ALBERT

Description: Due to its high degree of contagiousness and like almost no other virus, SARS-CoV-2 has put the health of the world population on alert. COVID-19 can provoke an acute inflammatory process and uncontrolled oxidative stress, which predisposes one to respiratory syndrome, and in the worst case, death. Recent evidence suggests the mechanistic role of mitochondria and vitamin D in the development of COVID-19. Indeed, mitochondrial dynamics contribute to the maintenance of cellular homeostasis, and its uncoupling involves pathological situations. SARS-CoV-2 infection is associated with altered mitochondrial dynamics with consequent oxidative stress, pro-inflammatory state, cytokine production, and cell death. Furthermore, vitamin D deficiency seems to be associated with increased COVID-19 risk. In contrast, vitamin D can normalize mitochondrial dynamics, which would improve oxidative stress, pro-inflammatory state, and cytokine production. Furthermore, vitamin D reduces renin–angiotensin–aldosterone system activation and, consequently, decreases ROS generation and improves the prognosis of SARS-CoV-2 infection. Thus, the purpose of this review is to deepen the knowledge about the role of mitochondria and vitamin D directly involved in the regulation of oxidative stress and the inflammatory state in SARS-CoV-2 infection. As future prospects, evidence suggests enhancing the vitamin D levels of the world population, especially of those individuals with additional risk factors that predispose to the lethal consequences of SARS-CoV-2 infection.

Description: Alzheimer’s disease (AD) is the main cause of dementia and cognitive impairment. It has been associated with a significant diminution of omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) levels in the brain. Clinical trials with DHA as a treatment in neurological diseases have shown inconsistent results. Previously, we reported that the presence of phytanic acid (PhA) in standard DHA compositions could be blunting DHA’s beneficial effects. Therefore, we aimed to analyze the effects of a low PhA-concentrated DHA and a standard PhA-concentrated DHA in Apolipoprotein E knockout (ApoE−/−) mice. Behavioral tests and protein expression of pro-inflammatory, pro-oxidant, antioxidant factors, and AD-related mediators were evaluated. Low PhA-concentrated DHA decreased Aβ, ß-amyloid precursor protein (APP), p-tau, Ca2+/calmodulin-dependent protein kinase II (CAMKII), caspase 3, and catalase, and increased brain derived neurotrophic factor (BDNF) when compared to standard PhA-concentrated DHA. Low PhA-concentrated DHA decreased interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) protein expression in ApoE−/− mice when compared to standard PhA-concentrated DHA. No significant differences were found in p22phox, inducible nitric oxide synthase (iNOS), glutathione peroxidase (GPx), superoxide dismutase 1 (SOD-1), and tau protein expression. The positive actions of a low PhA-concentrated DHA were functionally reflected by improving the cognitive deficit in the AD experimental model. Therefore, reduction of PhA content in DHA compositions could highlight a novel pathway for the neurodegeneration processes related to AD.

Description: Abstract 810FN2 Increasing evidence shows that a small fraction of MM patients (pts) treated with high-dose therapy followed by autologous stem cell transplantation achieve long-term remission. Interestingly, this is not restricted to pts in complete response (CR), since those that revert to a monoclonal gammopathy of undetermined significance (MGUS) profile may also achieve long-term remission, despite the persistence of residual myeloma plasma cells (PCs). These results suggest that in addition to the anti-myeloma therapy, other factors may play a role in the control of the disease. Herein, we used 8-color MFC for detailed characterization of the structural components of the immune system and