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Multiple myeloma (MM) is a genetically heterogeneous disease and the management of relapses is one of the biggest clinical challenges. TP53 alterations are established high-risk markers and are included in the current disease staging criteria. KRAS is the most frequently mutated gene affecting around 20% of MM patients. Applying Clonal Competition Assays (CCA) by co-culturing color-labeled genetically modified cell models, we recently showed that mono- and biallelic alterations in TP53 transmit a fitness advantage to the cells. Here, we report a similar dynamic for two mutations in KRAS (G12A and A146T), providing a biological rationale for the high frequency of KRAS and TP53 alterations at MM relapse. Resistance mutations, on the other hand, did not endow MM cells with a general fitness advantage but rather presented a disadvantage compared to the wild-type. CUL4B KO and IKZF1 A152T transmit resistance against immunomodulatory agents, PSMB5 A20T to proteasome inhibition. However, MM cells harboring such lesions only outcompete the culture in the presence of the respective drug. To better prevent the selection of clones with the potential of inducing relapse, these results argue in favor of treatment-free breaks or a switch of the drug class given as maintenance therapy. In summary, the fitness benefit of TP53 and KRAS mutations was not treatment-related, unlike patient-derived drug resistance alterations that may only induce an advantage under treatment. CCAs are suitable models for the study of clonal evolution and competitive (dis)advantages conveyed by a specific genetic lesion of interest, and their dependence on external factors such as the treatment.

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This study discusses the importance of minimal residual disease (MRD) detection in acute myeloid leukemia (AML) patients using liquid biopsy and next-generation sequencing (NGS). AML prognosis is based on various factors, including genetic alterations. NGS has revealed the molecular complexity of AML and helped refine risk stratification and personalized therapies. The long-term survival rates for AML patients are low, and MRD assessment is crucial in predicting prognosis. Currently, the most common methods for MRD detection are flow cytometry and quantitative PCR, but NGS is being incorporated into clinical practice due to its ability to detect genomic aberrations in the majority of AML patients. Typically, bone marrow samples are used for MRD assessment, but using peripheral blood samples or liquid biopsies would be less invasive. Leukemia originates in the bone marrow, along with the cfDNA obtained from peripheral blood. This study aimed to assess the utility of cell-free DNA (cfDNA) from peripheral blood samples for MRD detection in AML patients. A cohort of 20 AML patients was analyzed using NGS, and a correlation between MRD assessment by cfDNA and circulating tumor cells (CTCs) in paired samples was observed. Furthermore, a higher tumor signal was detected in cfDNA compared to CTCs, indicating greater sensitivity. Challenges for the application of liquid biopsy in MRD assessment were discussed, including the selection of appropriate markers and the sensitivity of certain markers. This study emphasizes the potential of liquid biopsy using cfDNA for MRD detection in AML patients and highlights the need for further research in this area.

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BACKGROUND/AIM: Extracellular vesicle DNA (EV-DNA) has emerged as a novel biomarker for tumor mutation detection using liquid biopsies, exhibiting biological advantages compared to cell-free DNA (cfDNA). This study assessed the feasibility of EV-DNA and cfDNA extraction and sequencing in old serum samples of patients with breast cancer (BC). PATIENTS AND METHODS: A total of 28 serum samples of 27 patients with corresponding clinical information were collected between 1983 and 1991. EV-DNA was extracted using Exo-GAG kit (Nasabiotech) and cfDNA using QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen), respectively. Subsequently, 10 matched samples (EV-DNA n=5, cfDNA n=5) of five patients were subjected to sequencing using the Oncomine™ Breast cfDNA Research Assay v2 (Thermo Fisher Scientific). RESULTS: Samples were collected on median 1.9 years after primary diagnosis [interquartile range (IQR)=0.2-7.2]. Median follow-up was 9.5 years (IQR=5.2-14.2). Median age of serum samples was 36.1 years (IQR=34.5-37.3). EV-DNA and cfDNA were extracted from 100% (28/28) of the included samples. Both, DNA quantity and concentration were comparable between EV-DNA and cfDNA. Sequencing was successfully performed in 100% (10/10) of the included samples. Two matched analyses yielded equivalent results in EV-DNA and cfDNA (no mutations, n=1; PIK3CA mutation, n=1), whilst in two analyses, PIK3CA mutation was only found in cfDNA, and in one analysis, a TP53 mutation was only found in EV-DNA. CONCLUSION: EV-DNA extraction and sequencing in old serum samples of patients with BC is feasible and has the potential to address clinically relevant questions in longitudinal studies.

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The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.

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[This corrects the article DOI: 10.3389/fimmu.2023.1188818.].

