The evolving pattern of the monoclonal protein improves the IMWG 2/20/20 classification for patients with smoldering multiple myeloma.

The 2/20/20 International Myeloma Working Group (IMWG) score is the most employed risk score in clinical practice to evaluate the risk of progression from smoldering multiple myeloma (SMM) to symptomatic multiple myeloma. However, it faces a serious limitation: The risk score is applied at diagnosis and cannot be reapplied. Since a dynamic accurate patient risk assessment for progression is necessary, we aimed to investigate whether the detection of an evolving pattern in serum M-protein (SMP) improves the identification of high-risk patients. Eighty-three patients diagnosed with SMM between 2011 and 2020 were included. Patients were initially classified applying the 2/20/20 IMWG score at baseline and later reclassified depending on the presence of an SMP evolving pattern into six groups. We regrouped the patients into three final risk groups: low-risk, intermediate-risk, and high-risk. The risk of progression at two years for the high-risk group was 88% and all patients had progressed at 4 years. The performance measurements were superior for the new 2/20/20-Evolving score independently for the detection of high-risk patients. We show that the sequential measurement of the SMP is a noninvasive and widely available test that improves the 2/20/20 IMWG risk score.

Membrane Protein Detection and Morphological Analysis of Red Blood Cells in Hereditary Spherocytosis by Confocal Laser Scanning Microscopy.

In hereditary spherocytosis (HS), genetic mutations in the cell membrane and cytoskeleton proteins cause structural defects in red blood cells (RBCs). As a result, cells are rigid and misshapen, usually with a characteristic spherical form (spherocytes), too stiff to circulate through microcirculation regions, so they are prone to undergo hemolysis and phagocytosis by splenic macrophages. Mild to severe anemia arises in HS, and other derived symptoms like splenomegaly, jaundice, and cholelithiasis. Although abnormally shaped RBCs can be identified under conventional light microscopy, HS diagnosis relies on several clinical factors and sometimes on the results of complex molecular testing. It is specially challenging when other causes of anemia coexist or after recent blood transfusions. We propose two different approaches to characterize RBCs in HS: (i) an immunofluorescence assay targeting protein band 3, which is affected in most HS cases and (ii) a three-dimensional morphology assay, with living cells, staining the membrane with fluorescent dyes. Confocal laser scanning microscopy (CLSM) was used to carry out both assays, and in order to complement the latter, a software was developed for the automated detection of spherocytes in blood samples. CLSM allowed the precise and unambiguous assessment of cell shape and protein expression.

Gene Expression Analysis of the Bone Marrow Microenvironment Reveals Distinct Immunotypes in Smoldering Multiple Myeloma Associated to Progression to Symptomatic Disease.

Background: We previously reported algorithms based on clinical parameters and plasma cell characteristics to identify patients with smoldering multiple myeloma (SMM) with higher risk of progressing who could benefit from early treatment. In this work, we analyzed differences in the immune bone marrow (BM) microenvironment in SMM to better understand the role of immune surveillance in disease progression and to identify immune biomarkers associated to higher risk of progression. Methods: Gene expression analysis of BM cells from 28 patients with SMM, 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and 22 patients with symptomatic MM was performed by using Nanostring Technology. Results: BM cells in SMM compared to both MGUS and symptomatic MM showed upregulation of genes encoding for key molecules in cytotoxicity. However, some of these cytotoxic molecules positively correlated with inhibitory immune checkpoints, which may impair the effector function of BM cytotoxic cells. Analysis of 28 patients with SMM revealed 4 distinct clusters based on immune composition and activation markers. Patients in cluster 2 showed a significant increase in expression of cytotoxic molecules but also inhibitory immune checkpoints compared to cluster 3, suggesting the presence of cytotoxic cells with an exhausted phenotype. Accordingly, patients in cluster 3 had a significantly longer progression free survival. Finally, individual gene expression analysis showed that higher expression of TNF superfamily members (TNF, TNFAIP3, TNFRSF14) was associated with shorter progression free survival. Conclusions: Our results suggest that exhausted cytotoxic cells are associated to high-risk patients with SMM. Biomarkers overexpressed in patients with this immune gene profile in combination with clinical parameters and PC characterization may be useful to identify SMM patients with higher risk of progression.

Immunoparesis defined by heavy/light chain pair suppression in smoldering multiple myeloma shows initial isotype specificity and involves other isotypes in advanced disease.

Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous plasma cell disorder, with a highly variable clinical course. Immunoparesis, defined by total immunoglobulin measurements, has been shown to be an independent risk factor for progression to symptomatic disease. The heavy/light chain (HLC) assay allows precise measurement of the polyclonal immunoglobulin of the same isotype, enabling the evaluation of isotype-matched immunoparesis (IMI). In this study, we prospectively characterized immunoparesis, as determined by HLC measurements, in 53 SMM patients. Severe IMI was present in 51% of patients, while severe IP of uninvolved isotypes (HLC IP) was present in 39%. Most of the patients with severe HLC IP presented with severe IMI, but not the other way around. Isotype specificity of immune suppression was suggested by lower relative values of isotype-matched HLC pairs, both for IgG and IgA SMM. Severe IMI was associated with other risk factors for progression while patients with severe IMI and severe HLC IP showed an even higher risk profile. Both severe IMI and severe IgM HLC IP showed a significantly shorter time to progression. Finally, gene expression analysis demonstrated differences in the bone marrow microenvironment between patients with IMI and IMI plus HLC IP, with an increased expression of genes associated with cytolytic cells. In conclusion, our data supports isotype specificity of early immunoglobulin suppression mechanisms. While suppression of both involved and uninvolved isotypes is associated with risk of progression, the later appears to develop with more advanced disease and could be mediated by different mechanisms.

Defining an Ultra-Low Risk Group in Asymptomatic IgM Monoclonal Gammopathy.

We analyzed 171 patients with asymptomatic IgM monoclonal gammopathies (64 with IgM monoclonal gammopathy of undetermined significance-MGUS and 107 with smoldering Waldenström macroglobulinemia - SWM) who had a bone marrow (BM) evaluation performed at diagnosis. Abnormal free-light chain ratio (53% vs. 31%) and MYD88 mutation prevalence (66% vs. 30%) were higher in patients with SWM. No other differences were found among groups. With a median follow-up of 4.3 years, 14 patients progressed to Waldenström macroglobulinemia, 1 to amyloidosis, and 28 died without progression. The MYD88 mutation was found in 53% of patients (available in 160 patients). Multivariate analysis showed that immunoparesis (subhazard ratio-SHR 10.2, 95% confidence interval-CI: 4.2-24.8; p < 0.001) and BM lymphoplasmacytic infiltration ≥ 20% (SHR: 6, 95% CI: 1.6-22.1; p = 0.007) were associated with higher risk of progression. We developed a risk model based on these two risk factors. In the absence of both variables, an ultra-low risk group was identified (SHR 0.1, 95% CI 0.02-0.5; p = 0.004), with 3% and 6% of cumulative incidence of progression at 10 and 20 years, respectively. Bootstrap analysis confirmed the reproducibility of these results. This study finds immunoparesis and BM infiltration as biomarkers of progression as well as a low-risk group of progression in asymptomatic IgM monoclonal gammopathies.

Spectroscopic Evaluation of Red Blood Cells of Thalassemia Patients with Confocal Microscopy: A Pilot Study.

Hemoglobinopathies represent the most common single-gene defects in the world and pose a major public health problem, particularly in tropical countries, where they occur with high frequency. Diagnosing hemoglobinopathies can sometimes be difficult due to the coexistence of different causes of anemia, such as thalassemia and iron deficiency, and blood transfusions, among other factors, and requires expensive and complex molecular tests. This work explores the possibility of using spectral confocal microscopy as a diagnostic tool for thalassemia in pediatric patients, a disease caused by mutations in the globin genes that result in changes of the globin chains that form hemoglobin-in pediatric patients. Red blood cells (RBCs) from patients with different syndromes of alpha-thalassemia and iron deficiency (including anemia) as well as healthy (control) subjects were analyzed under a Leica TCS SP8 confocal microscope following different image acquisition protocols. We found that diseased RBCs exhibited autofluorescence when excited at 405 nm and their emission was collected in the spectral range from 425 nm to 790 nm. Three experimental descriptors calculated from the mean emission intensities at 502 nm, 579 nm, 628 nm, and 649 nm allowed us to discriminate between diseased and healthy cells. According to the results obtained, spectral confocal microscopy could serve as a tool in the diagnosis of thalassemia.

Platelet Dysfunction in Noonan and 22q11.2 Deletion Syndromes in Childhood.