hematopoietic precursor cells (HPC) in paired bone marrow (BM) and peripheral blood (PB) samples from 26 MM patients in long-term disease control (LTDC): 9 in continuous CR and 17 who reverted to an MGUS profile and that subsequently showed stable disease without treatment for ≥5 years (median of 9 years; range, 5–19). As controls, paired BM and PB samples from 23 newly-diagnosed MGUS and 16 MM pts, together with 10 healthy adults (HA), were studied in parallel. In all BM and PB samples the distribution of the major T- (CD4, CD8, Tregs and γδ), NK- (CD56dim and CD56bright) and B-cell subsets (Pro-B, Pre-B, naïve and memory), in addition to normal PCs, dendritic cell (DC) subsets (plasmacytoid, myeloid and monocytic), monocytes, and CD34+ HPC (myeloid and lymphoid), were studied. The percentage and absolute count of each cell population was analysed in the BM and PB, respectively. Comparison of the two groups of MM pts with LTDC (9 CR vs. 17 MGUS-like) showed similar (p〉.05) cellular profiles in PB and BM, except for an increased number of BM and PB normal PCs in CR patients (P≤.04). Consequently, for all subsequent analyses, LTDC myeloma pts were pooled together. When compared to HA, patients with LTDC had increased numbers of CD8 T-cells and CD56dim NK-cells in both the BM and PB (p≤.03 and p≤.01, respectively). Despite this, the distribution of BM and PB CD4, CD8 and γδ T-cells among LTDC patients was similar (p〉.05) to that of both newly-diagnosed MM and MGUS cases; in contrast, BM and PB Tregs were significantly decreased vs newly-diagnosed MM (P=.03) and MGUS (P=.04). Regarding B-cells and normal PCs, LTDC patients showed increased numbers of BM B-cell precursors (both Pro-B and Pre-B cells) and normal PCs vs. newly diagnosed MM (P≤.05), but not MGUS, together with increased numbers of naïve B-cells vs. both MM and MGUS pts (P≤.01); all such cell populations returned to levels similar (p〉.05) to those of HA. As expected, this also included the number of CD34+ B-cell HPC which was increased among patients who achieved LTDC vs MM (p=.02), at levels similar (p〉.05) to those of MGUS and HA. Regarding DC, LTDC patients showed normal DC numbers in PB (but with higher PB myeloid-DC numbers vs. MM; p=.02), in association with decreased numbers of plasmacytoid DC and increased monocytic-DC in the BM vs. HA (p≤.04). No differences were found for the numbers of BM and PB monocytes. In summary, here we investigated for the first time the immune cell profile of MM patients who achieve long-term disease control. Our results show that, as newly-diagnosed MM, patients that achieve long-term disease control also show increased numbers of cytotoxic T-cells and CD56dim NK-cells; however, in contrast to newly-diagnosed MM, among LTDC patients such increase is associated with lower numbers of T-regs and an almost complete recovery of the normal PC, B-cell precursor and naïve B-cell compartments both in BM and PB. Further investigations on the activation and functional status of these cell populations are warranted.MO (%)/SP (cels./μl)HA N= 10MGUS N= 23MM N= 16LTDC-MM N= 26T cells9.588110.6117313113711926    CD4+4.85004.6624^6*5085463    CD8+3.7∼216∼4.63865.32645.3431    TCR γδ.2426.3230.2428.3421    Treg.4137.4141^.54*38.3432NK cells.7∼87∼1.51982.11721.6212    CD56 dim.65∼79∼1.41922.21681.6202B cells2.81471.8104.97*68*1.9160    Pro B.11—.06—.02*—.07—    Pre B.6—.4—.08—.23—    Naive SP—80—57^—36*—118    Normal-PCS.18.9.11.7.008.72*.11.84DCs.3449.3653.6848.558    Monocytes2.22472.42853.43023.1315    m-DC SP—11—14—8*—12    MO-DC.11∼29.2036.434.2837    p-DC.2∼4.1.145.112.8.123.8CD34+.9∼1.46.61.1.261.4.431.4    Mie-HPC.8∼—.53—.26—.36—    Linfo-HPC.1—.07—.03*—.05—*p≤.05 LTDC vs MM: ^ p≤.05 LTDC vs MGUS; ∼ p≤.05 LTDC vs HA Disclosures: Paiva: Jansen-Cillag: Honoraria; Celgene: Honoraria. Martinez:Janssen: Honoraria; Celgene: Honoraria. Maiolino:Centocor Ortho Biotech Research & Development: Research Funding.

