Anemia in patients receiving anticancer treatments: focus on novel therapeutic approaches.

Anemia is common in cancer patients and impacts on quality of life and prognosis. It is typically multifactorial, often involving different pathophysiological mechanisms, making treatment a difficult task. In patients undergoing active anticancer treatments like chemotherapy, decreased red blood cell (RBC) production due to myelosuppression generally predominates, but absolute or functional iron deficiency frequently coexists. Current treatments for chemotherapy-related anemia include blood transfusions, erythropoiesis-stimulating agents, and iron supplementation. Each option has limitations, and there is an urgent need for novel approaches. After decades of relative immobilism, several promising anti-anemic drugs are now entering the clinical scenario. Emerging novel classes of anti-anemic drugs recently introduced or in development for other types of anemia include activin receptor ligand traps, hypoxia-inducible factor-prolyl hydroxylase inhibitors, and hepcidin antagonists. Here, we discuss their possible role in the treatment of anemia observed in patients receiving anticancer therapies.

Red Blood Cell Morphologic Abnormalities in Patients Hospitalized for COVID-19.

Peripheral blood smear is a simple laboratory tool, which remains of invaluable help for diagnosing primary and secondary abnormalities of blood cells despite advances in automated and molecular techniques. Red blood cells (RBCs) abnormalities are known to occur in many viral infections, typically in the form of mild normo-microcytic anemia. While several hematological alterations at automated complete blood count (including neutrophilia, lymphopenia, and increased red cell distribution width-RDW) have been consistently associated with severity of COVID-19, there is scarce information on RBCs morphological abnormalities, mainly as case-reports or small series of patients, which are hardly comparable due to heterogeneity in sampling times and definition of illness severity. We report here a systematic evaluation of RBCs morphology at peripheral blood smear in COVID-19 patients within the first 72 h from hospital admission. One hundred and fifteen patients were included, with detailed collection of other clinical variables and follow-up. A certain degree of abnormalities in RBCs morphology was observed in 75 (65%) patients. Heterogenous alterations were noted, with spiculated cells being the more frequent morphology. The group with >10% RBCs abnormalities had more consistent lymphopenia and thrombocytopenia compared to those without abnormalities or <10% RBCs abnormalities (p < 0.018, and p < 0.021, respectively), thus underpinning a possible association with an overall more sustained immune-inflammatory "stress" hematopoiesis. Follow-up analysis showed a different mortality rate across groups, with the highest rate in those with more frequent RBCs morphological alterations compared to those with <10% or no abnormalities (41.9%, vs. 20.5%, vs. 12.5%, respectively, p = 0.012). Despite the inherent limitations of such simple association, our results point out towards further studies on erythropoiesis alterations in the pathophysiology of COVID-19.

Novel serum paraoxonase activity assays are associated with coronary artery disease.

BACKGROUND: Serum paraoxonase (PON1) exerts antiatherogenic effects. Novel PON1 enzymatic tests have been recently developed: 5-thiobutyl butyrolactone (TBBL) estimates PON1 lactonase activity, whereas 7-O-diethylphosphoryl-3-cyano-4-methyl-7-hydroxycoumarin (DEPCyMC) is considered a surrogate marker of PON1 concentration. The TBBL to DEPCyMC ratio provides the normalized lactonase activity (NLA), which may reflect the degree of PON1 lactonase catalytic stimulation. The aim of this study was to evaluate for the first time TBBLase and DEPCyMCase activity in patients with coronary artery disease (CAD). METHODS: An angiography-based case-control study was conducted, including 300 sex- and age-matched subjects [100 CAD-free, 100 CAD without myocardial infarction (MI) and 100 CAD with MI]. RESULTS: A low DEPCyMCase activity (lowest vs. highest tertile: OR 2.96, 95% CI 1.18-7.43) and a high NLA (highest vs. lowest tertile: OR 3.25, 95% CI 1.28-8.26) were both associated with CAD, independent of classical atherosclerosis risk factors, lipid-lowering therapy and PON1 genotype. Total TBBLase activity was, however, not different in CAD compared to CAD-free subjects. CONCLUSIONS: Novel PON1 activity assays may be associated with CAD. In this study, CAD patients had low DEPCyMCase activity, a possible marker of low PON1 concentration, but showed a high stimulation of PON1 lactonase activity.

