ALBERT

Description: 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.

Description: Introduction: Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous clonal plasma cell disorder. A number of prognostic factors to identify patients at a higher risk of progression have been described, such as the size of the M protein, proportion of abnormal bone marrow plasma cells (BMPCs), immunoparesis and serum free light chain (FLC) k/l ratio. More recently, isotype-specific uninvolved heavy and light chain (HLC) pair suppression measured with the Hevylite assay was also associated with an increased risk of progression. Recent studies have evaluated the key prognostic impact of an increase in M-protein levels during follow-up ("evolving" pattern). However, an important limitation could be the evaluation of M-protein level variations based on serum protein electrophoresis (SPE) in patients with a small size M-spike. The aim of this study was to prospectively analyze the changes in M-protein according to SPE and HLC measurements, as well as other risk factors for progression, in patients with SMM. Methods: Thirty patients newly diagnosed with SMM at a single institution from January 2014 through September 2017 were prospectively included in the study. For each patient, baseline levels of known prognostic factors (serum M-protein, serum and urine immunofixation, clonal BMPCs percentage, total immunoglobulins, involved/uninvolved FLC and involved/uninvolved HLC pairs) were recorded. During the follow up, M-protein level, FLC and isotype specific HLC pairs were also analyzed. Evolving change in M-protein level according to SPE was defined as ³ 10% increase within the first 6 months of diagnosis (if M-protein was ³ 30 g/L) and/or ³ 25% increase within the first 12 months (for any level of M-protein); evolving change according to HLC was defined as a ³ 10% increase in the involved pair. A sequential increase in each of three or more consecutive measurements from diagnosis was considered an evolving change regardless of its magnitude. Results: The clinical characteristics of the total of patients, as well as of the patients with evolving changes in M-protein according to HLC are summarized in Table 1. During the study period, 5/30 (17%) of patients demonstrated an evolving behavior of the M-protein according to SPE. Four of these patients (4/5) also showed a progressive increase in the M-protein in the HLC measurements. One patient showed stable HLC levels even though both the M-protein and the involved FLC progressively increased. This patient was of intermediate and low risk according to Mayo Clinic and PETHEMA scores, respectively. On follow up, no progressive suppression of the isotype-specific uninvolved HLC pair or increase in the FLC ratio was noted, and there have been no signs of progression after a follow up of 3 years. According to involved HLC-pair levels, 12/30 (40%) of patients demonstrated an evolving behavior. Five out of 7 patients that were not classified as evolving by SPE, were IgA isotype. Eight out of 12 patients showed severe isotype-specific suppression of the uninvolved HLC-pair (〉 50% below lower level of normal) as well as a highly abnormal FLC ratio (8). Three out of the 4 remaining patients showed either severe isotype-specific HLC pair suppression or highly abnormal FLC ratio in follow up measurements. Compared to patients with no "HLC-evolving pattern", evolving patients were more likely to have highly abnormal FLC ratios (90 vs. 33%, p=0.009), severe suppression of the other isotypes (64 vs. 19%, p=0,024), highly abnormal isotype-specific HLC ratios (67 vs. 33%, p=NS), severe isotype-specific HLC-pair suppression (75 vs. 50%, p=NS), and immunoparesis (67 vs. 39%p=NS). Five patients progressed to symptomatic multiple myeloma during follow up; 4 of them showed a progressive increase in the involved HLC pair from diagnosis. The remaining patient demonstrated a progressive increase in the involved HLC pair that started 19 months prior to progression, followed 4 months later with an increase in M-protein as measured by SPE. Conclusions: In our series, the Hevylite assay allowed us to identify patients with a progressive increase in M-protein (clonal heavy/light chain pair) that was not evident with SPE measurements. This "HLC evolving pattern" was associated with other risk factors for progression to symptomatic disease and with worsening of other prognostic parameters during follow up. Disclosures Rosinol: Janssen, Celgene, Amgen, Takeda: Honoraria. Bladé:Janssen: Honoraria.

