Immune reconstitution after single-unit umbilical cord blood transplantation using anti-thymoglobulin and myeloablative conditioning in adults with hematological malignancies.

This study aimed to investigate the kinetics of immune recovery following umbilical cord blood transplantation (UCBT) in adults who received a myeloablative conditioning (MAC) regimen and antithymocyte globulin (ATG). While the immune recovery kinetics has been extensively studied in pediatric UCBT recipients, limited data exist for adults. We conducted a comprehensive analysis of 221 consecutive adult patients who underwent UCBT with MAC and ATG at a single institution. Our objective was to evaluate the influence of patient, disease, and transplant factors, along with acute graft-versus-host disease (aGVHD), on immune reconstitution and overall survival. Our findings confirm a delayed recovery of T cells, while B and NK cell reconstitution exhibited rapid progress, with NK cell counts reaching normal levels within 3 months post-transplantation and B cells within 6 months. Within CD3+ T cells, CD8+ T cells also experienced a delayed recovery (12 months), but to a lesser extent compared to CD4+ T cells (18 months). Delayed immune recovery of T-cell subsets was associated with the development of aGVHD grade II-IV, older age, CMV negativity, and a female donor. Patients with lymphoproliferative diseases showed slower NK cell recovery. Our study demonstrates that adult patients undergoing MAC with ATG and receiving a single unit UCBT for hematologic malignancies experienced rapid reconstitution of NK and B cells. However, T cell recovery, particularly CD4+ T cells, was significantly delayed. To enhance T cell recovery, it may be crucial to consider UCB units with higher cellularity and optimize ATG doses in conditioning.

Bladder cancer patients have increased NETosis and impaired DNaseI-mediated NET degradation that can be therapeutically restored in vitro.

Background: Neutrophils, key players of the immune system, also promote tumor development through the formation of neutrophil extracellular traps (NETs) in a process called NETosis. NETs are extracellular networks of DNA, histones and cytoplasmic and granular proteins (calprotectin, myeloperoxidase, elastase, etc.) released by neutrophils upon activation. NETs regulate tumor growth while promoting angiogenesis and invasiveness, and tumor cells also stimulate NETosis. Although NETosis seems to be increased in cancer patients, an increase of NETs in plasma may also be mediated by an impaired degradation by plasma DNaseI, as evidenced in several immunological disorders like lupus nephritis. However, this has never been evidenced in bladder cancer (BC) patients. Herein, we aimed to evaluate the occurrence of increased NETosis in plasma and tumor tissue of BC patients, to ascertain whether it is mediated by a reduced DNaseI activity and degradation, and to in vitro explore novel therapeutic interventions. Methods: We recruited 71 BC patients from whom we obtained a plasma sample before surgery and a formalin-fixed paraffin embedded tumor tissue sample, and 64 age- and sex-matched healthy controls from whom we obtained a plasma sample. We measured NETs markers (cell-free fDNA, calprotectin, nucleosomes and neutrophil elastase) and the DNaseI activity in plasma with specific assays. We also measured NETs markers in BC tissue by immunofluorescence. Finally, we evaluated the ability of BC and control plasma to degrade in vitro-generated NETs, and evaluated the performance of the approved recombinant human DNaseI (rhDNaseI, Dornase alfa, Pulmozyme®, Roche) to restore the NET-degradation ability of plasma. In vitro experiments were performed in triplicate. Statistical analysis was conducted with Graphpad (v.8.0.1). Results: NETosis occurs in BC tissue, more profusely in the muscle-invasive subtype (P<0.01), that with the worst prognosis. Compared to controls, BC patients had increased NETosis and a reduced DNaseI activity in plasma (P<0.0001), which leads to an impairment to degrade NETs (P<0.0001). Remarkably, this can be therapeutically restored with rhDNaseI to the level of healthy controls. Conclusion: To the best of our knowledge, this is the first report demonstrating that BC patients have an increased NETosis systemically and in the tumor microenvironment, in part caused by an impaired DNaseI-mediated NET degradation. Remarkably, this defect can be therapeutically restored in vitro with the approved Dornase alfa, thus Pulmozyme® could become a potential therapeutic tool to locally reduce BC progression.