Clinical, pathological, and molecular correlates in ferroportin disease: a study of two novel mutations.

BACKGROUND/AIMS: Clinico-pathological manifestations of ferroportin (Fpn) disease (FD) are heterogeneous, with some patients presenting with iron overload predominantly in macrophages ("M" phenotype), others predominantly in hepatocytes ("H" phenotype). This appears to reflect functional heterogeneity of Fpn mutants, with loss-of-function generally resulting in the M type. METHODS: Two unrelated probands with "non-HFE" hemochromatosis were screened for Fpn mutations. Mutants were functionally characterized by immunofluorescence microscopy, evaluation of their ability to bind hepcidin and export iron, and by expressing them in zebrafish. RESULTS: Two novel Fpn mutations were identified: I152F in patient-1, presenting with typical M phenotype; and L233P in patient-2, presenting with ambiguous features (massive overload in both macrophages and hepatocytes). Molecular studies suggested loss of function in both cases. The I152F, normally localized on cell membrane and internalized by hepcidin, showed a unique "primary" deficit of iron export capability. The L233P did not appropriately traffic to cell surface. Loss of function was confirmed by expressing both mutants in vivo in zebrafish, resulting in iron limited erythropoiesis. Clinical manifestations were likely enhanced in both patients by non-genetic factors (HCV, alcohol). CONCLUSIONS: The combination of careful review of clinico-pathological data with molecular studies can yield compelling explanations for phenotype heterogeneity in FD.

Combined effect of hemostatic gene polymorphisms and the risk of myocardial infarction in patients with advanced coronary atherosclerosis.

BACKGROUND: Relative little attention has been devoted until now to the combined effects of gene polymorphisms of the hemostatic pathway as risk factors for Myocardial Infarction (MI), the main thrombotic complication of Coronary Artery Disease (CAD). The aim of this study was to evaluate the combined effect of ten common prothrombotic polymorphisms as a determinant of MI. METHODOLOGY/PRINCIPAL FINDINGS: We studied a total of 804 subjects, 489 of whom with angiographically proven severe CAD, with or without MI (n = 307; n = 182; respectively). An additive model considering ten common polymorphisms [Prothrombin 20210G>A, PAI-1 4G/5G, Fibrinogen beta -455G>A, FV Leiden and "R2", FVII -402G>A and -323 del/ins, Platelet ADP Receptor P2Y12 -744T>C, Platelet Glycoproteins Ia (873G>A), and IIIa (1565T>C)] was tested. The prevalence of MI increased linearly with an increasing number of unfavorable alleles (chi(2) for trend = 10.68; P = 0.001). In a multiple logistic regression model, the number of unfavorable alleles remained significantly associated with MI after adjustment for classical risk factors. As compared to subjects with 3-7 alleles, those with few (/=8) alleles had an increased MI risk (OR 2.49, 95%CIs 1.03-6.01). The number of procoagulant alleles correlated directly (r = 0.49, P = 0.006) with endogenous thrombin potential. CONCLUSIONS: The combination of prothrombotic polymorphisms may help to predict MI in patients with advanced CAD.

Measurement of urinary hepcidin levels by SELDI-TOF-MS in HFE-hemochromatosis.

