Single-point and kinetics of peripheral residual disease by mass spectrometry to predict outcome in patients with high risk smoldering multiple myeloma included in the GEM-CESAR trial.

The value of quantitative immunoprecipitation mass spectrometry (QIP-MS) to identify the M-protein is being investigated in patients with monoclonal gammopathies but no data are yet available in high-risk smoldering myeloma (HRsMM). We have therefore investigated QIP-MS to monitor peripheral residual disease (PRD) in 62 HRsMM patients enrolled in the GEM-CESAR trial. After 24 cycles of maintenance, detecting the M-protein by MS or clonal plasma cells by NGF identified cases with a significantly shorter median PFS (mPFS; MS: not reached vs 1,4 years, p=0.001; NGF: not reached vs 2 years, p=0.0002) but reaching CR+sCR did not discriminate patients with different outcome. With NGF as a reference, the combined results of NGF and MS showed a high negative predictive value (NPV) of MS: 81% overall and 73% at treatment completion. When sequential results were considered, sustained negativity by MS or NGF was associated with a very favorable outcome with a mPFS not yet reached vs 1.66 years and 2.18 years in cases never attaining PRD or minimal residual disease (MRD) negativity, respectively. We can thus conclude that 1) the standard response categories of the IMWG do not seem to be useful for treatment monitoring in HRsMM patients, 2) MS could be used as a non-invasive, clinical valuable tool with the capacity of guiding timely bone marrow evaluations (based on its high NPV with NGF as a reference) and 3) similarly to NGF, sequential results of MS are able identify a subgroup of HRsMM patients with long-term disease control. This study was registered at www.clinicaltrials.gov (ClinicalTrials.gov identifier: NCT02415413).

Critical analysis of the stringent complete response in multiple myeloma: contribution of sFLC and bone marrow clonality.

Stringent complete response (sCR) criteria are used in multiple myeloma as a deeper response category compared with CR, but prospective validation is lacking, it is not always clear how evaluation of clonality is performed, and is it not known what the relative clinical influence is of the serum free light chain ratio (sFLCr) and bone marrow (BM) clonality to define more sCR. To clarify this controversy, we focused on 94 patients that reached CR, of which 69 (73%) also fulfilled the sCR criteria. Patients with sCR displayed slightly longer time to progression (median, 62 vs 53 months, respectively; P = .31). On analyzing this contribution to the prognosis of sFLCr or clonality, it was found that the sFLCr does not identify patients in CR at distinct risk; by contrast, low-sensitive multiparametric flow cytometry (MFC) immunophenotyping (2 colors), which is equivalent to immunohistochemistry, identifies a small number of patients (5 cases) with high residual tumor burden and dismal outcome; nevertheless, using traditional 4-color MFC, persistent clonal BM disease was detectable in 36% of patients, who, compared with minimal residual disease-negative cases, had a significantly inferior outcome. These results show that the current definition of sCR should be revised.

Host virus and pneumococcus-specific immune responses in high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia: implications for disease progression.

Patients diagnosed with chronic lymphocytic leukemia (CLL) display a high incidence of infections due to an associated immunodeficiency that includes hypogammaglobulinemia. A higher risk of infections has also been recently reported for high-count monoclonal B-cell lymphocytosis, while no information is available in low-count monoclonal B-cell lymphocytosis. Here, we evaluated the status of the humoral immune system in patients with chronic lymphocytic leukemia (n=58), as well as in low- (n=71) and high- (n=29) count monoclonal B-cell lymphocytosis versus healthy donors (n=91). Total free plasma immunoglobulin titers and specific levels of antibodies against cytomegalovirus, Epstein-Barr virus, influenza and S.pneumoniae were measured by nephelometry and ELISA-based techniques, respectively. Overall, our results show that both CLL and high-count monoclonal B-cell lymphocytosis patients, but not low-count monoclonal B-cell lymphocytosis subjects, present with relatively high levels of antibodies specific for the latent viruses investigated, associated with progressively lower levels of S.pneumoniae-specific immunoglobulins. These findings probably reflect asymptomatic chronic reactivation of humoral immune responses against host viruses associated with expanded virus-specific antibody levels and progressively decreased protection against other micro-organisms, denoting a severe humoral immunodeficiency state not reflected by the overall plasma immunoglobulin levels. Alternatively, these results could reflect a potential role of ubiquitous viruses in the pathogenesis of the disease. Further analyses are necessary to establish the relevance of such asymptomatic humoral immune responses against host viruses in the expansion of the tumor B-cell clone and progression from monoclonal B-cell lymphocytosis to CLL.