Description: Introduction: As in multiple myeloma, Waldenström macroglobulinemia (WM) is preceded by an asymptomatic phase, mainly as monoclonal gammopathy of undetermined significance (MGUS) or by a smoldering WM (SWM) phase. It has recently been reported that patients with IgM MGUS have a higher risk of progression in comparison to those with other MGUS isotypes. Moreover, it has been described that if the IgM MGUS is associated with adverse factors such as abnormal serum free light chains ratio and more than 15 g /L serum M-spike, the risk of progression at 20 years is as high as 55%. The aim of this study was to evaluate the risk of progression of asymptomatic IgM monoclonal gammopathies, using bone marrow aspiration as the main criteria for classification between SWM and IgM MGUS. Methods: We reviewed retrospectively the medical records of 206 patients with asymptomatic IgM monoclonal gammopathies, 143 (69.4%) with IgM MGUS and 63 (30.6%) with SWM diagnosed in our institution from May 1982 to December 2017. In the absence of clinical manifestations, IgM MGUS was defined as the presence of less than 30 g/L of serum IgM monoclonal protein and less than 10% lymphoplasmacytic bone marrow (BM) infiltration. The diagnosis of SWM required the presence of any size IgM M spike associated with ≥10% lymphoplasmacytic bone marrow infiltration with no target organ involvement, constitutional symptoms, hyperviscosity, lymphadenopathies or peripheral neuropathy. Progression to WM was defined according to the 2014 International Consensus. Results: The median age of the series was 70 years (range, 29 to 98) and the male/female distribution was 106/100. SWM group had a significantly higher median serum M spike than the MGUS group (9.2 vs. 4.6 g/L; p

Description: Introduction: Multiple myeloma (MM) is considered an incurable disease. Best results are obtained with autologous stem cell transplantation (ASCT). The achievement of complete remission (CR) is the crucial step for a prolonged survival. Unfortunately, even with the use of new regimens, the vast majority of patients will ultimately relapse. In fact, with conventional chemotherapy, ASCT or allogeneic stem-cell transplantation (alloSCT), only 1‰, 10% and 15%, respectively are alive and in continued CR beyond 10 years in the best circumstances. The aim of the present research was to evaluate the proportion of patients in long-term CR after stem-cell transplantation, describing the different aspects related to minimal residual disease (MRD) and immunological correlative studies. Methods: We analyzed the medical records of 160 patients who underwent single melphalan-based ASCT from March 31th 1994 to December 31th 2010, allowing at least 5 years of follow up from diagnosis (median follow-up of 8 years), as well as 58 patients who had received an alloSCT over a 28-year period. Bone marrow (BM) samples from the alive CR patients beyond 5 years were analyzed to assess MRD using multiparameter flow cytometry (MFC) with Euroflow 8-color consensus panel. Serum free-light chains (FLC) and immunoglobulin heavy/light chains (HLC) (The Binding Site) were assayed by immunonephelometry. A whole-body PET/CT (Siemens) was also performed to evaluate extramedullary and bone disease. Furthermore, a detailed panel of surface cell markers by MFC was also developed to evaluate B, T and NK cell compartments, among other immune cell subpopulations in BM and/or peripheral blood. Results: Twenty patients (20/160; 12.5%) were alive and in continuous CR after ASCT beyond 5 years; nine (5.7%) remained in CR for more than 10 years. In the alloSCT group, 6 out 58 patients (10.3%) were in CR beyond 5 years (3 of them for more than 10 years). Remarkably, two patients were in CR after 20 and 28 years after ASCT and alloSCT, respectively. Fourteen long survivors after ASCT (70%) had received maintenance therapy. These regimens were mainly interferon (8 patients), followed by thalidomide (3) or thalidomide plus bortezomib (3). None patient showed increased uptake of FDG by PET/CT suspicious of MM activity, neither in bone lesions nor previous plasmacytomas. However, in the vast majority of patients (16/22; 72.7%) there were stable lytic lesions, usually with sclerotic borders. Unspecific findings, particularly lymph nodes with unspecific FDG uptake, were described in 28.6%. Fourteen out of 20 patients after ASCT (70%) and 2 after alloSCT (33.3%) showed oligoclonal bands (OB) in serum and/or urine, differents from the original monoclonal paraprotein by immunofixation. Among the 22 