INTRODUCTION: Insufficient production of hepcidin, the master regulator of iron metabolism, is recognized as the key pathogenetic feature of HFE-related hereditary hemochromatosis (HH). There is a growing interest in measuring the hepcidin levels, which may improve the diagnosis, prognostic evaluation and clinical management of HH. Nevertheless, few investigative tools are available: an immunodot method for urinary hepcidin developed by a single centre (UCLA), not yet ready for large-scale diffusion, and mass spectrometry (MS) based assays, such as surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF-MS). The latter is well suited to small peptides like hepcidin, and can rapidly analyze crude samples with high throughput. This study measured urinary hepcidin levels by SELDI-TOF-MS in a large group of HH patients at diagnosis and during treatment, including both C282Y homozygous and C282Y/H63D compound heterozygotes. METHODS: We used a protocol based on PBSIIc mass spectrometer and Normal Phase chips. Urinary samples from 30 control subjects were compared to those obtained from 80 HH patients (57 C282Y homozygotes, 23 C282Y/H63D compound heterozygotes). Eighteen C282Y homozygotes and 11 C282Y/H63D compound heterozygotes were analyzed at diagnosis, the remainder during maintenance phlebotomy. RESULTS: C282Y homozygotes either at diagnosis, or after phlebotomy had significantly lower urinary hepcidin levels than controls (P<0.05). C282Y/H63D compound heterozygotes had hepcidin levels at diagnosis higher than controls, while the hepcidin/ferritin ratio was significantly decreased (P<0.001) suggesting inadequate hepcidin production. After phlebotomy, mean hepcidin levels in the compound heterozygotes were significantly lower than in controls (P<0.001). Samples from 12 randomly selected control subjects were sent to UCLA for duplicate measurement by the immunodot method, yielding a significant correlation (rho=0.64; P=0.024). CONCLUSIONS: This study supports the use of SELDI-TOF-MS for measuring hepcidin levels in research and clinical applications. Our results in phlebotomized patients suggest that the depletion of iron stores may further exacerbate the HFE-related hepcidin defect.

Blunted hepcidin response to oral iron challenge in HFE-related hemochromatosis.

Inadequate hepcidin synthesis leads to iron overload in HFE-related hemochromatosis. We explored the regulation of hepcidin by iron in 88 hemochromatosis patients (61 C282Y/C282Y, 27 C282Y/H63D) and 23 healthy controls by analyzing urinary hepcidin before and 24 hours after a 65-mg oral iron dose. Thirty-four patients were studied at diagnosis and had iron overload, and 54 patients were iron depleted. At diagnosis, hepcidin values in C282Y homozygotes were similar to controls, whereas values in C282Y/H63D heterozygotes were higher (P = .02). However, the hepcidin/ferritin ratio was decreased in both homozygotes (P < .001) and heterozygotes (P = .017), confirming the inadequate hepcidin production for the iron load with both genotypes. In iron-depleted patients of both genotypes studied at a time remote from phlebotomy, basal hepcidin was still lower than in controls (P < .001 for C282Y/C282Y and P = .002 for heterozygotes). After an iron challenge, mean urinary hepcidin excretion increased in controls (P = .001) but not patients, irrespective of genotype and iron status. Significant hepcidin increase ( > or = 10 ng/mg creatinine) was observed in 74% of controls, 15% of homozygotes, and 32% of heterozygotes. The hepcidin response to oral iron is blunted in HFE-related hemochromatosis and not improved after iron depletion. The findings support the involvement of HFE in iron sensing and subsequent regulation of hepcidin.

Hyperhomocysteinemia and mortality after coronary artery bypass grafting.

BACKGROUND: The independent prognostic impact, as well as the possible causal role, of hyperhomocysteinemia (HHcy) in coronary artery disease (CAD) is controversial. No previous study specifically has addressed the relationship between HHcy and mortality after coronary artery bypass grafting (CABG) surgery. The aim of this study is to evaluate the prognostic impact of HHcy after CABG surgery. METHODOLOGY AND PRINCIPAL FINDINGS: We prospectively followed 350 patients who underwent elective CABG between May 1996 and May 1999. At baseline, fasting total homocysteine (tHcy) levels were measured in all participants, and a post-methionine loading (PML) test was performed in 77.7% of them (n = 272). After a median follow-up of 58 months, 33 patients (9.4%) had died, 25 because of cardiovascular events. HHcy, defined by levels higher than the 90th percentile (25.2 micromol/L) of the population's distribution, was significantly associated to total and cardiovascular mortality (P = 0.018 [log-rank test 5.57]; P = 0.002 [log-rank test 9.76], respectively). The PML test had no prognostic value. After multiple adjustment for other univariate predictors by Cox regression, including statin therapy (the most powerful predictor in uni-/multivariate analyses), high-sensitivity C Reactive Protein (hs-CRP) levels, and all known major genetic (MTHFR 677C-->T polymorphism) and non-genetic (B-group vitamin status and renal function) tHcy determinants, HHcy remained an independent prognostic factor for mortality (HRs: 5.02, 95% CIs 1.88 to 13.42, P = 0.001). CONCLUSIONS: HHcy is an important prognostic marker after CABG, independent of modern drug therapy and biomarkers.