Mass Spectrometry as Alternative Method to Identify and Monitor Non-Secretory Progressive Disease in Patients with Multiple Myeloma.

INTRODUCTION: After receiving different lines of treatment, multiple myeloma patients tend to present with less secretory and more frequent extramedullary disease. These features make treatment monitoring and follow-up very complex since they have to be based on the use of imaging methods and/or bone marrow aspirations or biopsies. OBJECTIVE: To present the case of a patient with myeloma progressing with non-secretory bone disease and to discuss the potential impact of mass spectrometry as a new highly sensitive method able to identify the monoclonal protein (MP) in the serum of these types of patients. MATERIALS AND METHODS: Informed consent was signed by the patient prior to receiving each line of treatment. The clinical information and images were obtained from anonymized electronic files. The mass spectrometry was performed with the Immunoglobulin Isotypes (GAM) assay for the mass spectrometry EXENT® Analyser Technology from Binding Site, part of Thermofisher. RESULTS: A 73-year-old male with IgG kappa multiple myeloma progressing with a new lytic lesion after receiving 14 cycles of Talquetamab as a third line of therapy who, due to the non-secretory nature of the disease at this point, could not be enrolled in a clinical trial, thus limiting his therapeutic options. The mass spectrometry was able to identify and quantify the presence of the patient's MP when the serum protein electrophoresis and immunofixation were still negative and therefore could have been used to confirm the progression, to permit the inclusion of the patient in a clinical trial and to further monitor the disease response. CONCLUSIONS: The higher sensitivity of the mass spectrometry methods to detect the MP in patients with myeloma and other monoclonal gammopathies translates into better identification of the disease progression, permits the inclusion of more patients in clinical trials and facilitates treatment monitoring.

The combination of bortezomib and dexamethasone is an efficient therapy for relapsed/refractory scleromyxedema: a rare disease with new clinical insights.

Scleromyxedema (SM) is a rare primary cutaneous inflammatory mucinosis characterised by papular mucinosis, monoclonal gammopathy and extracutaneous involvement. Most therapeutic options have failed in SM but high-dose therapy followed by autologous peripheral blood stem cell transplantation (APBSCT) appears to be highly effective, although SM normally relapses. We report the case of a 29-yr-old patient with severe SM who achieved stringent complete response with Bortezomib plus Dexamethasone after an early relapse subsequent to a high-dose melphalan regimen followed APBSCT. It is of particular note that dermatological lesions responded to both therapies before M-component modifications, suggesting that SM is independent of M-component characteristics. However, treatment should be directed towards the underlying plasma cell malignancy with typical anti-myeloma agents.

Predictive Model for the Effect of Environmental Conditions on the Postharvest Development of Colletotrichum gloeosporioides Strains Isolated from Papaya (Carica papaya L.).

ABSTRACT: Colletotrichum species are the most important postharvest spoilage fungi of papaya fruit. The objective of this research was to evaluate the effect of temperature and relative humidity on growth rate and time for growth to become visible of five strains of Colletotrichum gloeosporioides isolated from papaya fruit in a complex medium. As a primary model, the radial growth rates were estimated using the Baranyi and Roberts model in papaya agar. The Solver MS Excel function was used to obtain the time to visible mycelium (tv). Secondary models obtained with the Rosso et al. cardinal model of inflection were applied to describe the effect of temperature on the growth rate (µ). The Arrhenius-Davey model was used to model tv. The obtained models seem to be satisfactory for describing both µ and tv. The relative humidity had an effect on µ and tv for all tested C. gloeosporioides isolates, but no model accurately described the behavior of the fungus. External validation of models was performed with papaya fruit. Growth models were developed with the same models used in vitro. The bias and the accuracy factors as indices for performance evaluation of predictive models in food microbiology as a function of temperature and RH were 1.22 and 1.33, respectively, for µ and 1.18 and 1.62, respectively, for tv, indicating accurate predictions. The supply chain of papaya is complex and requires constant conditions, and poor conditions can result in damage to the fruit. Knowledge of the behavior of C. gloeosporioides on papaya fruit and application of the developed models in the supply chain will help to establish transport control strategies to combat these fungi. This research has contributed to development of the first models of growth for C. gloeosporioides in Mexico.

Influence of fibrinogen levels on erythrocyte aggregation determined with the Myrenne aggregometer and the Sefam erythro-aggregometer.