Cytogenetic Assessment and Risk Stratification in Myelofibrosis with Optical Genome Mapping.

Cytogenetic assessment in myelofibrosis is essential for risk stratification and patient management. However, an informative karyotype is unavailable in a significant proportion of patients. Optical genome mapping (OGM) is a promising technique that allows for a high-resolution assessment of chromosomal aberrations (structural variants, copy number variants, and loss of heterozygosity) in a single workflow. In this study, peripheral blood samples from a series of 21 myelofibrosis patients were analyzed via OGM. We assessed the clinical impact of the application of OGM for disease risk stratification using the DIPSS-plus, GIPSS, and MIPSS70+v2 prognostic scores compared with the standard-of-care approach. OGM, in combination with NGS, allowed for risk classification in all cases, compared to only 52% when conventional techniques were used. Cases with unsuccessful karyotypes (n = 10) using conventional techniques were fully characterized using OGM. In total, 19 additional cryptic aberrations were identified in 9 out of 21 patients (43%). No alterations were found via OGM in 4/21 patients with previously normal karyotypes. OGM upgraded the risk category for three patients with available karyotypes. This is the first study using OGM in myelofibrosis. Our data support that OGM is a valuable tool that can greatly contribute to improve disease risk stratification in myelofibrosis patients.

Definition and Clinical Significance of the Monoclonal Gammopathy of Undetermined Significance-Like Phenotype in Patients With Monoclonal Gammopathies.

PURPOSE: The existence of patients with multiple myeloma (MM) and light-chain (AL) amyloidosis who present with a monoclonal gammopathy of undetermined significance (MGUS)-like phenotype has been hypothesized, but methods to identify this subgroup are not standardized and its clinical significance is not properly validated. PATIENTS AND METHODS: An algorithm to identify patients having MGUS-like phenotype was developed on the basis of the percentages of total bone marrow (BM) plasma cells (PC) and of clonal PC within the BM PC compartment, determined at diagnosis using flow cytometry in 548 patients with MGUS and 2,011 patients with active MM. The clinical significance of the algorithm was tested and validated in 488 patients with smoldering MM, 3,870 patients with active MM and 211 patients with AL amyloidosis. RESULTS: Patients with smoldering MM with MGUS-like phenotype showed significantly lower rates of disease progression (4.5% and 0% at 2 years in two independent series). There were no statistically significant differences in time to progression between treatment versus observation in these patients. In active newly diagnosed MM, MGUS-like phenotype retained independent prognostic value in multivariate analyses of progression-free survival (PFS; hazard ratio [HR], 0.49; P = .001) and overall survival (OS; HR, 0.56; P = .039), together with International Staging System, lactate dehydrogenase, cytogenetic risk, transplant eligibility, and complete remission status. Transplant-eligible patients with active MM with MGUS-like phenotype showed PFS and OS rates at 5 years of 79% and 96%, respectively. In this subgroup, there were no differences in PFS and OS according to complete remission and measurable residual disease status. Application of the algorithm in two independent series of patients with AL predicted for different survival. CONCLUSION: We developed an open-access algorithm for the identification of MGUS-like patients with distinct clinical outcomes. This phenotypic classification could become part of the diagnostic workup of MM and AL amyloidosis.

IgG antibody response to pneumococcal-conjugated vaccine (Prevenar®13) in children with immunodeficiency disorders.

Measurement of anti-pneumococcal capsular polysaccharides (anti-PnPs) IgG titers is an important tool in the immunologic assessment of patients with suspected immunodeficiency disorders (ID) to reduce the morbi-mortality and minimize severe infections. Retrospectively, we studied the relationship among anti-PnPs IgG response to 3 doses of Prevenar®13, levels of immune system components, leukocyte populations, and clinical data in children with ID. Serum samples were collected at least 4 weeks post vaccination. Subsequently, multi-serotype enzyme-linked immunosorbent assay (ELISA) was performed. Eighty-seven children (under 12 years) were enrolled. Primary immunodeficiency disorder (PID) was the most common disorder (45) followed by possible immunodeficiency disorder (POID) (19), secondary immunodeficiency disorder (SID) (15), and mixed immunodeficiency disorder (MID) (8). The median age was 3 (1.50-5.33) years, 65% of patients were male. Deficient production of anti-PnPs IgG (titer ≤ 50 mg/L) was detected in 47 patients (54%), especially in the MID group, all of them under immunosuppressive therapy. In PCV13 responders, the mean of leukocyte population levels was higher with statistically significance differences in CD4 + /CD8 + T lymphocytes (p = 0.372, p = 0.014) and CD56 + /CD16 + NK (p = 0.016). Patients with previous bone marrow transplantation were the worst PCV13 responders. Pneumococcal IgG antibody titers (post-vaccination) along with clinical and analytical markers represented.