INTRODUCTION: An underlying thrombocytopathy seems to be responsible for hemorrhagic symptoms in patients diagnosed with 22q11.2 deletion syndrome (22q11DS) or Noonan syndrome (NS). In 22q11DS, it is explained by a defect in the membrane glycoprotein (GP) complex Ib-V-IX. The cause of thrombocytopathy in NS remains unclear. AIM: The objective is to study the incidence of thrombocytopathy in pediatric patients diagnosed with 22q11DS or NS assessing the utility of ISTH-BAT questionnaire and laboratory techniques. MATERIALS AND METHODS: Prospective study between March and December 2018 in children (2-18 years old) diagnosed with 22q11DS or NS. Hemorrhagic symptoms using ISTH-BAT score, total cell blood count, platelet indices, PFA-200 closure times, and platelet aggregation were evaluated in all patients and membrane GP expression in 22q11DS patients. RESULTS: Nearly 70% of NS patients (n = 22) had a platelet aggregation defect without thrombocytopenia. A defect of platelet aggregation with adenosine diphosphate (ADP) and epinephrine was the most frequent pattern. A statistically significant inverse correlation between closure times and aggregation with arachidonic acid (p = 0.049, p = 0.043) and epinephrine (p = 0.021, p = 0.035), and ADP (p = 0.117, p = 0.05) was found. Total 5 out of 29 patients diagnosed with 22q11DS had macrothrombocytopenia; more noteworthy in older patients. Twenty-six patients showed an impairment in ristocetin-induced platelet aggregation that correlated with prolonged collagen/epinephrine (p = 0.034) and collagen/ADP (p = 0.01). A significant association between ISTH-BAT score >3 and closure times (p = 0.022, p = 0.002) and aggregation defect with ristocetin (p = 0.043) was also demonstrated. CONCLUSION: Most NS and 22q11DS patients show an impairment of platelet aggregation that correlates with closure times. In 22q11DS patients, these results were also related to hemorrhagic symptoms.

Pomalidomide-dexamethasone for treatment of soft-tissue plasmacytomas in patients with relapsed / refractory multiple myeloma.

OBJECTIVE: The presence of plasmacytomas (Ps) in patients with multiple myeloma (MM) is associated with a poor outcome, both in patients treated conventionally and in patients treated with novel agents. Two types of plasmacytomas have being recognized: paraskeletal plasmacytomas (PPs) and extramedullary plasmacytomas (EMPs), being the incidence of EMPs lower but with worse prognosis. Our aim has been to analyze the efficacy of the pomalidomide-dexamethasone combination in this patient profile. METHOD: In the present study, the efficacy of pomalidomide and dexamethasone in 21 patients from nine hospitals of Catalonia (Spain), with relapsed or refractory MM and Ps, was analyzed. For this purpose, we describe the evolution of paraprotein in serum and urine and the size of plasmacytomas during treatment with pomalidomide-dexamethasone. RESULTS: While 34% of the patients achieved a paraprotein response, only two patients with PPs (9%) responded (RC and PR). There were no responses among patients with EMPs. The median progression-free survival from the start of treatment with pomalidomide/dexamethasone was only 1.7 months and the median overall survival of 4.5 months. CONCLUSION: In conclusion, pomalidomide and dexamethasone has limited efficacy in patients with advanced MM and soft-tissue plasmacytomas.

Loss of the Immune Checkpoint CD85j/LILRB1 on Malignant Plasma Cells Contributes to Immune Escape in Multiple Myeloma.

Mechanisms of immune regulation may control proliferation of aberrant plasma cells (PCs) in patients with monoclonal gammopathy of undetermined significance (MGUS) preventing progression to active multiple myeloma (MM). We hypothesized that CD85j (LILRB1), an inhibitory immune checkpoint for B cell function, may play a role in MM pathogenesis. In this study, we report that patients with active MM had significantly lower levels of CD85j and its ligand S100A9. Decreased CD85j expression could also be detected in the premalignant condition MGUS, suggesting that loss of CD85j may be an early event promoting tumor immune escape. To gain insight into the molecular mechanisms underlying CD85j functions, we next enforced expression of CD85j in human myeloma cell lines by lentiviral transduction. Interestingly, gene expression profiling of CD85j-overexpressing cells revealed a set of downregulated genes with crucial functions in MM pathogenesis. Furthermore, in vitro functional assays demonstrated that CD85j overexpression increased susceptibility to T cell- and NK-mediated killing. Consistently, ligation of CD85j decreased the number of PCs from individuals with MGUS but not from patients with MM. In conclusion, downregulation of inhibitory immune checkpoints on malignant PCs may provide a novel mechanism of immune escape associated with myeloma pathogenesis.

Evolving M-protein pattern in patients with smoldering multiple myeloma: impact on early progression.