Fibrinogen is one of the plasmatic proteins which has a major influence on erythrocyte aggregation. The level of fibrinogen at which erythrocyte aggregation does not further increase is not well established. Therefore we aim to determine erythrocyte aggregation with two devices: Myrenne aggregometer (M0 and M1) and Sefam erythro-aggregometer (Ta, AI10 and gammaD) in relation with fibrinogen levels, in patients with several diseases with fibrinogen levels ranging between 200-1100 mg/dl. With the Myrenne aggregometer a plateau can be observed for fibrinogen levels higher than 400 mg/dl, while with the Sefam erythro-aggregometer, aggregation increases proportionally with fibrinogen levels higher than 400 mg/dl. In addition, for fibrinogen values higher than 400 mg/dl, only statististically significant correlations could be observed between fibrinogen and erythrocyte aggregation parameters with the Sefam erythro-aggregometer: r(Ta)=-0.463; r(AI10)=0.624, r(gammaD)=0.817, p<0.01, but not with the Myrenne: r(M0)=0.01, r(M1)=0.02, ns. Although the Sefam erythro-aggregometer is a better tool for determining erythrocyte aggregation both at low and high fibrinogen levels, the Myrenne aggregometer may be useful for assessing the erythrocyte aggregation as an indicator of cardiovascular risk, as in such circumstances fibrinogen levels do not usually exceed values higher than 400 mg/dl.

Increased circulating endothelial cells and microparticles in patients with psoriasis.

INTRODUCTION: Psoriasis is a chronic pathology characterized by increased inflammation that can be associated with changes in the vascular endothelium. We quantified the levels of circulating endothelial cells (CECs) and microparticles (MPs) in patients with psoriasis in order to analyze their relationship with endothelial and inflammation markers, subclinical atherosclerosis and microcirculation. METHODS: We studied 20 patients and 20 controls. Circulating markers of endothelial damage (CEC, MPs and von Willebrand factor, vWF) and inflammation (E-selectin, E-sel; Interleukin-6, IL-6 and C-reactive protein, CRP) were determined. Subclinical atherosclerosis was assessed by carotid ultrasound to obtain intima-media thickness. Microcirculation was evaluated by nailfold capillaroscopy. RESULTS: CECs, MPs, vWF, CRP and E-sel levels were significantly elevated in patients when compared with controls (p <  0.05). Ninety-four and fifty-three percentage of patients had CEC and MP levels higher than 99th percentile in controls. Forty-seven percent of patients simultaneously showed increased CEC and MP levels. MPs correlate with the inflammatory markers and with the intima-media thickness. CECs correlate with the capillaries loops per mm (p <  0.05). CONCLUSION: Psoriasis patients show elevated CECs and MPs, as a sign of endothelial dysfunction, which correlates with inflammatory markers as well as subclinical atherosclerosis and some capillaroscopy findings.

Erythrocyte aggregation determined with the Myrenne aggregometer at two modes (M0, M1) and at two times (5 and 10 sec).

Erythrocyte aggregation index determined with the Myrenne aggregometer gives a wide range of values both in healthy and disease, as observed in the literature, making results less comparable. This is due to the fact that it gives two aggregation indexes depending on the rotation speed of the cone, M0 (at stasis) and M1 (at 3 s(-1)) and time elapsed (5 or 10 sec), after the cone is stopped abruptly. We determined in 112 healthy volunteers both indexes at two modes and at two times, along with fibrinogen, plasmatic lipids and hematimetric indexes. The correlations between both the 5 and 10 sec mode, and the different variables were analysed and although a statistically significant correlation was observed in both modes, M1 at 5 sec gave the highest correlation values: 0.445, 0.485, 0.364, 0.460 for T-Chol, LDL-Chol, Apo B/A1 and fibrinogen, respectively; p<0.01. Therefore, we suggest that all the erythrocyte aggregation measurements should be performed at a hematocrit adjusted to 45%, with autologous plasma, and that an aggregation time of 5 sec (as opposed to 10 sec) should be used in order to avoid confusion and misunderstanding and to allow for comparability of results with the de Myrenne aggregometer between laboratories.

Effect of a hypocaloric diet on lipids and rheological profile in subjects with severe and morbid obesity. A follow-up study.