evaluated patients, all but 2 were negative by MFC for MRD in BM. These two patients were in stable CR, with evidence of OB in serum, considering the clone found unrelated to the original one. The aberrant plasma cell population (CD38+/++, CD56+, CD27-, CD19-, CD45-, CD81-, CD117+) was found to a frequency of 0.09% and 0.004% BM cells. Other 4 patients showed a population of plasma cells with some aberrant markers expression; two of them also showed OB. All patients showed normal serum FLC ratio except one patient after alloSCT. Levels of IgG and IgA HLC were also normal, except in one patient each. Six patients had higher IgM HLC ratio than normal, all of them showing OB. We also investigated whether T cell clonal expansion could be also involved. Indeed, patients with OB showed a higher diversity of TCR Vb over-represented, preferentially 13.1 and 17, suggesting a potential oligoclonal expansion of the T cell response. Characterization of TCR repertoire of the long survivors also indicated a higher involvement of the CD8+ compared to CD4+ T cells. In BM, the expression of the co-inhibitory receptor PD-1 on CD8+ T cells was elevated in active MM compared to MGUS and long survivors suggesting that exhausted PD1+CD8+ T cells increase in active MM and recover to normal levels in long survivors. Conclusions: There are a small proportion of long-term survivors after SCT in MM. MRD studies are negatives, but some false positives could be observed by MFC when OB are present. PET invariably showed metabolic CR with stable bone lesions. Our results support that oligoclonal humoral and cellular responses may play an important role in the disease control over the years. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Smoldering multiple myeloma (SMM) is a plasma cell dyscrasia defined by the presence of a monoclonal protein (MP) (≥ 30 g/L in serum or 〉1g/24-hours in urine) and/or plasma cell bone marrow involvement (BMPC) ≥ 10%, in the absence of symptoms due to the gammopathy. The risk of progression to symptomatic disease in patients with SMM is highly variable. Several biomarkers and prognostic index associated with risk of early progression based on tumoral load (M-protein size and percentage of BMPC), M-protein behaviour (evolving vs. non-evolving) and/or immunological status (heavy chain isotype, isotype suppression of uninvolved immunoglobulins and serum free light-chain (FLC) κ/λ ratio) have been recently identified. The identification of patients at risk for early progression is crucial when considering the current possibility of prompt therapeutic intervention. The aim of this study was to analyze the factors associated with early progression to multiple myeloma (MM) in patients diagnosed with SMM and long follow-up in a single institution. Methods: Medical records of the 207 patients (76M/131M; median age 65 years, range 33 to 92) diagnosed with SMM (International Myeloma Working Group criteria, 2003) at our institution between January 1973 and December 2012 were systematically reviewed. Progressive increase in the value of MP was defined as "evolving" when at least 10% increase was observed within the first 6 months from diagnosis when MP was ≥ 30 g/L (Rosiñol et al, Br J Haematol. 2003) or progressive increase in MP in each of the annual consecutive measurements during a period of 3 years in patients with an initial MP 〈 30 g/L (Rosiñol et al, Mayo Clin Proc. 2007). Immunoparesis was defined as any value below normal in not involved immunoglobulins. Bone marrow aspirates obtained at diagnosis were reviewed independently by 2 observers. Plasma cell percentages were estimated from a 500-cell count by each examiner and the mean values were considered. Results: Sixty-seven patients (33%) accomplished both SMM criteria (MC and BMPCs), while the remaining 140 patients only had one of them. With a follow up of 1,692 years-person, 105 patients had progressed (50.7%) to MM and one case to AL amyloidosis (0.5%). The estimated probability of progression at 2 and 5 years was 19.9% and 44.9% respectively, with a median time to progression (TTP) of 7.3 years (95% CI 3.9 to 10.6). At the time of progression, clinical manifestations were mainly anemia (52%) and skeletal lytic lesions (40%). The presence of renal insufficiency, extramedullary plasmacytomas or hypercalcemia was only identified in 12 patients (5.8%). The median survival after progression was 5 years (95% CI 3.8 to 6.2). Evolving type was recognized in 25% of the patients, and was associated with a probability of progression of 45% and 78.1% at 2 and 5 years and was higher than those with stable MP (HR 4.5; 95% CI 3 to 6.9; p

