Impacto clínico de las aberrancias inmunofenotípicas y perfil mutacional en síndromes mielodisplásicos

Los síndromes mielodisplásicos (SMD) constituyen un grupo heterogéneo de entidades clínicas. La citomorfología y la citogenética constituyen el estándar para el estudio de pacientes con sospecha de SMD. Sin embargo, todavía existen dificultades a la hora de establecer el diagnóstico de SMD, sobre todo en muestras con una sola citopenia, y sin exceso de blastos. Las anomalías citogenéticas representan un factor pronóstico fundamental y apoyo a la hora del diagnóstico, aunque sólo existen alteraciones en un 30-50% de los pacientes. Las nuevas herramientas diagnósticas de las que disponemos en la actualidad (en citometría de flujo y en biología molecular) pueden contribuir no sólo al diagnóstico, sino también al pronóstico en pacientes con SMD. OBJETIVO Desarrollar una metodología por citometría de flujo que nos permita realizar el diagnóstico diferencial entre pacientes con SMD y con citopenias de otro origen. Evaluar la aplicabilidad de la técnica de secuenciación masiva de nuevo generación y de alta sensibilidad (NGS) para el diagnóstico molecular y pronóstico de pacientes con SMD..

Ocena minimalnej choroby resztkowej w szpiczaku plazmocytowym

Minimal residual disease (MRD) in a patient with multiple myeloma (MM) is defined as the mini-mum levels of pathological plasma cells remaining after treatment when a patient is in complete response (CR). The ultimate aim of studying MRD is to identify patients with different prognosis and to tailor treatment for individual patients. MRD studies in MM should be recommended in young patients in CR after autologous hematopoietic stem cells transplantation and in older patients in CR after regimens including proteasome inhibitors. Bone marrow is the only recommend location to assess MRD in MM. The recommended methods of MRD testing include next generation sequencing of immunoglobulin genes or multiparametric flow cytometry (MFC), depending on the experience of each center and the possibility of study samples being available in the first 24 hours for MFC analysis. MRD should be considered as a therapeutic objective. However, there is not enough evidence for taking clinical decisions based on MRD status alone, and for this reason we encourage the design of new clinical studies to address these questions.Minimalną chorobę resztkową (MRD) u pacjenta z rozpoznaniem szpiczaka plazmocytowego defi-niuje się jako populację nowotworowych komórek plazmatycznych, która pozostała w organizmie chorego po osiągnięciu odpowiedzi całkowitej (CR). Ostatecznym celem badań MRD jest dążenie do identyfikacji chorych o odmiennym rokowaniu i indywidualizacji leczenia na tej podstawie. Ocenę MRD u chorych na szpiczaka plazmocytowego zaleca się u młodszych pacjentów, którzy osiągną CR po przeszczepieniu autologicznych krwiotwórczych komórek macierzystych oraz u chorych starszych osiągających CR po chemioterapii opartej na inhibitorach proteasomu. Powinno się oznaczać MRD wyłącznie w szpiku kostnym. Do rekomendowanych metod oceny MRD w szpiczaku plazmocytowym zalicza się sekwencjonowanie następnej generacji genów immunoglobulinowych oraz wieloparametryczną cytometrię przepływową, przy czym wybór jednej z tych metod powinien zależeć od doświadczenia danego ośrodka oraz możliwości wykonania badania cytometrycznego w czasie 24 godzin od pobrania próbki szpiku. Eradykację MRD powinna się obecnie uważać za istotny cel terapii szpiczaka plazmocytowego. Jednak, ze względu na brak wystarczających danych do podejmowania decyzji klinicznych wyłącznie na podstawie wyniku badania MRD, istnieje po¬trzeba dalszych, dobrze zaprojektowanych badań klinicznych w tym zakresie

Large T cell clones expressing immune checkpoints increase during multiple myeloma evolution and predict treatment resistance