Clinical heterogeneity of acquired hemophilia A: a description of 4 cases.

Acquired hemophilia A is a rare but severe auto-immune bleeding disorder characterized by the presence of autoantibodies directed against clotting factor VIII. Acquired hemophilia A may be idiopathic or associated with several conditions, such as postpartum, autoimmune diseases, malignancies or drugs. The treatment modalities of bleeding episodes and eradication of the factor VIII auto-antibody depend on the titer of anti-factor VIII:C and may include desmopressin (DDAVP), prednisolone, prednisolone-cyclophosphamide, high dose intravenous gammaglobulin, FVIII-VWF concentrate and/or recombinant FVIIa (rFVIIa). In this study we report four cases of autoimmune factor VIII inhibitors (2 associated with autoimmune disorders, 2 idiopathic) demonstrating the heterogeneity of this disease from pathogenic, clinical, therapeutic and prognostic points of view.

Influence of polymorphisms in the factor VII gene promoter on activated factor VII levels and on the risk of myocardial infarction in advanced coronary atherosclerosis.

In this study, we investigate the influence of three factor VII (FVII) gene polymorphisms on activated FVII levels (FVIIa), and also on the risk of myocardial infarction (MI) in patients with advanced coronary atherosclerotic disease (CAD). The -323A2 allele in the promoter is known to be associated with low FVII levels, and has been suggested to protect against MI in some studies. The -402GA promoter polymorphism, that in vitro has been associated with having opposite effect, is less well studied clinically. For this study, plasma FVIIa levels and three FVII gene polymorphisms were assessed in 934 subjects of both sexes, all with an angiographic documentation of coronary vessels. Our results show that two promoter polymorphisms, plasma cholesterol, and gender, were significant predictors of FVIIa levels. The -402A allele was associated to a significant increase of FVIIa levels in males (by 19.2%). In a selected clinical model including the patients with severe CAD, with or without a thrombotic complication (MI), male carriers of the -402A had an increased risk of MI (OR=1.79; 95% CI 1.15-2.80). The -323A2 allele was associated to a significant decrease in FVIIa (by 36.02% in males, and 39.7% in females). Male carriers of the -323A2 were protected from MI (OR=0.6; 95% CI 0.39-0.94), but only after correction for the confounding effect of combined heterozygosity for the promoter polymorphisms. We can conclude that FVII gene polymorphisms with an opposite effect on FVIIa levels may modulate the risk of MI in males with advanced CAD. This study highlights a "within-gene" interaction, and the need to explore polymorphisms in candidate gene(s) in detail.

Low plasma vitamin B-6 concentrations and modulation of coronary artery disease risk.