Description: Introduction: the frequency of soft-tissue plasmacytomas (EMPs) is high in patients with relapsed multiple myeloma (MM). There are two types of plasmacytomas: 1) paraskeletal: originating from focal bone involvement (vertebrae, ribs, sternum, skull) and 2) extramedullary: originating from hematogenous spread (skin, liver, CNS). The reported incidence in relapsed patients is 3-34% for paraskeletal and 3-10% for extramedullary plasmacytomas. The presence of soft-tissue masses is associated with poor prognosis and the efficacy of novel agents is not well established. There are some reports about the lack of efficacy of thalidomide (Bladé et al, Br J Haematol 2001; 113: 422-24) while some efficacy has been reported with bortezomib (Rosiñol et al, Eur J Haematol 2006;76:405-08) and pomalidomide (Detweiler et al, Leukemia 2011, 25; 906-908). Aim: to analyze the effectiveness of novel drugs (thalidomide, bortezomib, lenalidomide, pomalidomide, carfilzomib) in patients with relapsed MM and EMPs. Patients and Methods: patients with EMPs (paraskeletal or extramedullary) at the time of first or subsequent relapses from our database from Hospital Clínic of Barcelona who were treated with novel agents were analyzed. Only patients receiving the novel drugs in monotherapy or in combination with corticosteroids were included in the analysis. Patients receiving combination therapy including two novel drugs (i.e. VTD, VRD) were excluded. Results: 29 patients (median age 61, M 17/F 12) with relapsed myeloma and EMPs were treated with bortezomib. The median number of previous therapies was one. 22 patients had paraskeletal and 7 extramedullary plasmacytomas. The median number of cycles received was 4. The serological response rate was: 4% CR, 27% PR, 7% MR, 24% SD, 17% PD, 7% early death, 14% non evaluable. The response of the plasmacytomas were: 14% CR, 17% PR, 10% SD, 41% PD, 4% early death, 14% non evaluable. The median PFS from the initiation of bortezomib was 3.9 months. Sixteen patients (median age 49 years, M 6/F 10) were treated with lenalidomide. 13 patients had paraskeletal and 3 extramedullary plasmacytomas. The median number of previous therapies was two. The median number of cycles was 5.5. Serological response was: 38% PR, 12% MR, 19% SD, 19% PD, 12% non evaluable. The plasmacytoma response was: 25% PR, 19% SD, 44% PD, 12% non evaluable. The median PFS from initiantion of lenalidomide was 8.4 months. Nine patients (median age 61 years, 3M/6F) were treated with pomalidomide at relapse. The median number of previous therapies was 4. Two patients had paraskeletal and 7 extramedullary plasmacytomas. The median number of cycles was 2. Serological response rate was: 44% PR, 11% SD, 23% PD, 11% non evaluable. However, none of the patients showed response of the plasmacytomas (11% SD, 77% PD, 11% early death). The median PFS was 1.3 months. 8 patients (median age 49 years, 6M/2F) were treated with thalidomide at relapse. The median number of previous therapies was one. Six patients had paraskeletal and 2 extramedullary plasmacytomas. The median number of cycles of thalidomide received was one. None of the patients showed serological response (25% stable disease (SD), 50% progressive disease (PD), 25% early death) or reduction in the size of the plasmacytomas (50% SD, 50% PD). The median progressive free survival (PFS) from initiation of treatment with thalidomide was 1.6 months. Four patients (median age 62, 2M, 2F) were treated with carfilzomib. The median number of previous therapies was 3. The EMPs type was paraskeletal (1) and extramedullary (3). The median number of cycles administered was one. None patient responded to carfilzomib: serological response rate: 75% SD, 25% PD; plasmacytomas response: 100% PD. The median PFS was 0.7 months. The median survival in the overall series of patients with soft tissues masses at relapse treated with novel agents was 15.2 months. Conclusions: The efficacy of novel drugs in the treatment of EMPs is limited, being the most effective bortezomib and lenalidomide. None of the patients treated with thalidomide, pomalidomide or carfilzomib showed any response of the soft-tissue involvement (paraskeletal or extramedullary). Finally, the presence of plasmacytomas at relapse is associated with poor OS even in the era of novel agents. However, the efficacy of these novel agents as part of front-line therapy is unknown. Disclosures No relevant conflicts of interest to declare.