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PURPOSE: To assess the potential value of LiqBio as a complementary tool for diagnosis and surveillance of BCL. METHODS: This prospective multi-center study included 78 patients (25 follicular lymphomas (FL) and 53 large B-cell lymphomas (LBCL)). We performed next-generation sequencing (NGS) of cfDNA LiqBio and paired gDNA tissue biopsies at diagnosis and compared the mutational statuses. Also, through NGS of LiqBio, we identified MRD biomarkers and compared this novel LiqBio-MRD method with PET/CT in detecting MRD at follow-up. RESULTS: We identified mutations in 71% of LiqBio and 95% of tissue biopsies, and found a correlation between variant allele frequency of somatic mutations. Additionally, we identified mutations in 73% of LiqBio from patients with no available tissue samples or no mutations in them. Regarding the utility of LiqBio-MRD as a dynamic monitoring tool, when compared with the PET/CT method, a lower sensitivity was observed for LiqBio-MRD at 92.3% (vs. 100% for PET/CT), but a higher specificity of 91.3% (vs. 86.9% for PET/CT). CONCLUSION: Genetic profiling of tumor cfDNA in plasma LiqBio is a complementary tool for BCL diagnosis and MRD surveillance.

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Background: CART therapy has produced a paradigm shift in the treatment of relapsing FL patients. Strategies to optimize disease surveillance after these therapies are increasingly necessary. This study explores the potential value of ctDNA monitoring with an innovative signature of personalized trackable mutations. Method: Eleven FL patients treated with anti-CD19 CAR T-cell therapy were included. One did not respond and was excluded. Genomic profiling was performed before starting lymphodepleting chemotherapy to identify somatic mutations suitable for LiqBio-MRD monitoring. The dynamics of the baseline mutations (4.5 per patient) were further analyzed on 59 cfDNA follow-up samples. PET/CT examinations were performed on days +90, +180, +365, and every six months until disease progression or death. Results: After a median follow-up of 36 months, all patients achieved a CR as the best response. Two patients progressed. The most frequently mutated genes were CREBBP, KMT2D and EP300. Simultaneous analysis of ctDNA and PET/CT was available for 18 time-points. When PET/CT was positive, two out of four ctDNA samples were LiqBio-MRD negative. These two negative samples corresponded to women with a unique mesenteric mass in two evaluations and never relapsed. Meanwhile, 14 PET/CT negative images were mutation-free based on our LiqBio-MRD analysis (100%). None of the patients had a negative LiqBio-MRD test by day +7. Interestingly, all durably responding patients had undetectable ctDNA at or around three months after infusion. Two patients presented discordant results by PET/CT and ctDNA levels. No progression was confirmed in these cases. All the progressing patients were LiqBio-MRD positive before progression. Conclusion: This is a proof-of-principle for using ctDNA to monitor response to CAR T-cell therapy in FL. Our results confirm that a non-invasive liquid biopsy MRD analysis may correlate with response and could be used to monitor response. Harmonized definitions of ctDNA molecular response and pinpointing the optimal timing for assessing ctDNA responses are necessary for this setting. If using ctDNA analysis, we suggest restricting follow-up PET/CT in CR patients to a clinical suspicion of relapse, to avoid false-positive results.

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For the treatment of Multiple Myeloma, proteasome inhibitors are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients. The proteasome is a large and complex machinery. Here, we focus on the AAA ATPases of the 19S proteasome regulator (PSMC1-6) and their implication in PI resistance. As an example of cancer evolution and the acquisition of resistance, we conducted an in-depth analysis of an index patient by applying FISH, WES, and immunoglobulin-rearrangement sequencing in serial samples, starting from MGUS to newly diagnosed Multiple Myeloma to a PI-resistant relapse. The WES analysis uncovered an acquired PSMC2 Y429S mutation at the relapse after intensive bortezomib-containing therapy, which was functionally confirmed to mediate PI resistance. A meta-analysis comprising 1499 newly diagnosed and 447 progressed patients revealed a total of 36 SNVs over all six PSMC genes that were structurally accumulated in regulatory sites for activity such as the ADP/ATP binding pocket. Other alterations impact the interaction between different PSMC subunits or the intrinsic conformation of an individual subunit, consequently affecting the folding and function of the complex. Interestingly, several mutations were clustered in the central channel of the ATPase ring, where the unfolded substrates enter the 20S core. Our results indicate that PSMC SNVs play a role in PI resistance in MM.