Large T cell; Immune checkpoints; Multiple myelomaCèl·lules T grans; Punts de control immunitari; Mieloma múltipleCélulas T grandes; Puntos de control inmunitario; Mieloma múltipleTumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults (n = 4) and patients with precursor (n = 8) and full-blown MM (n = 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27− and CD27+ T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations.This work was supported by grants from the Instituto de Salud Carlos III/Subdireccion General de Investigacion Sanitaria and co-financed by FEDER funds (CB16/12/00233, CB16/12/00369, PI17/01243, PI19/00818 and PI20/00048), and together with Fundación Científica de la Asociación Española Contra el Cáncer (FCAECC) for iMMunocell Transcan-2 (AC17/00101), FCAECC Predoctoral Grant Junta Provincial Navarra, the Cancer Research UK (C355/A26819), FCAECC and Italian Association for Cancer Research (AIRC) under the Accelerator Award Program (EDITOR), 2017 Multiple Myeloma Research Foundation Immunotherapy Networks of Excellence, Black Swan Research Initiative of the International Myeloma Foundation, European Hematology Association nonclinical advanced research grant (3680644), European Research Council 2015 Starting Grant (MYELOMANEXT grant 680200), the Cancer Research Innovation in Science Cancer Foundation (PR_EX_2020-02), the Leukemia Lymphoma Society, unrestricted grants from Bristol-Myers Squibb/Celgene and Takeda, Roche imCORE program (NAV-4 and NAV-15), Fondazione Regionale per la Ricerca Biomedica (Regione Lombardia) (Project ID 065 JTC 2016), ERA-NET TRANSCAN-2, and by My First AIRC Grant 2020 (n. 24534, 2021/2026), and by the Riney Family Multiple Myeloma Research Program Fund

Mass spectrometry vs immunofixation for treatment monitoring in multiple myeloma

Monitoring of the monoclonal protein (M-protein) by electrophoresis and/or immunofixation (IFE) has long been used to assess treatment response in multiple myeloma (MM). However, with the use of highly effective therapies, the M-protein becomes frequently undetectable, and more sensitive methods had to be explored. We applied IFE and mass spectrometry (EXENT&FLC-MS) in serum samples from newly diagnosed MM patients enrolled in the PETHEMA/GEM2012MENOS65 obtained at baseline (n = 223), and after induction (n = 183), autologous stem cell transplantation (n = 173), and consolidation (n = 173). At baseline, the isotypes identified with both methods fully matched in 82.1% of samples; in the rest but 2 cases, EXENT&FLC-MS provided additional information to IFE with regards to the M-protein(s). Overall, the results of EXENT&FLC-MS and IFE were concordant in >80% of cases, being most discordances due to EXENT&FLC-MS+ but IFE− cases. After consolidation, IFE was not able to discriminate 2 cohorts with different median progression-free survival (PFS), but EXENT&FLC-MS did so; furthermore, among IFE− patients, EXENT&FLC-MS identified 2 groups with significantly different median PFS (P = .0008). In conclusion, compared with IFE, EXENT&FLC-MS is more sensitive to detect the M-protein of patients with MM, both at baseline and during treatment, and provides a more accurate prediction of patients’ outcome. This trial was registered at www.clinicaltrials.gov as #NCT01916252.This study was supported by grants from the Centro de Investigacion Biomédica en Red–Area de Oncología–del Instituto de Salud Carlos III CIBERONC, CB16/12/00369, CB16/12/00400, CB16/12/00233, and CB16/12/00284, Instituto de Salud Carlos III/Subdireccion General de Investigaci on Sanitaria FIS no. PI15/ 01956, PI15/02049, PI15/02062, PI18/01709, PI18/01673, and PI19/01451, the Cancer Research UK, FCAECC, and AIRC under the Accelerator Award Program (EDITOR).Peer reviewe