Discrepant results have been published regarding modifications of rheological parameters in obese subjects after a low caloric diet (LCD). In order to ascertain whether a decrease in BMI associated to a LCD, is accompanied by changes in the hemorheological parameters, we determined in 41 morbid obese subjects (32 female, 9 male aged 33+/-10 years) BMI, glucose, plasmatic lipids and apolipoproteins, fibrinogen, blood viscosity (Brookfield viscosimeter), plasma viscosity (Fresenius capillary viscosimeter), erythrocyte aggregation (Myrenne aggregometer), hematocrit and erythrocyte indexes, before starting on a LCD and 1 and 3 months after. During the first month obese subjects received a very low caloric diet (VLCD) (Modifast) providing 458 kcal per day. The second and third month they received a LCD providing 1500 kcal/day for men and 1200 kcal/day for women. One month after starting on a VLCD, a statistical significant decrease in glucose (p<0.001), Total-cholesterol (p<0.001), LDL-cholesterol (p<0.001), triglycerides (p=0.012), apoB (p<0.001) and erythrocyte aggregation (p<0.001) were observed together with a concomitant decrease in BMI (p<0.001). The expected decrease in HDL-cholesterol associated with a low fat diet was also noted in these individuals. No changes in fibrinogen, hematocrit, blood viscosity or plasma viscosity were observed. At 3 months only a slight increase in BMI was observed regarding the one month period, glucose being the only parameter which remained statistically lower. All the other significant parameters returned to their basal values at 3 months. VLCD (Modifast) is associated to a significant decrease in BMI with the corresponding improvement in glucose, lipids and erythrocyte aggregation at one month. However a LCD alone does not produce a further decrease in weight and both lipids and erythrocyte aggregation return to the basal situation at three months.

Do hemorheological alterations play any role in the development of thrombotic events in Behçet's disease?

Behçet's disease (BD) is associated with an increased thrombotic risk, although the prothrombotic mechanisms are not clearly defined. Alterations in blood rheology, specially increased erythrocyte aggregation has been suggested to play an important role in the development of thrombotic events in patients with Behçet's disease. In order to ascertain whether any rheological parameter could be involved in the pathogenesis of thrombotic events in Behçet's disease we have determined plasmatic lipids, fibrinogen, hematocrit, erythrocyte aggregation (Myrenne aggregometer), erythrocyte deformability (Rheodyn SSD), blood viscosity (Brookfield viscosimeter), plasma viscosity (Fresenius capillary viscosimeter) and erythrocyte indexes in Behçet's patients with a non-active disease when sampling, and a well matched control group. The patient group was made up of 40 Behçet's patients (20 male, 20 female aged 43+/-12 years) and the control group comprised 70 healthy volunteers (24 male, 46 female aged 45+/-13 years). Twelve of the 40 Behçet's patients have had a previous documented history of deep vein thrombosis at least six months before entering the study, and the other 28 did not. When patients and controls were compared, patients showed a statistically higher fibrinogen level (p=0.002), plasma viscosity (p=0.003), blood viscosity (p=0.021) and erythrocyte aggregation (p=0.049), the other rheological parameters not being statistically significant. No differences were observed in the rheological parameters when patients with and without a previous thrombotic episode were compared. Our results suggest that rheological alterations do not seem to play any role in the development of thrombotic events in patients with Behçet's disease.

Influence of plasmatic lipids on the hemorheological profile in healthy adults.

Some hemorheological parameters constitute risk factors for ischemic cardiovascular events. Most of these hemorheological factors are determined by the erythrocyte intrinsic properties and the high molecular weight plasmatic proteins, especially fibrinogen. The contribution of the plasmatic lipids to hemorheological factors is not well established. With this aim we determined hemorheological parameters in 112 healthy volunteers (62 males, 50 females) aged 35+/-10 years, range 19-54 years, members of our hospital staff. A complete set of rheological test was performed. Blood viscosity (BV) 230 sec(-1), plasma viscosity (PV), erythrocyte aggregation index (EAI), erythrocyte elongation index (EEI), hematocrit and fibrinogen. We also determined plasmatic lipids including total cholesterol (T-Ch) and its fractions (HDL-Ch, LDL-Ch, VLDL-Ch), triglycerides, lipoproteins (Apo B, Apo A(1), B/A(1)). Exclusion criteria were concomitant cardiovascular risk factors or any other associated pathology. Our results show a positive correlation between BV 230 sec(-1) and triglycerides (r=0.335) and negative with HDL-Ch (r=-0.451) (p=0.01), respectively; PV shows a positive correlation with T-Ch (r=0.297), LDL-Ch (r=0.298) and Apo B/A (r=0.290) (p=0.01). The EEI was negatively correlated with TG (p=0.05). Of all the rheological parameters evaluated, EAI is the factor which shows the highest significant correlation with plasmatic lipids: T-Ch (r=0.515), TG (r=0.303), LDL-Ch (r=0.507) and Apo B/A ratio (r=0.403); (p=0.01). These results suggest that plasmatic lipids contribute to modulate the blood rheological properties, slowing blood flow, favouring the development of atherothrombotics events, especially in stenotic areas or bifurcations in the vascular tree.