Protein-Functionalized Microgel for Multiple Myeloma Cells' 3D Culture.

Multiple myeloma is a hematologic neoplasm caused by an uncontrolled clonal proliferation of neoplastic plasma cells (nPCs) in the bone marrow. The development and survival of this disease is tightly related to the bone marrow environment. Proliferation and viability of nPCs depend on their interaction with the stromal cells and the extracellular matrix components, which also influences the appearance of drug resistance. Recapitulating these interactions in an in vitro culture requires 3D environments that incorporate the biomolecules of interest. In this work, we studied the proliferation and viability of three multiple myeloma cell lines in a microgel consisting of biostable microspheres with fibronectin (FN) on their surfaces. We also showed that the interaction of the RPMI8226 cell line with FN induced cell arrest in the G0/G1 cell cycle phase. RPMI8226 cells developed a significant resistance to dexamethasone, which was reduced when they were treated with dexamethasone and bortezomib in combination.

Novel microgel culture system as semi-solid three-dimensional in vitro model for the study of multiple myeloma proliferation and drug resistance.

Multiple myeloma (MM) is a hematological malignancy in which the patient's drug resistance is one of the main clinical problems. As 2D cultures do not recapitulate the cellular microenvironment, which has a key role in drug resistance, there is an urgent need for better biomimetic models. Here, a novel 3D platform is used to model MM. The semi-solid culture consists of a dynamic suspension of microspheres and MM cells, termed as microgel. Microspheres are synthesized with acrylic polymers of different sizes, compositions, and functionalities (fibronectin or hyaluronic acid). Optimal conditions for the platform in terms of agitation speed and microsphere size have been determined. With these parameters the system allows good proliferation of the MM cell lines RPMI8226, U226, and MM1.S. Interestingly, when used for drug resistance studies, culture of the three MM cell lines in microgels showed close agreement in revealing the role of acrylic acid in resistance to anti-MM drugs such as dexamethasone and bortezomib. This work presents a unique platform for the in vitro modeling of non-solid tumors since it allows keeping non-adherent cells in suspension conditions but in a 3D context that can be easily tuned with different functionalizations.

Concurrent Zrsr2 mutation and Tet2 loss promote myelodysplastic neoplasm in mice.

RNA splicing and epigenetic gene mutations are the most frequent genetic lesions found in patients with myelodysplastic neoplasm (MDS). About 25% of patients present concomitant mutations in such pathways, suggesting a cooperative role in MDS pathogenesis. Importantly, mutations in the splicing factor ZRSR2 frequently associate with alterations in the epigenetic regulator TET2. However, the impact of these concurrent mutations in hematopoiesis and MDS remains unclear. Using CRISPR/Cas9 genetically engineered mice, we demonstrate that Zrsr2m/mTet2-/- promote MDS with reduced penetrance. Animals presented peripheral blood cytopenia, splenomegaly, extramedullary hematopoiesis, and multi-lineage dysplasia, signs consistent with MDS. We identified a myelo-erythroid differentiation block accompanied by an expansion of LT-HSC and MPP2 progenitors. Transplanted animals presented a similar phenotype, thus indicating that alterations were cell-autonomous. Whole-transcriptome analysis in HSPC revealed key alterations in ribosome, inflammation, and migration/motility processes. Moreover, we found the MAPK pathway as the most affected target by mRNA aberrant splicing. Collectively, this study shows that concomitant Zrsr2 mutation and Tet2 loss are sufficient to initiate MDS in mice. Understanding this mechanistic interplay will be crucial for the identification of novel therapeutic targets in the spliceosome/epigenetic MDS subgroup.