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

This article belongs to the Special Issue Advances in Multiple Myeloma Research and Treatment.[Simple Summary] Although the majority of patients with myeloma who achieve undetectable minimal residual disease show prolonged survival, some of them relapse shortly afterwards. False-negative results due to hemodiluted bone marrow samples could explain this inconsistency, but there is no guidance on how to evaluate them. We analyzed three cell populations normally absent in peripheral blood in 1404 aspirates obtained in numerous disease settings and in 85 healthy adults. Pairwise comparisons according to age and treatment showed significant variability, thus suggesting that hemodilution should be preferably evaluated with references obtained after receiving identical regimens. Leveraging the minimal residual disease results from 118 patients, we showed that a comparison with age-matched healthy adults could also inform on potential hemodilution. Our study supports the routine assessment of bone marrow cellularity to evaluate hemodilution, using as reference values either treatment-specific or from healthy adults if the former are unavailable.[Abstract] 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.This study was supported by grants from the Centro de Investigación Biomédica en Red—Área de Oncología—del Instituto de Salud Carlos III (CIBERONC; CB16/12/00369, CB16/12/00400, CB16/12/00233 and CB16/12/00284); Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria and co-financed by FEDER funds (FIS No. PI15/01956, PI15/02049, PI15/02062, PI18/01709, PI18/01673 and PI19/01451); the Cancer Research UK (C355/A26819), FCAECC and AIRC under the Accelerator Award Programme (EDITOR); the Black Swan Research Initiative of the International Myeloma Foundation and the European Research Council (ERC) 2015 Starting Grant (Contract 680200 MYELOMANEXT). This study was supported by the Riney Family Multiple Myeloma Research Program Fund

Mass spectrometry vs immunofixation for treatment monitoring in multiple myeloma

Monitoring of the monoclonal protein (M-protein) by electrophoresis and/or immunofixation (IFE) has long been used to assess treatment response in multiple myeloma (MM). However, with the use of highly effective therapies, the M-protein becomes frequently undetectable, and more sensitive methods had to be explored. We applied IFE and mass spectrometry (EXENT&FLC-MS) in serum samples from newly diagnosed MM patients enrolled in the PETHEMA/GEM2012MENOS65 obtained at baseline (n = 223), and after induction (n = 183), autologous stem cell transplantation (n = 173), and consolidation (n = 173). At baseline, the isotypes identified with both methods fully matched in 82.1% of samples; in the rest but 2 cases, EXENT&FLC-MS provided additional information to IFE with regards to the M-protein(s). Overall, the results of EXENT&FLC-MS and IFE were concordant in >80% of cases, being most discordances due to EXENT&FLC-MS+ but IFE cases. After consolidation, IFE was not able to discriminate 2 cohorts with different median progression-free survival (PFS), but EXENT&FLC-MS did so; furthermore, among IFE patients, EXENT&FLC-MS identified 2 groups with significantly different median PFS (P = .0008). In conclusion, compared with IFE, EXENT&FLC-MS is more sensitive to detect the M-protein of patients with MM, both at baseline and during treatment, and provides a more accurate prediction of patients' outcome

A novel deep targeted sequencing method for minimal residual disease monitoring in acute myeloid leukemia

A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for minimal residual disease assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-single nucleotide variants. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by sequencing, evaluating the level of mutations detected at diagnosis. The predictive value of minimal residual disease status by sequencing, multiparameter flow cytometry, or quantitative polymerase chain reaction analysis was determined by survival analysis. The sequencing method achieved a sensitivity of 10-4 for single nucleotide variants and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnostic data set). Sequencing-determined minimal residual disease positive status was associated with lower disease-free survival (hazard ratio 3.4, P=0.005) and lower overall survival (hazard ratio 4.2, P<0.001). Multivariate analysis showed that minimal residual disease positive status determined by sequencing was an independent factor associated with risk of death (hazard ratio 4.54, P=0.005) and the only independent factor conferring risk of relapse (hazard ratio 3.76, P=0.012). This sequencing-based method simplifies and standardizes minimal residual disease evaluation, with high applicability in acute myeloid leukemia. It is also an improvement upon flow cytometry- and quantitative polymerase chain reaction-based prediction of outcomes of patients with acute myeloid leukemia and could be incorporated in clinical settings and clinical trials.This study was supported by the Subdirección General de Investigación Sanitaria (Instituto de Salud Carlos III, Spain) grants PI13/02387 and PI16/01530, and the CRIS against Cancer foundation grant 2014/0120. ML holds a postdoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013- 16409). PRP holds a postdoctoral fellowship of the Spanish Instituto de Salud Carlos III: Contrato Predoctoral de Formación en Investigación en Salud i-PFIS (IFI 14/00008).S