Description: Introduction: Over half of patients with chronic myeloid leukemia (CML) in sustained deep molecular remission do not lose the major molecular response (MMR) after stopping treatment with tyrosine kinase inhibitors (TKI). This strategy is safe in controlled clinical trials, but there is scarce information on its applicability in the real-life setting. We aimed to assess if treatment cessation was feasible in clinical practice in a large nationwide series of CML patients from Spain. Methods: This retrospective study comprised a series of 236 patients in chronic-phase CML who discontinued TKI treatment outside of clinical trials between April 2009 and February 2018 in 33 Spanish institutions. Inclusion criteria were: a) TKI treatment duration 〉3 years; b) sustained MR4.5 in 〉4 consecutive determinations (one single point in MR4 was acceptable) during 〉2 years; c) molecular monitoring in a reference laboratory expressing the results on the International Scale (IS). Patients who had undergone allogeneic hematopoietic stem-cell transplantation were excluded. Molecular relapse was defined as consecutively detectable BCR-ABL1 transcripts showing a ≥1 log increase or loss of MMR in any single sample. Treatment-free remission (TFR) was estimated by the method of Kaplan-Meier and defined as the time from TKI discontinuation to the date of restarting therapy for any reason or, if treatment was not restarted, the date of last contact. Incidence of molecular relapse was calculated using the cumulative incidence function with resumption of TKI treatment in the absence of molecular relapse and death in MMR as competing events. Analysis of factors predicting molecular relapse was done by the method of Fine and Gray. Results: Table 1 shows the main characteristics of the series. Median follow-up from treatment discontinuation was 21.5 months, and 5 patients died in MMR due to CML unrelated causes. TKI therapy was reinitiated due to molecular relapse (MMR loss: n=52, increase 〉1 log in BCR-ABL transcript level at two consecutive assessments without losing MMR: n=12), patient preference (n=2), and severe withdrawal syndrome (n=1). One additional patient lost MMR after 20 months from treatment cessation but decided not to be retreated, with spontaneous recovery of MMR. The probability of TFR at 4 years was 64% (95% Confidence Interval [CI]: 55%-72%)(Figure 1). The cumulative incidence of molecular recurrence was 33% (95% CI: 26%-38%) at 3 years (Figure 2). Forty-nine relapses (75% of total) occurred in the first 6 months. The latest MMR loss was detected 30 months after treatment stop. One patient restarted treatment 44 months after TKI discontinuation due to ≥1 log increase in BCR-ABL1 transcripts in two consecutive samples without losing MMR. In univariate analysis, duration of TKI treatment of less than 5 years (P=0.005) and time in RM4.5 shorter than 4 years before TKI discontinuation (P=0.003) were both significantly associated with a higher incidence of molecular recurrence. No patient progressed to the advanced phases of CML. At the time of restarting treatment, the median BCR-ABL1 IS was 0.3%, with this value being 〉5% in only 7 instances. Most patients (81%) received the same TKI that they were taking before the trial of treatment cessation. Median follow-up after treatment resumption was 20 months. Among the 64 patients who restarted treatment due to molecular relapse, 46 of 52 cases regained MMR after a median time of 3 months, and 47 of 64 regained MR4.5 after a median time of 5 months. Response status at last control was: MR4.5 (n=196), MR4 (n=15), MMR (n=14), complete cytogenetic response (n=10), and other (n=1). Fifty-one patients (22%) developed musculoskeletal or joint pain after treatment cessation. In patients stopping imatinib, a significant increase in Hb levels, leukocyte counts, total lymphocyte counts, platelet counts, and cholesterol levels was observed. At 6 months, an increase in Hb level 〉2 g/dL was observed in 47% of patients with anemia. By contrast, nilotinib discontinuation was not followed by any relevant change in laboratory values. Conclusions: Our results confirm that treatment discontinuation is feasible and safe in clinical practice in Spain. Duration of TKI treatment of less than 5 years and a time in RM4.5 shorter than 4 years before TKI discontinuation were significantly associated with a higher incidence of molecular recurrence. Disclosures Hernandez Boluda: Incyte: Consultancy; Novartis: Consultancy. García Gutiérrez:Incyte: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ferrer Marin:Incyte: Consultancy; Novartis: Consultancy, Research Funding. Cervantes:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hospital Clinic Barcelona: Employment.