Circulating Tumor Cells for the Staging of Patients With Newly Diagnosed Transplant-Eligible Multiple Myeloma.

PURPOSE: Patients with multiple myeloma (MM) may show patchy bone marrow (BM) infiltration and extramedullary disease. Notwithstanding, quantification of plasma cells (PCs) continues to be performed in BM since the clinical translation of circulating tumor cells (CTCs) remains undefined. PATIENTS AND METHODS: CTCs were measured in peripheral blood (PB) of 374 patients with newly diagnosed MM enrolled in the GEM2012MENOS65 and GEM2014MAIN trials. Treatment included bortezomib, lenalidomide, and dexamethasone induction followed by autologous transplant, consolidation, and maintenance. Next-generation flow cytometry was used to evaluate CTCs in PB at diagnosis and measurable residual disease (MRD) in BM throughout treatment. RESULTS: CTCs were detected in 92% (344 of 374) of patients with newly diagnosed MM. The correlation between the percentages of CTCs and BM PCs was modest. Increasing logarithmic percentages of CTCs were associated with inferior progression-free survival (PFS). A cutoff of 0.01% CTCs showed an independent prognostic value (hazard ratio: 2.02; 95% CI, 1.3 to 3.1; P = .001) in multivariable PFS analysis including the International Staging System, lactate dehydrogenase levels, and cytogenetics. The combination of the four prognostic factors significantly improved risk stratification. Outcomes according to the percentage of CTCs and depth of response to treatment showed that patients with undetectable CTCs had exceptional PFS regardless of complete remission and MRD status. In all other cases with detectable CTCs, only achieving MRD negativity (and not complete remission) demonstrated a statistically significant increase in PFS. CONCLUSION: Evaluation of CTCs in PB outperformed quantification of BM PCs. The detection of ≥ 0.01% CTCs could be a new risk factor in novel staging systems for patients with transplant-eligible MM.

Biomimetic 3D Environment Based on Microgels as a Model for the Generation of Drug Resistance in Multiple Myeloma.

The development of three-dimensional environments to mimic the in vivo cellular response is a problem in the building of disease models. This study aimed to synthesize and validate three-dimensional support for culturing monoclonal plasma cells (mPCs) as a disease model for multiple myeloma. The three-dimensional environment is a biomimetic microgel formed by alginate microspheres and produced on a microfluidic device whose surface has been functionalized by a layer-by-layer process with components of the bone marrow's extracellular matrix, which will interact with mPC. As a proof of concept, RPMI 8226 cell line cells were cultured in our 3D culture platform. We proved that hyaluronic acid significantly increased cell proliferation and corroborated its role in inducing resistance to dexamethasone. Despite collagen type I having no effect on proliferation, it generated significant resistance to dexamethasone. Additionally, it was evidenced that both biomolecules were unable to induce resistance to bortezomib. These results validate the functionalized microgels as a 3D culture system that emulates the interaction between tumoral cells and the bone marrow extracellular matrix. This 3D environment could be a valuable culture system to test antitumoral drugs efficiency in multiple myeloma.

Reference Values to Assess Hemodilution and Warn of Potential False-Negative Minimal Residual Disease Results in Myeloma.

BACKGROUND: Whereas, in most patients with multiple myeloma (MM), achieving undetectable MRD anticipates a favorable outcome, some others relapse shortly afterwards. Although one obvious explanation for this inconsistency is the use of nonrepresentative marrow samples due to hemodilution, there is no guidance on how to evaluate this issue. METHODS: Since B-cell precursors, mast cells and nucleated red blood cells are normally absent in peripheral blood, we analyzed them in 1404 bone marrow (BM) aspirates obtained in numerous disease settings and in 85 healthy adults (HA). RESULTS: First, we confirmed the systematic detection of the three populations in HA, as well as the nonreduced numbers with aging. Pairwise comparisons between HA and MM patients grouped according to age and treatment showed significant variability, suggesting that hemodilution should be preferably evaluated with references obtained from patients treated with identical regimens. Leveraging the MRD results from 118 patients, we showed that a comparison with HA of similar age could also inform on potential hemodilution. CONCLUSIONS: Our study supports the routine assessment of BM cellularity to evaluate hemodilution, since reduced BM-specific cell types as compared to reference values (either treatment-specific or from HA if the former are unavailable) could indicate hemodilution and a false-negative MRD result.