Novel deep targeted sequencing method for minimal residual disease monitoring in acute myeloid leukemia

A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease (MRD) negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for MRD assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-SNVs. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by NGS, evaluating the level of mutations detected at diagnosis. The predictive value of MRD status by NGS, multiparameter flow cytometry, or quantitative PCR was determined by survival analysis. The method achieved a sensitivity of 10-4 for SNV mutations and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnosis data set). NGS-determined MRD positive status was associated with lower disease-free survival (hazard ratio [HR] 3.4, p=0.005) and lower overall survival (HR 4.2, p<0.001). Multivariate analysis showed that MRD positive status by NGS was an independent factor associated with risk of death (HR 4.54, p =0.005) and the only independent factor conferring risk of relapse (HR 3.76, p =0.012). This NGS based method simplifies and standardizes MRD evaluation, with high applicability in acute myeloid leukemia. It also improves upon flow cytometry and quantitative PCR to predict acute myeloid leukemia outcome and could be incorporated in clinical settings and clinical trials.This study was supported by the Subdirección General de Investigación Sanitaria (Instituto de Salud Carlos III, Spain) grants PI13/02387 and PI16/01530, and the CRIS against Cancer foundation grant 2014/0120. M.L. holds a postdoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409). P.R.P. holds a postdoctoral fellowship of the Spanish of Instituto de Salud Carlos III: Contrato Predoctoral de Formación en Investigación en Salud i-PFIS (IFI 14/00008).S

Validation of the International Myeloma Working Group standard response criteria in the PETHEMA/GEM2012MENOS65 study: are these times of change?

Induction and consolidation based on proteasome inhibitors, immunomodulatory drugs, and corticoids integrated with high-dose therapy (HDT) and autologous stem cell transplantation (ASCT), are showing complete response (CR) rates >50% in multiple myeloma (MM).1-3 The addition of anti-CD38 monoclonal antibodies may increase these unprecedented CR rates.4-6 When more than half of transplant-eligible patients with MM achieve CR with frontline therapy, it is reasonable to ask, what other tests are clinically relevant after negative immunofixation. The achievement of deep responses with modern therapy led the International Myeloma Working Group (IMWG) to propose new guidelines that included definitions of negative minimal residual disease (MRD) for standard response criteria.7 Indeed, recent studies have reported nearly 50% MRD− rates,5,8,9 and, more importantly, the prognostic value of MRD criteria was validated in clinical trials8,10-12 and routine practice...

Depth of response in multiple myeloma: A pooled analysis of three PETHEMA/GEM clinical trials

On behalf of the GEM (Grupo Español de Mieloma)/PETHEMA (Programa para el Estudio de la Terapéutica en Hemopatías Malignas) Cooperative Study Group.[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 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.Supported by the Centro de Investigación Biomédica en Red – Area de Oncologia - del Instituto de Salud Carlos III (CIBERONC; CB16/12/00369; CB16/12/00400; CB16/12/00233; CB16/12/00284), formerly named as Cooperative Research Thematic Network (Grants No. RD12/0036/0058, RD12/0036/0048, RD12/0036/0046, and RD12/0036/0061) of the Red de Cancer (Cancer Network of Excellence); Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria; funded in part by the European Regional Development Fund (FIS No. 98/1239, 00/10160, 01/0089, 02/0089, 02/0905, G03/136, PI051284, PI06033906/1354, PS09/01897/01370, PI12/01761, PI12/02311, PI13/01469, PI14/01867, G03/136); Sara Borrell (No. CD13/00340); Asociación Española Contra el Cáncer (No. GCB120981SAN); and Federación Española de Enfermedades Raras. Also supported internationally by the Black Swan Research Initiative of the International Myeloma Foundation and the European Research Council 2015 Starting Grant (MYELOMANEXT).Peer Reviewe