Rheological profile in severe and morbid obesity. Preliminary results.

The association of hemorheological alterations with morbid obesity remains a question of debate. In order to ascertain whether morbid obese subjects show certain hemorheological alterations which might be involved in the higher thrombotic risk which characterizes these subjects, we determine glucose, plasma lipids, apolipoproteins, fibrinogen, hematocrit, blood viscosity (Brookfield DVIII viscosimeter), both at native and corrected hematocrit of 45%, plasma viscosity (Fresenius capillary viscosimeter), erythrocyte aggregation (Myrenne aggregometer), both at stasis and at 3 s(-1) at 45% hematocrit and erythrocyte indexes in 41 morbid obese subjects (32 female, 9 male aged 33+/-10 years), and in a well matched non-obese control group (40 female, 15 male, aged 32+/-10 years). Mean BMI in the morbid obese group was 44.9+/-6.7 kg/m2 vs 23.5+/-4.8 kg/m2 in the control group (p<0.001). Morbid obese subjects when compared with the control group showed a statistically higher glucose level (p<0.001), LDL-cholesterol (p=0.019), triglycerides (p<0.001), apoB (p=0.019), apoB/A1 (p<0.001), fibrinogen (p<0.001), erythrocyte aggregation (p<0.001), and a statistically lower HDL-cholesterol (p<0.001). No differences between both groups were observed regarding total-cholesterol, plasma viscosity, blood viscosity and hematocrit (p=0.109; p=0.690; p=0.510; p=0.950), respectively. After the adjustment for BMI, differences in glucose, LDL-cholesterol, triglycerides, apoB, apoB/A1, and erythrocyte aggregation did not reach the statistical significance, and differences in fibrinogen were borderline significant (p=0.051), showing a direct effect of BMI on the detected differences between obese and non-obese. Our results suggest that in morbid obese subjects the increased fibrinogen levels and the altered lipid profile associated with their higher BMI, could in addition to its known mechanisms on haemostasis, favour both venous and arterial thrombotic events by enhancing erythrocyte aggregation.

Circulating endothelial cells in patients with heart failure and left ventricular dysfunction.

INTRODUCTION AND AIMS: Acute and chronic heart failure may manifest different degrees of endothelial damage and angiogenesis. Circulating endothelial cells (CEC) have been identified as marker of vascular damage. The aim of our study was to evaluate the evolution of the CEC at different stages of patients with heart failure. We also investigated a potential correlation between CEC and markers of vascular damage and angiogenesis. METHODS: We studied 32 heart failure patients at hospital admission (acute phase) and at revision after 3 months (stable phase) and 32 controls. Circulating markers of endothelial damage (CEC; von Willebrand factor, vWF and soluble E-selectin, sEsel) and angiogenesis (vascular endothelial growth factor, VEGF and thrombospondin-1) were quantified. RESULTS: Levels of CEC, vWF, sEsel and VEGF are significantly higher in heart failure patients than in controls. Levels of CEC (36.9 ± 15.3 vs. 21.5 ± 10.0 cells/ml; p< 0.001), vWF (325 ± 101 vs. 231 ± 82%; p< 0.001) and VEGF (26.3 ± 15.2 vs. 21.9 ± 11.9 ng/ml; p< 0.001) are significantly higher in the acute phase than in the stable phase of heart failure. CEC levels correlate with vWF and VEGF. RESULTS show than 100% of patients in acute phase and 37.5% in stable phase have levels of CEC higher than the 99th percentile of the distribution of controls (16 cells/ml). Therefore, increases in CEC represent a relative risk of 9.5 for heart failure patients suffering from acute phase. CONCLUSIONS: CEC, in addition to being elevated in heart failure, correlate with vWF levels, providing further support for CEC as markers of endothelial damage. Levels of CEC are associated with the acute phase of heart failure and could be used as a marker of the worsening in heart failure.