BACKGROUND: Low concentrations of pyridoxal-5'-phosphate (PLP), the active metabolite of vitamin B-6, are associated with high C-reactive protein (CRP) concentrations. Both low PLP and elevated inflammatory markers, such as high-sensitivity CRP (hs-CRP) and fibrinogen, are related to higher risk of coronary artery disease (CAD). OBJECTIVES: The objectives were to evaluate the relation between PLP and acute-phase reactants in affecting CAD risk and to estimate the risk of CAD related to low plasma PLP, either alone or in combination with high concentrations of acute-phase reactants and other classic risk factors for CAD. DESIGN: A case-control study was conducted with 742 participants: 475 with severe multivessel CAD and 267 free from coronary atherosclerosis (CAD-free). We measured plasma PLP, fibrinogen, hs-CRP, and serum lipid concentrations and all major biochemical CAD risk factors, including total homocysteine. RESULTS: A significant, inverse, graded relation was observed between PLP and both hs-CRP and fibrinogen (P < 0.001). The prevalence of PLP concentrations in the lower half of the population (<50th percentile: 36.3 nmol/L) was significantly higher among CAD patients than among CAD-free subjects (P < 0.001). The odds ratio for CAD risk related to low PLP concentrations after adjustments for the major classic CAD risk factors, including hs-CRP and fibrinogen, was 1.89 (95% CI: 1.18, 3.03; P = 0.008). The CAD risk as a result of low PLP was additive when considered in combination with elevated hs-CRP concentrations or with an increased ratio of LDL to HDL. CONCLUSION: Low plasma PLP concentrations are inversely related to major markers of inflammation and independently associated with increased CAD risk.

Clinical and pathologic findings in hemochromatosis type 3 due to a novel mutation in transferrin receptor 2 gene.

BACKGROUND & AIMS: Although most patients with hereditary hemochromatosis are homozygous for a single mutation of the HFE gene on chromosome 6p, accumulating evidence indicates that the disease is genetically heterogeneous. Type 3 hemochromatosis, recently described in 4 families, is linked to mutations of the gene encoding transferrin receptor 2 on chromosome 7q22. Here we report data from a family carrying a new mutation of the transferrin receptor 2 gene. METHODS: Detailed clinical and histopathologic documentation was available for most family members. The entire coding sequence and exon/intron boundaries of the transferrin receptor 2 gene were analyzed by direct sequencing. RESULTS: A 12-nucleotide deletion in exon 16, causing the loss of 4 amino acids (AVAQ 594-597 del), was detected at the homozygous state in the 3 patients with histologically proven iron overload. The deletion segregated with the disease within the family and was not found in 100 healthy controls. Some clinical and pathologic characteristics, such as low penetrance in the premenopausal woman, and early iron deposition in periportal hepatocytes resembled those of classic, HFE-related hemochromatosis. CONCLUSIONS: Our data support the role of the transferrin receptor 2 gene in hemochromatosis type 3 as well as its critical involvement in the maintenance of iron homeostasis in humans.

Biochemical and genetic markers of iron status and the risk of coronary artery disease: an angiography-based study.

BACKGROUND: Iron may promote coronary atherosclerotic disease (CAD) by increasing lipid peroxidation. Studies on biochemical or genetic markers of body iron stores as risk factors for CAD have yielded conflicting results. METHODS: We studied 849 individuals with a clear-cut definition of the CAD phenotype, i.e., with (CAD; n = 546) or without (CAD-free; n = 303) angiographically documented disease. We determined serum ferritin, as a biochemical estimate of iron stores, and the C282Y mutation in the HFE gene, i.e., the main cause of hemochromatosis in Caucasians. The relationships of ferritin with serum markers of either inflammation [C-reactive protein (CRP)] or lipid peroxidation (malondialdehyde) were also investigated. RESULTS: Mean ferritin concentrations were slightly higher in CAD vs CAD-free individuals, but this difference disappeared after adjusting for sex and CRP. Ferritin was significantly correlated with CRP (Spearman's test, rho = 0.129; P <0.001). Heterozygotes for Cys282Tyr were 4.8% among the CAD group and 6.6% among the CAD-free group (P = 0.26). The prevalence of high concentrations of stored iron, defined as ferritin concentrations above the sex-specific upper quintiles of the control distribution, was also similar in the two groups. There was a higher prevalence of "iron depletion" in CAD-free vs CAD females (20% vs 8.8%, respectively), but this difference disappeared after adjustment for age and other cardiovascular risk factors (odds ratio, 0.66; 95% confidence interval, 0.21-2.08). No differences in iron markers were found in CAD patients with or without myocardial infarction. CONCLUSIONS: Our results do not support a role for biochemical or genetic markers of iron stores as predictors of the risk of CAD or its thrombotic complications.