Description: Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 617 Disappearance of normal bone marrow (BM) plasma cells (N-PC) predicts malignant transformation of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) into symptomatic myeloma (MM). The homing, behavior and survival of N-PC, but also CD34+ hematopoietic stem/precursor cells (HPC), B-cell precursors, and (mono)clonal/aberrant PC (M-PC) largely depends on their interaction with SDF-1 expressing BM stromal cell niches. Accordingly, it can be hypothesized that a certain degree of competition among BM B-cell precursors, CD34+ HPC, and PC exist for the same BM niches. Thus, progressive replacement of normal cells by M-PC could help to explain the occurrence of cytopenias and hypogammaglobulinemia in MM patients. However, this hypothesis has not been investigated in depth neither in MM nor in SMM and MGUS. In this study we analyze by 8-color multiparameter flow cytometry the distribution and competitive migration capacity of B-cell precursors, CD34+ HPC, N-PC and M-PC in the BM and PB of patients with MGUS (n=60), SMM (n=47) and MM (n=87) at diagnosis plus 12 MM cases studied after high-dose therapy/autologous stem cell transplantation (MM POST-HDT/ASCT) vs. healthy adults aged 〉60 years (HA; n=26). The percentage of BM M-PC as well as the number of M-PC from all BMPC found at diagnosis significantly (p.05) and SMM (p〉.05). Despite the number of N-PC in the BM was significantly lower among MM and SMM vs. MGUS cases (p.05 vs. HA). Interestingly however, the proportion of CXCR4+ PB N-PC progressively increased from HA (11%) to MGUS (14%), SMM (15%) and MM (21%; p=.05 vs. HA), while MM POST-HDT/ASCT cases showed the lowest median percentage of CXCR4+ PB N-PC (5%). CD34+ HPC were found to be depleted in the BM of MM (0.3%; p=.001) and SMM (0.4%; p=.002) patients vs. HA (0.9%) while MGUS (0.8%) and MM POST-HDT/ASCT (1.1%) patients showed normal CD34+ HPC numbers. Conversely, PB CD34+ HPC progressively increase from HA to MGUS, SMM and MM cases (p=.008 and p=.06 vs. HA, respectively). By contrast, in MM POST-HDT/ASCT patients the number of PB CD34+ HPC returned to normal/lower levels. Ex-vivo competition assays between BM B-cells, PC and CD34+ HPC for SDF-1 induced migration showed that in HA, CD34+ HPC displayed the highest migration potential in the presence of SDF-1, followed by pre-B cell precursors; conversely N-PC barely migrated. No significant differences were found for the migration of all cell populations analyzed between MGUS and SMM patients vs. HA, except for M-PC that showed an impaired migration in the presence of SDF-1. In turn, the migration potential of CD34+ HPC (p=.04), and pre-B cell precursors (p=.02) was markedly reduced in symptomatic MM, particularly at lower SDF-1 concentrations (30nM). Most interestingly, the migration of M-PC from symptomatic MM was markedly increased at both concentrations of SDF-1 used: median of 2.9% and 1.0% for SDF-1 concentrations of 30nM and 70nM, respectively. Overall, these findings provide evidence about the role of progressive competition and replacement of normal BM cells by M-PC in determining transformation of pre-malignant MGUS and SMM into symptomatic MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1717 Background: Aberrant DNA methylation of tumor suppressor genes is a common event in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). Therefore hypomethylating agents like azacytidine (AZA) and decitabine seem to be a good therapeutic approach for the treatment of these diseases. Clinical experience and recent published data have demonstrated that AZA is effective for the treatment of MDS and AML patients. However, the prognostic impact of the aberrant hypermethylation on response and outcome to AZA treatment remains to be determined. For this reason the influence of the methylation status of a selected set of tumor suppressor genes on the overall survival and clinical response in MDS and AML patients, prior to treatment with AZA was studied. Patients and Methods: A total of 78 patients with MDS or AML who had been treated with AZA were evaluated. Among these, 25 were excluded because they had received less than 4 cycles, AZA was used after allogeneic stem cell transplantation, response was not assessable, or there was not enough quality DNA available. So finally, the study was focused in 53 patients: 36 MDS and 17 AML. Most of the AML included in the study had low blast count (20–30%). Responses were assessed according to the IWG MDS criteria in accordance to Fenaux et.al, and IWG AML criteria following European LeukemiaNet recommendations. DNA methylation status was analyzed using the Methylation Specific Multiplex Ligation Probe Amplification (MS-MLPA), with a panel of 24 different tumor suppressor genes related to cell cycle control, apoptosis regulation, DNA repair, cell adhesion and cell growth. Results: In the study cohort 47% of patients had cytogenetic alterations prior to AZA treatment, 4 with isolated -5/del(5q), 7 with isolated −7/del(7q), 4 with trisomy 8, 4 with not isolated -5/del(5q), 1 with trisomy 14, and 5 with complex cytogenetics. Methylation analysis showed that most patients (74%) had at least one methylated gene, but only 10% of patients displayed more than 3 methylated genes. The most frequently methylated genes were IGSF4 (28.3%), CDKN2B (24.5%), ESR1 (22.6%), CDH13 (17%) and CDKN1B (11.3%). The presence of a high number (≥2) of methylated genes (p=0.02), an adverse cytogenetics (p=0.03) or anemia (p=0.04) were independent prognostic factors associated with shorter overall survival. Moreover, the analysis of those patients displaying “no methylation”, patients with 1 methylated gene, patients with 2 methylated genes and those with more than 3 methylated genes, showed that as the number of methylated genes increases, the survival was shorter. The patients displaying the highest level of methylation (more than 3 genes), had a very short survival (median OS of 9.3 months, p

Description: Abstract 2871 Deletion at 13q14 (13q) is the most common genomic aberration in CLL. It is present in more than 50% of cases, and is the sole documented cytogenetic abnormality in 36% of the patients. These latter cases are known to have a more favorable clinical course. However, recent data from our group and others, suggest that patients with CLL and 13q deletion as the only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus CLL patients with a high number of 13q cells usually had both shorter overall survival and shorter time to first therapy. However, to the best of our knowledge the molecular characteristics of these patients have not been so far analyzed in detail. A total of 102 samples were selected for the study, 32 of which served as a validation cohort. A complete immunophenotypic analysis by flow cytometry and FISH studies were carried out in all cases. The median age was 68 years (range, 35 to 90 years). For the purpose of the study, only samples with one cytogenetic abnormality were included. For the gene expression profile analysis, according to our previous results, two groups of patients with 13q were compared: those in whom 80% or more of cells showed 13q (13qH) and those in whom fewer than 80% of cells showed 13q losses (13qL). The distribution of cases in the study cohort was: 13qH (n=25; 36%), 13qL (n=27; 39%), normal FISH (nCLL, n=8; 11%) and 17p/11q (n= 10; 14%); and in the validation cohort: 13qH (n=7; 22%), 13qL (n=11; 34%) and nCLL (n=9; 28%). Peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral blood samples using Ficoll gradient, snapfrozen and stored at 80°C. For the validation cohort, CD19positive B cells were purified by magnetically activated cell sorting (MACS) CD19 MicroBeads resulting in a 〉98% purity, as analyzed by flow cytometry. CD19positive normal B cells from peripheral blood of five healthy donors served as controls. All samples were hybridized with the Affymetrix Human Exon arrays 1.0 ST. A total of 3 450 genes significantly distinguished 13qH from 13qL patients, defining the 13qH signature. To determine the biological significance of the deregulated genes, a further analysis was carried out, revealing that apoptosis, BCR and NFkB signaling were the most significant affected pathways in 13qH CLL patients. Moreover, 13qH CLL patients were also characterized by a striking overrepresentation of deregulated miRNAs. A total of 15 miRNAs were deregulated in 13qH relative to 13qL patients. HsamiR155 was the most highly upregulated miRNA (Rfold=3.70), while hsamiR223 was the most significantly downregulated (Rfold=0.10). The posttranscriptional regulatory network of miRNA and genes in CLL patients with more than 80% of 13q cells was carried out by analyzing the miRNAmRNA relationships and the pathway analysis demonstrated that B cell receptor signaling, PI3K signaling and NFkB signaling were among the most strongly affected pathways in 13qH patients, highlighting the importance of miRNA regulation in CLL. The influence of other factors with prognostic relevance in CLL, such as IGVH mutational status, was discarded. We also analyzed the gene signature of CLL high risk cytogenetic subgroups in comparison with 13q patients. Surprisingly, our results suggest that some of the biological characteristics of 13qH CLL patients were similar to those of highrisk cytogenetic subgroups, since they share the deregulation of several key signaling pathways. To validate the differences observed between the subgroups of 13q CLL patients and get a visualization of these, we applied the Principal Component Analysis (PCA) in an independent series of patients. The expression pattern of CD19+ cells from CLL patients was notably different from the gene expression profile of CD19+ cells from healthy donors. Thus, CLL patients with a high number of 13q cells can be differentiated based on their expression profile. By contrast, the gene expression of B lymphocytes from 13qL and normal FISH subgroups was similar. Therefore, this study provides new evidences regarding the heterogeneity of 13q deletion in CLL patients. Thus an overexpression of BCR and NFKB patways and as well as a deregulation of the balance between the proliferative and apoptotic signals and miRNA expression are involved in cases with higher percentages of 13q- cells. Disclosures: No relevant conflicts of interest to declare.