Recommendations for the Clinical Approach to Immune Thrombocytopenia: Spanish ITP Working Group (GEPTI)

Glucocorticoids; Intravenous immunoglobulins; Primary immune thrombocytopeniaGlucocorticoides; Immunoglobulines intravenoses; Trombocitopènia immune primàriaGlucocorticoides; Inmunoglobulinas intravenosas; Trombocitopenia inmune primariaPrimary immune thrombocytopenia (ITP) is a complex autoimmune disease whose hallmark is a deregulation of cellular and humoral immunity leading to increased destruction and reduced production of platelets. The heterogeneity of presentation and clinical course hampers personalized approaches for diagnosis and management. In 2021, the Spanish ITP Group (GEPTI) of the Spanish Society of Hematology and Hemotherapy (SEHH) updated a consensus document that had been launched in 2011. The updated guidelines have been the reference for the diagnosis and management of primary ITP in Spain ever since. Nevertheless, the emergence of new tools and strategies makes it advisable to review them again. For this reason, we have updated the main recommendations appropriately. Our aim is to provide a practical tool to facilitate the integral management of all aspects of primary ITP management

Recovery of polyclonal immunoglobulins during treatment in patients ineligible for autologous stem-cell transplantation is a prognostic marker of longer progression-free survival and overall survival

Immunoparesis is the suppression of normal polyclonal immunoglobulins and is present in most patients with newly diagnosed multiple myeloma (MM). The association of immunoparesis at diagnosis, and particularly its recovery along with treatment, with survival in patients ineligible for autologous stem-cell transplantation (ASCT) has not been well established. This retrospective study evaluated the impact of immunoparesis in 431 patients diagnosed with MM, ineligible for ASCT, with a median overall survival of 36 months [95% confidence interval (CI): 31–40]. Immunoparesis was present in 81.2% of patients at diagnosis and was associated with a trend to a worse overall response rate (ORR: 84.8% vs. 74.9%; OR 1.88 (95% CI: 0.97–3.63), shorter progression-free survival (PFS) [22.0 vs. 18.2 months; hazard ratio (HR) 0.775; 95%CI: 0.590–1.018; p = 0.066], and overall survival (OS) (45.9 vs. 34.2 months; HR 0.746; 95% CI: 0.551–1.010; p = 0.057). Twenty-four per cent of patients who had immunoparesis at diagnosis recovered polyclonal immunoglobulins in the follow-up period. Interestingly, these patients had a better ORR (96.3% vs. 68.2%; OR 12.29 (95% CI: 3.77–40.06), PFS (HR 0.703; 95CI%: 0.526–0.941; p = 0.018) and OS (HR 0.678; 95 CI%: 0.503–0.913; p = 0.011) than patients who did not recover it. In summary, restoring a healthy immune system along with first-line treatment in patients with MM, not receiving ASCT, is associated with better outcomes

B-cell regeneration profile and minimal residual disease status in bone marrow of treated multiple myeloma patients

© 2021 by the authors.B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (≥50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naïve B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19− nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome.This work has been supported by the International Myeloma Foundation-Black Swan Research Initiative, the EuroFlow Consortium (grant LSHB-CT-2006-018708); Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC; Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER), numbers: CB16/12/00400, CB16/12/00233, CB16/12/00369, CB16/12/00489 and CB16/12/00480; grant from Bilateral Cooperation Program between Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES (Brasília/Brazil) and Dirección General de Políticas Universitárias (DGPU)-Ministério de Educación, Cultura y Deportes (Madrid/Spain) number DGPU 311/15; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro of Brazil (FAPERJ) numbers: E26/110.105/2014 and E26/102.191/2013; grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil (CNPQ), number: 400194/2014-7. R.M.d.P. was supported by a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/DGPU), number: 000281/2016-06 and CAPES/PROEX 641/2018, Brazil; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro of Brazil (FAPERJ) number: E01/200/537/2018

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

The number of tumor infiltrating T-cell subsets in lymph nodes from patients with Hodgkin lymphoma is associated with the outcome after first line ABVD therapy