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In the present study, we screened 84 Follicular Lymphoma patients for somatic mutations suitable as liquid biopsy MRD biomarkers using a targeted next-generation sequencing (NGS) panel. We found trackable mutations in 95% of the lymph node samples and 80% of the liquid biopsy baseline samples. Then, we used an ultra-deep sequencing approach with 2 · 10-4 sensitivity (LiqBio-MRD) to track those mutations on 151 follow-up liquid biopsy samples from 54 treated patients. Positive LiqBio-MRD at first-line therapy correlated with a higher risk of progression both at the interim evaluation (HRINT 11.0, 95% CI 2.10-57.7, p = 0.005) and at the end of treatment (HREOT, HR 19.1, 95% CI 4.10-89.4, p < 0.001). Similar results were observed by PET/CT Deauville score, with a median PFS of 19 months vs. NR (p < 0.001) at the interim and 13 months vs. NR (p < 0.001) at EOT. LiqBio-MRD and PET/CT combined identified the patients that progressed in less than two years with 88% sensitivity and 100% specificity. Our results demonstrate that LiqBio-MRD is a robust and non-invasive approach, complementary to metabolic imaging, for identifying FL patients at high risk of failure during the treatment and should be considered in future response-adapted clinical trials.

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PURPOSE: Proteasome inhibitors (PI) are the backbone of various treatment regimens in multiple myeloma. We recently described the first in-patient point mutations affecting the 20S subunit PSMB5 underlying PI resistance. Notably, in vivo, the incidence of mutations in PSMB5 and other proteasome encoding genes is too low to explain the development of resistance in most of the affected patients. Thus, additional genetic and epigenetic alterations need to be explored. EXPERIMENTAL DESIGN: We performed DNA methylation profiling by Deep Bisulfite Sequencing in PSMB5, PSMC2, PSMC5, PSMC6, PSMD1, and PSMD5, a subset of proteasome subunits that have hitherto been associated with PI resistance, recruited from our own previous research, the literature, or a meta-analysis on the frequency of somatic mutations. Methylation was followed up on gene expression level and by dual-luciferase reporter assay. The KMS11 cell line served as a model to functionally test the impact of demethylating agents. RESULTS: We identified PSMD5 promoter hypermethylation and subsequent epigenetic gene silencing in 24% of PI refractory patients. Hypermethylation correlated with decreased expression and the regulatory impact of this region was functionally confirmed. In contrast, patients with newly diagnosed multiple myeloma, along with peripheral blood mononuclear cells and CD138+ plasma cells from healthy donors, generally show unmethylated profiles. CONCLUSIONS: Under the selective pressure of PI treatment, multiple myeloma cells acquire methylation of the PSMD5 promoter silencing the PSMD5 gene expression. PSMD5 acts as a key orchestrator of proteasome assembly and its downregulation was described to increase the cell's proteolytic capacity. PSMD5 hypermethylation, therefore, represents a novel mechanism of PI tolerance in multiple myeloma.

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[This corrects the article DOI: 10.3389/fimmu.2023.1188818.].

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Next-generation sequencing (NGS) has greatly improved our ability to detect the genomic aberrations occurring in multiple myeloma (MM); however, its transfer to routine clinical labs and its validation in clinical trials remains to be established. We designed a capture-based NGS targeted panel to identify, in a single assay, known genetic alterations for the prognostic stratification of MM. The NGS panel was designed for the simultaneous study of single nucleotide and copy number variations, insertions and deletions, chromosomal translocations and V(D)J rearrangements. The panel was validated using a cohort of 149 MM patients enrolled in the GEM2012MENOS65 clinical trial. The results showed great global accuracy, with positive and negative predictive values close to 90% when compared with available data from fluorescence in situ hybridization and whole-exome sequencing. While the treatments used in the clinical trial showed high efficacy, patients defined as high-risk by the panel had shorter progression-free survival (p = 0.0015). As expected, the mutational status of TP53 was significant in predicting patient outcomes (p = 0.021). The NGS panel also efficiently detected clonal IGH rearrangements in 81% of patients. In conclusion, molecular karyotyping using a targeted NGS panel can identify relevant prognostic chromosomal abnormalities and translocations for the clinical management of MM patients.

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The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E-4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E-4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.

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The study of cell-free DNA (cfDNA) and other peripheral blood components (known as "liquid biopsies") is promising, and has been investigated especially in solid tumors. Nevertheless, it is increasingly showing a greater utility in the diagnosis, prognosis, and response to treatment of hematological malignancies; in the future, it could prevent invasive techniques, such as bone marrow (BM) biopsies. Most of the studies about this topic have focused on B-cell lymphoid malignancies; some of them have shown that cfDNA can be used as a novel way for the diagnosis and minimal residual monitoring of B-cell lymphomas, using techniques such as next-generation sequencing (NGS). In myelodysplastic syndromes, multiple myeloma, or chronic lymphocytic leukemia, liquid biopsies may allow for an interesting genomic representation of the tumor clones affecting different lesions (spatial heterogeneity). In acute leukemias, it can be helpful in the monitoring of the early treatment response and the prediction of treatment failure. In chronic lymphocytic leukemia, the evaluation of cfDNA permits the definition of clonal evolution and drug resistance in real time. However, there are limitations, such as the difficulty in obtaining sufficient circulating tumor DNA for achieving a high sensitivity to assess the minimal residual disease, or the lack of standardization of the method, and clinical studies, to confirm its prognostic impact. This review focuses on the clinical applications of cfDNA on the minimal residual disease in hematological malignancies.

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The assessment of measurable residual disease (MRD) in bone marrow has proven of prognostic relevance in patients with multiple myeloma (MM). Nevertheless, and unlike other hematologic malignancies, the use of MRD results to make clinical decisions in MM has been underexplored to date. In this retrospective study, we present the results from a multinational and multicenter series of 400 patients with MRD monitoring during front-line therapy with the aim of exploring how clinical decisions made based on those MRD results affected outcomes. As expected, achievement of MRD negativity at any point was associated with improved PFS versus persistent MRD positivity (median PFS 104 vs. 45 months, p < 0.0001). In addition, however, 67 out of 400 patients underwent a clinical decision (treatment discontinuation, intensification or initiation of a new therapy) based on MRD results. Those patients in whom a treatment change was made showed a prolonged PFS in comparison with those 333 patients in which MRD results were not acted upon (respectively, mPFS 104 vs. 62 months, p = 0.005). In patients who achieved MRD negativity during maintenance (n = 186) on at least one occasion, stopping therapy in 24 patients vs. continuing in 162 did not alter PFS (mPFS 120 months vs. 82 months, p = 0.1). Most importantly, however, in patients with a positive MRD during maintenance (n = 214), a clinical decision (either intensification or change of therapy) (n = 43) resulted in better PFS compared to patients in whom no adjustment was made (n = 171) (mPFS NA vs. 39 months, p = 0.02). Interestingly, there were no significant differences when MRD was assessed by flow cytometry or by next-generation sequencing. Herein, we find that MRD is useful in guiding clinical decisions during initial therapy and has a positive impact on PFS in MM patients. This potentially opens a new dimension for the use of MRD in MM, but this role still remains to be confirmed in prospective, randomized clinical trials.

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Many studies over the last 20 years have investigated the role of mitochondrial DNA (mtDNA) alterations in carcinogenesis. However, the status of the mtDNACN in MM and its implication in the pathogenesis of the disease remains unclear. We examined changes in plasma cell mtDNACN across different stages of MM by applying RT-PCR and high-throughput sequencing analysis. We observed a significant increase in the average mtDNACN in myeloma cells compared with healthy plasma cells (157 vs. 40 copies; p = 0.02). We also found an increase in mtDNACN in SMM and newly diagnosed MM (NDMM) paired samples and in consecutive relapses in the same patient. Survival analysis revealed the negative impact of a high mtDNACN in progression-free survival in NDMM (p = 0.005). Additionally, we confirmed the higher expression of mitochondrial biogenesis regulator genes in myeloma cells than in healthy plasma cells and we detected single nucleotide variants in several genes involved in mtDNA replication. Finally, we found that there was molecular similarity between "rapidly-progressing SMM" and MM regarding mtDNACN. Our data provide evidence that malignant transformation of myeloma cells involves the activation of mitochondrial biogenesis, resulting in increased mtDNA levels, and highlights vulnerabilities and potential therapeutic targets in the treatment of MM. Accordingly, mtDNACN tracking might guide clinical decision-making and management of complex entities such as high-risk SMM.

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Cereblon is the direct binding target of the immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM), the second most frequent hematologic malignancy. Patients respond well to initial treatment with IMiDs, but virtually all patients develop drug resistance over time, and the underlying mechanisms are poorly understood. We identified an as yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found to be increased in healthy plasma cells, but was more pronounced in IMiD-refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNA methyltransferase inhibitor (DNTMi) in vitro experiments induced CRBN enhancer demethylation, and sensitizing effects on lenalidomide treatment were observed in 2 MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in MM and may predict IMiD response prior to treatment.

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Mitochondria are involved in the development and acquisition of a malignant phenotype in hematological cancers. Recently, their role in the pathogenesis of multiple myeloma (MM) has been suggested to be therapeutically explored. MYC is a master regulator of b-cell malignancies such as multiple myeloma, and its activation is known to deregulate mitochondrial function. We investigated the impact of mitochondrial activity on the distinct entities of the disease and tested the efficacy of the mitochondrial inhibitor, tigecycline, to overcome MM proliferation. COXII expression, COX activity, mitochondrial mass, and mitochondrial membrane potential demonstrated a progressive increase of mitochondrial features as the disease progresses. In vitro and in vivo therapeutic targeting using the mitochondrial inhibitor tigecycline showed promising efficacy and cytotoxicity in monotherapy and combination with the MM frontline treatment bortezomib. Overall, our findings demonstrate how mitochondrial activity emerges in MM transformation and disease progression and the efficacy of therapies targeting these novel vulnerabilities.

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