Smoldering multiple myeloma (SMM) is a biologically heterogeneous, clinically defined entity with a variable rate of progression to symptomatic multiple myeloma (MM). Reliable markers for progression are critical for the development of potential therapeutic interventions. We retrospectively evaluated the predictive value of the evolving pattern of serum M-protein among other progression risk factors in 206 patients with SMM diagnosed between 1973 and 2012. Median time from recognition of evolving type to progression into symptomatic MM was 1.1 years (95% CI 0.5-2.0) and progression rate at 3 years was 71%. Development of the evolving type drastically worsened the prognostic estimation made at diagnosis for every covariate predictive of progression (serum M-protein size, bone marrow plasma cell infiltration, immunoparesis and Mayo Clinic risk). On average, the hazard ratio for progression to symptomatic MM increased to 5.1 (95% CI 3.4-7.6) after recognition of the evolving type. In conclusion, in patients with SMM the evolving pattern accurately predicts the risk of early progression to symptomatic disease, thereby allowing the identification of ultra-high risk patients who would be candidates for immediate therapy.

Bone marrow plasma cell infiltration in light chain amyloidosis: impact on organ involvement and outcome.

OBJECTIVES: Prognosis of immunoglobulin light-chain (AL) amyloidosis depends mainly on the presence of cardiac involvement and the disease burden. A higher bone marrow plasma cell (BMPC) burden has been recognized as an adverse prognostic factor. The aim of our study was to analyze the correlation between the BMPC infiltration, clinical features and outcomes in patients with AL amyloidosis. METHODS: The clinical records of 79 patients with AL amyloidosis treated at a single institution. RESULTS: Median BMPC infiltration at diagnosis was 11% and significantly correlated with the serum free light-chain difference (p < .001). Patients with more than 10% BMPCs had more frequent cardiac involvement (86 vs. 63%; p = .015), a trend towards a higher early mortality (27 vs. 11%; p = .08) and a significantly shorter progression-free survival (PFS) (median of 18 vs. 48 months, p = .02) and overall survival (median of 33 months vs. not reached; p = .046). In the multivariate analysis, a BMPC infiltration over 10% retained its adverse prognostic value for PFS (HR = 2.26; 95% CI, 1.048-4.866; p = .038). The use of new drugs seemed to overcome the negative prognostic impact of a higher BMPC infiltration. CONCLUSION: Higher BMPC infiltration in AL amyloidosis might be associated with increased systemic organ damage, particularly cardiac involvement and is rarely related to the development of myeloma features.

Prognostic impact of immunoparesis at diagnosis and after treatment onset in patients with light-chain amyloidosis.

OBJECTIVES: Immunoparesis (IP) is a risk factor associated with an unfavourable outcome in several plasma cell disorders. It has been suggested that its presence in light-chain (AL) amyloidosis could be associated with worse prognosis. However, the relevance of IP after treatment has not been evaluated to date. The aim of this study was to determine the prognostic impact of IP at diagnosis and one year after treatment onset in patients with AL amyloidosis. METHODS: The clinical records of 69 patients with AL amyloidosis treated at a single institution from January 2006 to January 2016 were included in the study. RESULTS: IP was observed in 27.5% of patients at diagnosis. The presence of IP was associated with a lower probability to achieve very good partial response or better after first-line treatment (37.8% versus 62.2%; p = .04). However, only in the group of patients treated with autologous stem cell transplantation (ASCT), the presence of IP resulted in a shorter progression-free survival (PFS) (30.2 months versus not reached [NR]; p = .02) but not in overall survival (OS). Persistence of IP at one year after treatment onset was identified in only four (9.8%) of the 41 evaluable patients. In the ASCT group, the absence of IP at one year after treatment onset resulted in a longer median PFS and OS (NR versus 22.6 months; p = .006; and NR versus 35.2 months; p < .001; respectively). In the multivariate analysis, the absence of IP at one year after treatment onset was independently associated with longer survival. CONCLUSION: IP at diagnosis has a negative impact on survival while its absence at one year after treatment is an independent marker for long-term survival.

Danazol therapy for the anemia of myelofibrosis: assessment of efficacy with current criteria of response and long-term results.

The efficacy of danazol was assessed in 50 patients with myelofibrosis and anemia using the recently revised criteria of the International Working Group for Myelofibrosis Research and Treatment. According to them, anemia response (clinical improvement) is defined as transfusion cessation in transfusion-dependent patients or an Hb increase >2 g/dl in patients without transfusion requirements, both maintained for at least 12 weeks. Median follow-up from danazol start was 36 months (interquartile range [IQR] 19.2-58.8). Anemia response was achieved in 15 patients (30 %), including 5 of the 27 with transfusion dependency (18.5 %) and 10 of the 23 without transfusion requirements (43.5 %). Median time to response was 5 months (IQR 4-7) and median duration of the response 14 months (IQR 10-21). Among responder patients, 5 discontinued therapy due to toxicity or personal decision and 1 died from spleen rupture while being in response. A trend for worse response was seen in transfusion-dependent patients (p = 0.055). A platelet increase >50 × 10(9)/l was observed in 3 of 13 thrombocytopenic patients, all of whom had moderate thrombocytopenia. Toxicity was usually moderate, leading to treatment withdrawal in only 4 patients. Danazol is effective in 30 % of patients with anemia-associated myelofibrosis. The responses are less frequent in patients with transfusion dependency.