Prognostic factors in Hodgkin lymphoma (HL) still fail to accurately identify high-risk patients. Tumor microenvironment in HL is a current focus of research for risk definition but few studies have focused on infiltrating lymphocytes. Here, we analyzed the number of tumor infiltrating lymphocytes by flow cytometry in diagnostic biopsies from 96 HL homogeneously treated patients with ABVD with or without radiotherapy. Most lymph node cells were lymphocytes (90 ± 17), with a median T/B/NK distribution of 74%/26%/0.7%, and CD4 T-cell predominance. The amount of CD19 B cells, and NK cells did not show association with disease features. However, high numbers of CD8 and CD4 cells were associated with better and poorer outcomes, respectively. Patients with ≥15% cytotoxic CD8 cells among the total cell population had a longer 10-year freedom from treatment failure (FFTF) (93% vs. 73%, p=.04). In turn, cases with ≥75% of CD4 infiltrating cells showed a significantly decreased FFTF (73% vs. 96%, p=.021). Consequently, CD4/CD8 ratio ≥5 associated with a poorer 10-year FFTF (69.5% vs. 94%, p=.02). This deleterious effect was particularly prominent in advanced disease (n = 58, p=.01). In multivariate analysis, a CD4/CD8 ratio ≥5 was the only independent variable to predict for treatment failure (HR = 4.5, 95% confidence interval, 1.2–16.8). In conclusion, our study shows that high CD4 and low CD8 T-cells infiltrates of tumor specimens associate with poor prognosis in HL patients, and CD4/CD8 ratio might be potentially useful for tailoring therapy.Peer Reviewe

Recommendations on the management of patients with immune thrombocytopenia (ITP) in the context of SARS-CoV-2 infection and vaccination: Consensus guidelines from a Spanish ITP expert group

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease with highly variable presentation, characteristics, and clinical course. Thrombocytopenia is a common complication of many viral infections, including SARS-CoV-2. In addition, both de novo ITP and exacerbation of ITP after vaccination against SARS-CoV-2 have been reported. Patients infected with SARS-CoV-2 develop a prothrombotic coagulopathy called COVID-19-associated coagulopathy (CAC). In addition, autoimmune hematological disorders secondary to SARS-CoV-2 infection, mainly ITP and autoimmune hemolytic anemia (AIHA), have been described. Furthermore, SARS-CoV-2 infection has been associated with exacerbation of autoimmune processes, including ITP. In fact, there is evidence of a high relapse rate in patients with preexisting ITP and COVID-19. As for vaccination against SARS-CoV-2, hematological adverse events (HAE) are practically anecdotal. The most common HAE is thrombocytopenia-associated thrombosis syndrome (TTS) linked to vectored virus vaccines. Other HAEs are very rare, but should be considered in patients with previous complement activation disease or autoimmunity. In patients with ITP who are vaccinated against SARS-CoV-2, the main complication is exacerbation of ITP and the bleeding that may result. In fact, this complication occurs in 12% of patients, with splenectomized and refractory patients with more than five lines of previous treatment and platelet counts below 50 × 109/L being the most vulnerable. We conclude that, in general, there is no greater risk of severe SARS-CoV-2 infection in ITP patients than in the general population. Furthermore, no changes are advised in patients with stable ITP, the use of immunosuppressants is discouraged unless there is no other therapeutic option, and patients with ITP are not contraindicated for vaccination against COVID-19

Identification of relapse‐associated gene mutations by next‐generation sequencing in low‐risk acute myeloid leukaemia patients

Recommended genetic categorization of acute myeloid leukaemias (AML) includes a favourable‐risk category, but not all these patients have good prognosis. Here, we used next‐generation sequencing to evaluate the mutational profile of 166 low‐risk AML patients: 30 core‐binding factor (CBF)‐AMLs, 33 nucleophosmin (NPM1)‐AMLs, 4 biCEBPα‐AMLs and 101 acute promyelocytic leukaemias (APLs). Functional categories of mutated genes differed among subgroups. NPM1‐AMLs showed frequent variations in DNA‐methylation genes (DNMT3A, TET2, IDH1/2) (79%), although without prognostic impact. Within this group, splicing‐gene mutations were an independent factor for relapse‐free (RFS) and overall survival (OS). In CBF‐AML, poor independent factors for RFS and OS were mutations in RAS pathway and cohesin genes, respectively. In APL, the mutational profile differed according to the risk groups. High‐risk APLs showed a high mutation rate in cell‐signalling genes (P = 0·002), highlighting an increased incidence of FLT3 internal tandem duplication (ITD) (65%, P < 0·0001). Remarkably, in low‐risk APLs (n = 28), NRAS mutations were strongly correlated with a shorter five‐year RFS (25% vs. 100%, P < 0·0001). Overall, a high number of mutations (≥3) was the worst prognostic factor RFS (HR = 2·6, P = 0·003). These results suggest that gene mutations may identify conventional low‐risk AML patients with poor prognosis and might be useful for better risk stratification and treatment decisions.This work was partially supported by the ‘Instituto de Salud Carlos III’ (ISCIII), Spanish Ministry of Economy and Competitiveness PI15/01706, PI16/00517, PI18/01946, CIBERONC‐CB16/12/00233 and ‘Una manera de hacer Europa’ (Innocampus; CEI‐2010‐1‐0010). MIPC and MGA are supported by the Fundación Española de Hematología y Hemoterapia (FEHH). MCC has been supported by the Spanish Association Against Cancer Scientific Foundation, (AECC). MES is supported by Miguel Servet programme (CP13/00080) from the ISCIII, Ministerio de Ciencia e Innovación, Madrid, Spain. CJ is supported by the ‘Beca de investigación FEHH‐CRIS 2018’.Peer reviewe

Post-transcriptional modifications contribuye to the overexpression of cyclin D2 in multiple myeloma

[Purpose]: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma. [Experimental Design]: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR. [Results]: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. [Conclusions]: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma.This study was partially supported by Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias: PI08/0568 and PI13/00111), Asociacion Española Contra el Cancer (AECC, GCB120981SAN), >Gerencia Regional de Salud, Junta de Castilla y Leon> (GRS 702/A/11, BIO/SA57/13 and BIO/SA35/14) grants, the Spanish Myeloma Network Program (RD12/0036/0058) and the INNOCAMPUS Program (CEI10-1-0010). I. Misiewicz-Krzeminska was supported by Instituto de Salud Carlos III (PS09/01897). M.E. Sarasquete was supported by Contrato Miguel Servet (CP13/00080). Research in the I. Sanchez-García group was supported by FEDER and MINECO (SAF2012-32810, and Red de Excelencia Consolider OncoBIO SAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), NIH (R01 CA109335-04A1), >Junta de Castilla y Leon> (BIO/SA32/14 and CSI001U14) grants, the Fundacion Inocente Inocente, the German Carreras Foundation (DJCLS R13/26) and by the ARIMMORA project (European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 282891). Research in the C.V. Duenas group was supported by >Miguel Servet> Grant (CP14/00082 AES 2013-2016) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad). I. Sanchez-García is an API lab of the EuroSyStem project and a partner within the Marie Curie Initial Training Network DECIDE.Peer Reviewe

Methylation is an inactivating mechanism of the p16 gene in multiple myeloma associated with high plasma cell proliferation and short survival

[EN]In order to gain further insights into the role of the p16 gene in cell cycle regulation and the prognostic implications of its inactivation, we investigated the methylation status of the p16 gene in 98 untreated patients using a polymerase chain reaction assay based on the inability of some restriction enzymes to digest methylated sequences. Forty-one patients showed a p16 methylated gene (42%). The percentage of S-phase plasma cells (PC) in these patients was almost three times higher than in those with an unmethylated p16 gene (4Æ16% ± 3Æ37% vs 1Æ5% ± 1Æ41%, P < 0Æ001). The presence of p16 methylation also correlated with both elevated b2-microglobulin serum levels and high C-reactive protein values. Patients with a p16 methylated gene had shorter overall and progression-free survival than those patients without p16 methylation. However, this feature did not retain independent prognostic influence on multivariate analysis, probably due to its association with the S-phase PC, which had more potent statistical significance in the Cox model. These findings showed methylation of the p16 gene was a frequent event in MM patients at diagnosis, and was associated with an increased proliferative rate of plasma cells and a poor prognosis, indicating an important role for p16 gene in the cell cycle regulation of multiple myeloma tumour cells, and thus in the clinical outcome of the disease