Description: Background: Crosstalk between malignant plasma cells and surrounding cells in the bone marrow (BM), such as mesenchymal stromal cells (MSCs), endothelial cells and immune cells, is crucial for pathogenesis of multiple myeloma (MM) and in asymptomatic monoclonal gammopathies. In these diseases, microRNAs (miRNAs) could be useful as biomarkers for diagnosis, prognosis and evaluation of treatment response. miRNAs can be released to the serum and transferred among MM cells and BM-MSCs as cell-cell communication. Previously, we have showed a serum 14-miRNA signature associated with complete remission (CR) after autologous stem-cell transplantation (ASCT). In this sense, patients in CR with partial recovery of two normal serum miRNA levels, similar to those with monoclonal gammopathy of undetermined significance (MGUS), was associated with better prognosis. The aim of this study was to analyze the miRNAs profile in mesenchymal stromal cells derived from bone marrow of patients with multiple myeloma in different status of the disease, comparing with MGUS controls. Methods: We analyzed samples from 95 patients with MGUS (N=23), MM at diagnosis (N=14), relapsed/refractory MM (N=14), MM in partial response (PR) or very good partial response (VGPR) (N=15), MM in CR (N=24) and healthy donors (N=5). Mononuclear cells from BM samples were cultured in DMEM containing 10% FBS. After a week, non-adherent cells were removed, whereas BM-MSCs were selected by their adherence to the plastic and their phenotype was confirmed by multiparametric flow cytometry. In a first screening phase, we analyzed 670 microRNAs in 20 primary BM-MSC from patients with MGUS (N=4), symptomatic MM (N=8) and MM in CR (N=8). miRNAs differentially expressed were identified according to a supervised analysis using significance analysis of microarrays (SAM) and Student's t-test based on multivariate permutation (with random variance model). miRNAs differentially expressed between groups of patients were validated in the whole cohort of BM-MSC from patients. Paired malignant plasma cells (CD38+) miRNA expression from patients with symptomatic MM as well as miRNA in serum samples paired with BM-MSC samples were also compared. RmiR package was used to identify miRNA targets, cross-correlating the miRNA expression data from the present study with our findings on the gene expression signature (Affymetrix Human Genome U219 array) in 12 BM-MSCs from patients (4 MGUS, 4 symptomatic MM and 4 in CR), based on the predicted targets from TargetScan and miRBase databases. Results: In the screening phase, we identified a miRNA profile of 10 miRNAs (miR-663b, miR-654-3p, miR-206, miR-411*, miR-885-5p, miR-668, miR-638, miR-485-3p, miR-744* and miR-199a) differentially expressed between patients with symptomatic MM and MM in CR (adjusted p-value

Description: BACKGROUND Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous clonal plasma cell disorder. Prognostic factors to identify patients at higher risk of progression have been described, including the suppression of uninvolved polyclonal immunoglobulins (Ig) or immunoparesis (IP). Novel serum assays allow the quantification of each isotype-specific heavy and light chain pair (HLC), enabling the study of a new type of IP: the suppression of the uninvolved light chain counter part of the same heavy-chain Ig isotype (isotype matched immunoparesis). AIM: To prospectively characterize the prevalence and severity of immunoparesis as determined by HLC measurements, its association with other risk factors for progression and its prognostic significance, in patients with SMM diagnosed at a single institution. METHODS: Fifty-four patients newly diagnosed with SMM from January 2014 through March 2019 were prospectively included. Serum samples