Limitaciones de esfuerzos terapéuticos: Resultados de un registro prospectivo en una sala de clínica médica / Limitation of life-sustaining treatment: A prospective study developed in a clinical ward

El objetivo de este trabajo fue describir el proceso de limitación de los esfuerzos terapéuticos (LET) en los pacientes internados en una sala general. Para ello se realizó un estudio prospectivo descriptivo, desarrollado en la sala de internación general de un hospital universitario. Fueron evaluados pacientes que tuviesen alguna LET, asistidos por el servicio de clínica médica en un período de 60 días consecutivos. Durante el mismo se hospitalizaron 402 pacientes, 62 (15%) tuvieron algún tipo de LET. Este último grupo estaba compuesto por un 66% de mujeres, la mediana de edad fue de 86 años (78-90) y de la duración de hospitalización de 12 días (8-18). La mala calidad de vida fue la causa más frecuente de LET (69%). Se brindó información acerca de las limitaciones a 43 familias (69%) y 8 pacientes (13%). En la decisión participaron el médico de cabecera (50%), médicos de planta (50%), residentes (40%), la familia (42%) y los propios pacientes (11%). En 7 casos hubo constancia en la historia clínica (11%). Diecisiete pacientes (27%) con LET fallecieron durante la internación, mientras que 44 (71%) fueron dados de alta. En conclusión, la limitación de esfuerzos terapéuticos en nuestros pacientes constituyó un hecho frecuente. No se logró identificar un proceso uniforme o sistematizado para la toma de la decisión de LET. Resulta innegable la necesidad de normativas que guíen al equipo de salud en la toma de decisiones, tranquilicen a familiares y acompañen a los pacientes en sus reales necesidades.

The purpose of this study is to describe the limiting life-sustaining treatment process of patients admitted to a general ward. A prospective descriptive study was designed. The setting was the general ward of universitary hospital. Study participants were patients assisted by the internal medicine department during a 60- consecutive days period who had limitations of life sustaining treatments. During the study period, 402 patients were hospitalized, 62 (15%) of them had limitations of life support care. The median patient age of the last group was 86 years (78-90), 66% were women and the length of stay was 12 days (8-18). A low quality of life was the most frequent cause of limitation (69%). Information about the limitations was provided to 43 families (69%) and 8 patients (13%). The primary care physician participated in the decision in 50% of the cases, while the attending physician, the resident in charge, patient's family and patients themselves participated in 50%, 40%, 42% and 11% of the cases respectively. The decision of limiting life-sustaining treatments was recorded in seven patient's charts (11%). Seventeen (27%) patients with limitations died during the hospital stay while 44 (71%) were discharged. In conclusion, we found a frequent life sustaining treatment limitation in our patients. These decisions did not follow a uniform or systemized process. The need of guidelines to sort the medical and ethical challenges imposed to the medical team is undeniable.

[Limitation of life-sustaining treatment. A prospective study in a clinical ward]. / Limitaciones de esfuerzos terapéuticos. Resultados de un registro prospectivo en una sala de clínica médica.

The purpose of this study is to describe the limiting life-sustaining treatment process of patients admitted to a general ward. A prospective descriptive study was designed. The setting was the general ward of universitary hospital. Study participants were patients assisted by the internal medicine department during a 60-consecutive days period who had limitations of life sustaining treatments. During the study period, 402 patients were hospitalized, 62 (15%) of them had limitations of life support care. The median patient age of the last group was 86 years (78-90), 66% were women and the length of stay was 12 days (8-18). A low quality of life was the most frequent cause of limitation (69%). Information about the limitations was provided to 43 families (69%) and 8 patients (13%). The primary care physician participated in the decision in 50% of the cases, while the attending physician, the resident in charge, patient's family and patients themselves participated in 50%, 40%, 42% and 11% of the cases respectively. The decision of limiting life-sustaining treatments was recorded in seven patient's charts (11%). Seventeen (27%) patients with limitations died during the hospital stay while 44 (71%) were discharged. In conclusion, we found a frequent life sustaining treatment limitation in our patients. These decisions did not follow a uniform or systemized process. The need of guidelines to sort the medical and ethical challenges imposed to the medical team is undeniable.