Impact of measurable residual disease by decentralized flow cytometry: a PETHEMA real-world study in 1076 patients with acute myeloid leukemia.

The role of decentralized assessment of measurable residual disease (MRD) for risk stratification in acute myeloid leukemia (AML) remains largely unknown, and so it does which methodological aspects are critical to empower the evaluation of MRD with prognostic significance, particularly if using multiparameter flow cytometry (MFC). We analyzed 1076 AML patients in first remission after induction chemotherapy, in whom MRD was evaluated by MFC in local laboratories of 60 Hospitals participating in the PETHEMA registry. We also conducted a survey on technical aspects of MRD testing to determine the impact of methodological heterogeneity in the prognostic value of MFC. Our results confirmed the recommended cutoff of 0.1% to discriminate patients with significantly different cumulative-incidence of relapse (-CIR- HR:0.71, P < 0.001) and overall survival (HR: 0.73, P = 0.001), but uncovered the limited prognostic value of MFC based MRD in multivariate and recursive partitioning models including other clinical, genetic and treatment related factors. Virtually all aspects related with methodological, interpretation, and reporting of MFC based MRD testing impacted in its ability to discriminate patients with different CIR. Thus, this study demonstrated that "real-world" assessment of MRD using MFC is prognostic in patients at first remission, and urges greater standardization for improved risk-stratification toward clinical decisions in AML.

Deep MRD profiling defines outcome and unveils different modes of treatment resistance in standard- and high-risk myeloma.

Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of âˆ¼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species-mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.

Immunosuppressive profiles in liquid biopsy at diagnosis predict response to neoadjuvant chemotherapy in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterised by high pathological complete response to neoadjuvant chemotherapy (NAC). However, refractory and poor NAC responders still face very poor outcome, emphasising the urgent need for tools that facilitate identification of these patients, so that surgery or alternatives to NAC are considered early in the treatment protocol. MATERIALS AND METHODS: We combined metabolomics, exosome circulating miRNAs and flow cytometry experimental approaches in TNBC patients at diagnosis with immunohistochemistry in needle biopsy tumours to generate NAC-response predictive models. We also co-cultured and studied crosstalk between isolated patient-derived early myeloid-derived suppressor cells (eMDSCs) and TNBC cancer cell lines. RESULTS: Blood-derived liquid biopsy biomarkers display a novel immunosuppressive profile of tryptophan-derived metabolites and eMDSC levels that significantly predict NAC response. Notably, indoleamine 2,3-dioxygenase 1 (IDO1) expression in tumour cells inversely correlated with circulating tryptophan levels but directly correlated with the level of eMDSCs. In addition, a set of circulating exosome miRNAs that target pathways of immune maturation also predicted poor NAC response prior to chemotherapy. Interestingly, expression of IDO1 increased when TNBC cell lines were co-cultured with patient-derived eMDSCs and this, in turn, promoted proliferation of eMDSCs. CONCLUSION: Our findings demonstrate that the suppressive pathways of the immune system play a key role in modulating the NAC response in TNBC. We identify a crosstalk mechanism between tumour cells and eMDSCs that exacerbates immunosuppression. These results provide a potential new tool to identify poor NAC responders for alternative strategies of treatment, including early surgical resection of the tumour, and to explore in them alternative immune therapies.

Measurable Residual Disease by Next-Generation Flow Cytometry in Multiple Myeloma.

PURPOSE: Assessing measurable residual disease (MRD) has become standard with many tumors, but the clinical meaning of MRD in multiple myeloma (MM) remains uncertain, particularly when assessed by next-generation flow (NGF) cytometry. Thus, we aimed to determine the applicability and sensitivity of the flow MRD-negative criterion defined by the International Myeloma Working Group (IMWG). PATIENTS AND METHODS: In the PETHEMA/GEM2012MENOS65 trial, 458 patients with newly diagnosed MM had longitudinal assessment of MRD after six induction cycles with bortezomib, lenalidomide, and dexamethasone (VRD), autologous transplantation, and two consolidation courses with VRD. MRD was assessed in 1,100 bone marrow samples from 397 patients; the 61 patients without MRD data discontinued treatment during induction and were considered MRD positive for intent-to-treat analysis. The median limit of detection achieved by NGF was 2.9 × 10-6. Patients received maintenance (lenalidomide ± ixazomib) according to the companion PETHEMA/GEM2014MAIN trial. RESULTS: Overall, 205 (45%) of 458 patients had undetectable MRD after consolidation, and only 14 of them (7%) have experienced progression thus far; seven of these 14 displayed extraosseous plasmacytomas at diagnosis and/or relapse. Using time-dependent analysis, patients with undetectable MRD had an 82% reduction in the risk of progression or death (hazard ratio, 0.18; 95% CI, 0.11 to 0.30; P < .001) and an 88% reduction in the risk of death (hazard ratio, 0.12; 95% CI, 0.05 to 0.29; P < .001). Timing of undetectable MRD (after induction v intensification) had no impact on patient survival. Attaining undetectable MRD overcame poor prognostic features at diagnosis, including high-risk cytogenetics. By contrast, patients with Revised International Staging System III status and positive MRD had dismal progression-free and overall survivals (median, 14 and 17 months, respectively). Maintenance increased the rate of undetectable MRD by 17%. CONCLUSION: The IMWG flow MRD-negative response criterion is highly applicable and sensitive to evaluate treatment efficacy in MM.

Biomimetic microspheres for 3D mesenchymal stem cell culture and characterization.

Stem cells reside in niches, specialized microenvironments that sustain and regulate their fate. Extracellular matrix (ECM), paracrine factors or other cells are key niche regulating elements. As the conventional 2D cell culture lacks these elements, it can alter the properties of naïve stem cells. In this work we designed a novel biomimetic microenvironment for cell culture, consisting of magnetic microspheres, prepared with acrylates and acrylic acid copolymers and functionalized with fibronectin or hyaluronic acid as ECM coatings. To characterize cell proliferation and adhesion, porcine mesenchymal stem cells (MSCs) were grown with the different microspheres. The results showed that the 3D environments presented similar proliferation to the 2D culture and that fibronectin allows cell adhesion, while hyaluronic acid hinders it. In the 3D environments, cells reorganize the microspheres to grow in aggregates, highlighting the advantages of microspheres as 3D environments and allowing the cells to adapt the environment to their requirements.

Correction to: A phase I-II study of plerixafor in combination with fludarabine, idarubicin, cytarabine, and G-CSF (PLERIFLAG regimen) for the treatment of patients with the first early-relapsed or refractory acute myeloid leukemia.

The name of Pau Montesinos was inadvertently presented as Pau Montesinos Fernández in the original article.The original version of this article was revised: The name of Pau Montesinos was inadvertently presented as Pau Montesinos Fernández.

A phase I-II study of plerixafor in combination with fludarabine, idarubicin, cytarabine, and G-CSF (PLERIFLAG regimen) for the treatment of patients with the first early-relapsed or refractory acute myeloid leukemia.

Clinical outcomes of patients with acute myeloid leukemia (AML) showing the first primary refractory or early-relapsed disease remain very poor. The Programa Español de Tratamientos en Hematología (PETHEMA) group designed a phase I-II trial using FLAG-Ida (fludarabine, idarubicin, cytarabine, and G-CSF) plus high-dose intravenous plerixafor, a molecule inducing mobilization of blasts through the SDF-1α-CXCR4 axis blockade and potentially leading to chemosensitization of the leukemic cells. We aimed to establish a recommended phase 2 dose (RP2D) of plerixafor plus FLAG-Ida, as well as the efficacy and safety of this combination for early-relapsed (first complete remission (CR/CRi) < 12 months) or primary refractory AML. Between 2012 and 2015, 57 patients were enrolled, and 41 received the RP2D (median age 52 years [range, 18-64]). Among these patients, 20 (49%) achieved CR/CRi, and 3 (7%) died during induction. CR/CRi rate was 50% (13/26) among primary refractory and 47% (7/15) among early relapse. Overall, 25 patients (61%) were allografted. Median overall and disease-free survivals were 9.9 and 13 months, respectively. In summary, the combination of plerixafor plus FLAG-Ida resulted in a relatively high CR/CRi rate in adult patients with primary refractory or early relapsed AML, with an acceptable toxicity profile and induction mortality rate, bridging the majority of patients to allogeneic stem cell transplantation. ClinicalTrials.gov Identifier: NCT01435343.

Depth of Response in Multiple Myeloma: A Pooled Analysis of Three PETHEMA/GEM Clinical Trials.

Purpose To perform a critical analysis on the impact of depth of response in newly diagnosed multiple myeloma (MM). Patients and Methods Data were analyzed from 609 patients who were enrolled in the GEM (Grupo Español de Mieloma) 2000 and GEM2005MENOS65 studies for transplant-eligible MM and the GEM2010MAS65 clinical trial for elderly patients with MM who had minimal residual disease (MRD) assessments 9 months after study enrollment. Median follow-up of the series was 71 months. Results Achievement of complete remission (CR) in the absence of MRD negativity was not associated with prolonged progression-free survival (PFS) and overall survival (OS) compared with near-CR or partial response (median PFS, 27, 27, and 29 months, respectively; median OS, 59, 64, and 65 months, respectively). MRD-negative status was strongly associated with prolonged PFS (median, 63 months; P < .001) and OS (median not reached; P < .001) overall and in subgroups defined by prior transplantation, disease stage, and cytogenetics, with prognostic superiority of MRD negativity versus CR particularly evident in patients with high-risk cytogenetics. Accordingly, Harrell C statistics showed higher discrimination for both PFS and OS in Cox models that included MRD (as opposed to CR) for response assessment. Superior MRD-negative rates after different induction regimens anticipated prolonged PFS. Among 34 MRD-negative patients with MM and a phenotypic pattern of bone marrow involvement similar to monoclonal gammopathy of undetermined significance at diagnosis, the probability of "operational cure" was high; median PFS was 12 years, and the 10-year OS rate was 94%. Conclusion Our results demonstrate that MRD-negative status surpasses the prognostic value of CR achievement for PFS and OS across the disease spectrum, regardless of the type of treatment or patient risk group. MRD negativity should be considered as one of the most relevant end points for transplant-eligible and elderly fit patients with MM.

Minimal residual disease monitoring and immune profiling in multiple myeloma in elderly patients.

The value of minimal residual disease (MRD) in multiple myeloma (MM) has been more frequently investigated in transplant-eligible patients than in elderly patients. Because an optimal balance between treatment efficacy and toxicity is of utmost importance in patients with elderly MM, sensitive MRD monitoring might be particularly valuable in this patient population. Here, we used second-generation 8-color multiparameter-flow cytometry (MFC) to monitor MRD in 162 transplant-ineligible MM patients enrolled in the PETHEMA/GEM2010MAS65 study. The transition from first- to second-generation MFC resulted in increased sensitivity and allowed us to identify 3 patient groups according to MRD levels: MRD negative (<10(-5); n = 54, 34%), MRD positive (between <10(-4) and ≥10(-5); n = 20, 12%), and MRD positive (≥10(-4); n = 88, 54%). MRD status was an independent prognostic factor for time to progression (TTP) (hazard ratio [HR], 2.7; P = .007) and overall survival (OS) (HR, 3.1; P = .04), with significant benefit for MRD-negative patients (median TTP not reached, 70% OS at 3 years), and similar poorer outcomes for cases with MRD levels between <10(-4) and ≥10(-5) vs ≥10(-4) (both with a median TTP of 15 months; 63% and 55% OS at 3 years, respectively). Furthermore, MRD negativity significantly improved TTP of patients >75 years (HR, 4.8; P < .001), as well as those with high-risk cytogenetics (HR, 12.6; P = .01). Using second-generation MFC, immune profiling concomitant to MRD monitoring also contributed to identify patients with poor, intermediate, and favorable outcomes (25%, 61%, and 100% OS at 3 years, respectively; P = .01), the later patients being characterized by an increased compartment of mature B cells. Our results show that similarly to transplant candidates, MRD monitoring is one of the most relevant prognostic factors in elderly MM patients, irrespectively of age or cytogenetic risk. This trial was registered at www.clinicaltrials.gov as #NCT01237249.