obtained at baseline and during follow-up were tested for M-protein level, free light chain (FLC) and HLC concentrations (all three isotype pairs at baseline, involved isotype pair at follow up visits). Isotype matched immunoparesis (IMI) was defined by the HLC assay, based on the levels of the Ig with the same heavy chain of the monoclonal isotype but of the alternative light chain (e.g. IgG-kappa suppression in a patient with IgG-lambda SMM). Aditionally, HLC immunoparesis (IP) was defined by suppression of any Ig with a heavy chain different to the M-protein (e.g. IgA kappa/lambda pair suppression in a patient with IgG-lambda SMM). Severe immunoparesis was defined by Ig values suppressed by 50% or greater below the lower limit of normal (LLN). RESULTS: Median age at diagnosis was 73 years, with the following heavy chain isotype distribution: 31 IgG, 20 IgA, 2 biclonal IgG-IgA and 1 light-chain only SMM. Median follow up for the series was 2.5 years. Isotype specificity of immunoparesis was reflected in the greater prevalence of IMI vs. noninvolved isotype IP (Figure 1, A). HLC measurements identified IMI in 42 out of the 53 patients with IgG, IgA or biclonal SMM (82%), with severe IMI observed in 27 patients (53%). Suppression of at least one HLC pair of an uninvolved isotype was present in 37 patients (72%), with severe suppression observed in 20 patients (38%). Eleven patients presented severe IMI without severe IP of uninvolved isotypes. On the other hand, only one out of the 37 patients with IP of uninvolved isotypes presented without IMI. In the case of the 12 patients that showed IP of any IgM HLC pair, they all had concomitant severe IMI and IP of the other uninvolved heavy chain isotype. Analysis of other risk factors for progression between groups of patients with i) no severe immunoparesis (n=22), ii) severe IMI without severe IP of uninvolved isotypes (n=11) and iii) severe IMI and severe IP of one or more HLC pairs of uninvolved isotypes (n=18), showed significantly different FLC ratios and lower % of normal plasma cells in bone marrow (Figure 1, B). There was a trend, although statistically not significant, towards higher M-protein levels. Progression to malignant symptomatic gammopathy was observed in 12 patients. The suppression of any IgM HLC pair was associated with a shorter time to progression (p=0.007; HR=4.2; 95% CI, 0.84-21) (Figure 1, C). Severe IMI alone or severe IMI plus severe IP of a different isotype were associated only with a statistically not significant trend towards a shorter time to progression. Eight patients demonstrated an evolving behavior (≥ 10% increase in the involved HLC pair in 3 or more consecutive determinations from diagnosis), 7 of which presented with severe IMI at diagnosis. Of the five evolving patients that progressed, four had severe IMI and the remaining one developed severe IMI during follow up. Patients with severe IMI at diagnosis maintained the same level of HLC suppression throughout the follow up period. CONCLUSION: The significantly greater incidence of IMI over IP of uninvolved isotypes pairs supports an isotype specificity of early Ig suppression mechanisms in the case of IgG and IgA SMM. That would be of special interest at the time of initial evaluation of patients with SMM using HLC pairs. Both IMI and IP of uninvolved isotypes are associated with other recognized risk factors for progression, but the later appears to develop with more advanced disease and associate with a higher risk of progression. Disclosures Cibeira: Amgen: Honoraria, Other: Educational lectures; Celgene: Honoraria, Other: Educational lectures; Akcea Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational lectures. Bladé:Oncopeptides: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria.