Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC)

Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p < 0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p < 0.0001) and a shorter survival in MM patients with active disease requiring treatment (p <= 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM

Bortezomib plus melphalan and prednisone in elderly untreated patients with multiple myeloma: updated time-to-events results and prognostic factors for time to progression

New treatment options offering enhanced activity in elderly, newly diagnosed patients with multiple myeloma are required. One strategy is to combine melphalan and prednisone with novel agents. We previously reported an 89% response rate, including 32% complete responses and 11% near complete responses, in our phase 1/2 study of bortezomib plus melphalan and prednisone (VMP) in 60 newly diagnosed multiple myeloma patients with a median age of 75 years. Here, we report updated time-to-events data and the impact of poor prognosis factors on outcome

Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma

[EN]Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics.This work has been supported by the International Myeloma Foundation-Black Swan Research Initiative, the Red Temática de Investigación Cooperativa en Cáncer (RTICC); grant SA079U14 from the Consejería de Educación, Junta de Castilla y León, Valladolid, Spain and; grant DTS15/00119 from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain

Cut-offs and response criteria for the Hospital Universitario la Princesa Index (HUPI) and their comparison to widely-used indices of disease activity in rheumatoid arthritis

Objective To estimate cut-off points and to establish response criteria for the Hospital Universitario La Princesa Index (HUPI) in patients with chronic polyarthritis. Methods Two cohorts, one of early arthritis (Princesa Early Arthritis Register Longitudinal PEARL] study) and other of long-term rheumatoid arthritis (Estudio de la Morbilidad y Expresión Clínica de la Artritis Reumatoide EMECAR]) including altogether 1200 patients were used to determine cut-off values for remission, and for low, moderate and high activity through receiver operating curve (ROC) analysis. The areas under ROC (AUC) were compared to those of validated indexes (SDAI, CDAI, DAS28). ROC analysis was also applied to establish minimal and relevant clinical improvement for HUPI. Results The best cut-off points for HUPI are 2, 5 and 9, classifying RA activity as remission if =2, low disease activity if >2 and =5), moderate if >5 and <9 and high if =9. HUPI''s AUC to discriminate between low-moderate activity was 0.909 and between moderate-high activity 0.887. DAS28''s AUCs were 0.887 and 0.846, respectively; both indices had higher accuracy than SDAI (AUCs: 0.832 and 0.756) and CDAI (AUCs: 0.789 and 0.728). HUPI discriminates remission better than DAS28-ESR in early arthritis, but similarly to SDAI. The HUPI cut-off for minimal clinical improvement was established at 2 and for relevant clinical improvement at 4. Response criteria were established based on these cut-off values. Conclusions The cut-offs proposed for HUPI perform adequately in patients with either early or long term arthritis

X chromosome inactivation does not necessarily determine the severity of the phenotype in Rett syndrome patients

Rett syndrome (RTT) is a severe neurological disorder usually caused by mutations in the MECP2 gene. Since the MECP2 gene is located on the X chromosome, X chromosome inactivation (XCI) could play a role in the wide range of phenotypic variation of RTT patients; however, classical methylation-based protocols to evaluate XCI could not determine whether the preferentially inactivated X chromosome carried the mutant or the wild-type allele. Therefore, we developed an allele-specific methylation-based assay to evaluate methylation at the loci of several recurrent MECP2 mutations. We analyzed the XCI patterns in the blood of 174 RTT patients, but we did not find a clear correlation between XCI and the clinical presentation. We also compared XCI in blood and brain cortex samples of two patients and found differences between XCI patterns in these tissues. However, RTT mainly being a neurological disease complicates the establishment of a correlation between the XCI in blood and the clinical presentation of the patients. Furthermore, we analyzed MECP2 transcript levels and found differences from the expected levels according to XCI. Many factors other than XCI could affect the RTT phenotype, which in combination could influence the clinical presentation of RTT patients to a greater extent than slight variations in the XCI pattern

Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma

Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC)

Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p < 0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p < 0.0001) and a shorter survival in MM patients with active disease requiring treatment (p <= 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM

Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma

Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of