. The up‐regulation of CYP3A4 associated with disease control was reversed by clarithromycin, a potent inhibitor of CYP3A4. Our data suggest that minimal residual disease states are characterized by high levels of stromal drug metabolizing enzymes and thus, strong microenvironment‐mediated drug resistance. These results further suggest a potential role for clinically targeting drug metabolizing enzymes in the microenvironment.

Acute Myeloid Leukemia Stem Cell Heterogeneity and Its Clinical Relevance.

The failure of complete remissions to reliably translate into cures in acute myeloid leukemia (AML) can be explained by the leukemia stem cell (LSC) paradigm, which hypothesizes that rare leukemia cells with stem cell features, including self-renewal capacity and drug resistance, are primarily responsible for both disease maintenance and relapses. Traditionally, the ability to generate AML in immunocompromised mice werehow these so-called LSCs were identified. Only those rare AML cells characterized by a hematopoietic stem cell (HSC) CD34+CD38- phenotype were believed capable of generating leukemia in immunocompromised mice, but more recently, significant heterogeneity in the phenotypes of engrafting AML cells has been demonstrated. Moreover, AML cells that engraft immunocompromised mice do not necessarily represent either the founder clone or those cells responsible for relapse. A recent study found that the most immature phenotype present in an AML was heterogeneous, but correlated with genetically defined risk groups and outcomes. Patients with AML cells expressing a primitive HSC phenotype (CD34+CD38- with high aldehyde dehydrogenase activity) manifested significantly lower complete remission rates, as well as poorer event-free and overall survivals. AMLs in which the most primitive cells displayed more mature phenotypes were associated with better outcomes. The strong clinical correlations suggest that the most immature phenotype detectable within a patient's AML might serve as a biomarker for "clinically relevant" LSCs. The minimal residual disease state during first remission may be the optimal setting to study novel LSC-targeted therapies, since they may have limited activity against the bulk leukemia and will be utilized at lowest tumor burden as well as least tumor heterogeneity.

Diagnosis of Acute Lymphoblastic Leukemia Originating From T-Lineage Precursors and Approaches to Minimal Residual Disease Monitoring

Background. Minimal residual disease (MRD) is an independent prognostic factor in acute lymphoblastic leukemia (ALL) in children. The immunological assessment of MRD cell count is based on aberrant immunophenotype of tumor lymphoblasts. However, in the case of ALL originating from T-lineage precursor cells (T-ALL) no clear aberrancy criteria have been defined, yet. Flow-cytometric MRD assessment in T-ALL can be based on characteristics of normal T-cell ontogenesis, i.e. the absence of normal T-lineage precursor cells (T-LP) in bone marrow. Aim. To assess the feasibility of immunological method of flow cytometry for MRD detection based on T-LP immunophenotype on Days 15 and 33 of treatment of T-ALL children. Materials & Methods. The analysis included the data on primary immunophenotype and MRD assessment on Days 15 and 33 of treatment of 31 T-ALL patients in the age of 2-17 years. In the majority of cases (61.3 %) the cortical/ thymic immuno-subvariant of ALL was detected, in the rest of cases (38.7 %) it was the pre-T-cell one. Diagnosis was based on cumulative results of morphocytochemical and immunological bone marrow analyses. Assessing the MRD state the morphological and immunological analyses of bone marrow aspirate were carried out in parallel with one and the same tube. All patients enrolled in the trial were treated at Scientific Research Institute of Pediatric Oncology and Hematology of NN Blokhin National Medical Cancer Research Center according to the ALL IC-BFM 2009 protocol. Results. Our study demonstrated that at all therapy stages MRD can be assessed by the unified immunological method based on detecting cyCD3<sup>+</sup>CD7<sup>+/++</sup>smCD3<sup>-</sup> (T-LP) immunophenotype cells. It is important to ensure that the correct clones of monoclonal antibodies are used for detecting CD3 cytoplasmic and membrane molecules (UCHT1 and SK7, respectively). Standard risk group included no patients. The majority of patients (76.2 %) treated according to ALL IC-BFM 2009 protocol were assigned to medium risk group on Day 15 of treatment. By Day 33 a quarter of them (25 %) was included into high risk group. Conclusion. The capabilities of multicolor flow cytometry allow for the most complete characterization of primary immu-nophenotype of tumor T-cell lymphoblasts for further search of leukemia-associated immunophenotypes. Specific ontogenesis features of normal T-cells enable unification of immunological approaches to MRD assessment at all stages of T-ALL therapy.

PD-1 Blockade with Pembrolizumab for Classical Hodgkin Lymphoma after Autologous Stem Cell Transplantation

PD-1 Blockade with Pembrolizumab for Classical Hodgkin Lymphoma after Autologous Stem Cell Transplantation

PD-1 Blockade for Diffuse Large B-Cell Lymphoma after Autologous Stem Cell Transplantation

PD-1 Blockade for Diffuse Large B-Cell Lymphoma after Autologous Stem Cell Transplantation

Peripheral Immunotype Correlates with Minimal Residual Disease Status and Is Modulated by Immunomodulatory Drugs in Multiple Myeloma

Data indicate reversal of immune dysfunction with active treatment; however, the precise contribution of specific immune effector and immune suppressor components to achieve a minimal residual disease (MRD) state and immunomodulatory drug–mediated immunomodulatory effects in multiple myeloma (MM) patients remains poorly understood. In this prospective proof-of-principle study we sought to determine the dynamic alterations in natural killer (NK), NK-T, and T cells, including maturation and activating/inhibitory repertoire associated with MRDpos versus MRDneg status after autologous stem cell transplantation (ASCT) and during lenalidomide-based maintenance therapy. Of the 46MM patients enrolled, 36 had bone marrow MRD assessment 60+ days post-ASCT, 30 had longitudinal blood immunotyping during maintenance (pretherapy and after cycles 1, 3, and 6), and 20 had both MRD assessment and longitudinal immunotyping. Multicolor flow cytometry was used for MRD and immunotyping. Although the absolute number of NK cells was significantly lower in patients with MRDpos response, phenotypically NK cells in these patients displayed higher expression of activating receptors KIRDS4 and decreased expression of inhibitory molecules NKG2A compared with the MRDneg group. Furthermore, we observed significantly lower frequencies of T cells displaying KIR3DL1 in MRDpos versus MRDneg patients. Longitudinal immunotyping during lenalidomide maintenance showed loss of mature NK effector function, augmentation of NK-T effector function, and acquisition of PD1 independent anergic state. Our findings also suggest skewing of T cells toward an exhausted state during the maintenance phase in MRDpos patients. Put together, these observations provide a distinctive signature for MRDneg and MRDpos groups. These data support exploration of immune profiling in prospective clinical trials according to MRD-defined responses to identify patients that may benefit from maintenance intensification/modification or maintenance withdrawal.