Virginia Martínez

Alternative activation of macrophages in human peritoneum: implications for peritoneal fibrosis

BACKGROUND Depending on the cytokine microenvironment, macrophages (Mϕ) can adopt a proinflammatory (M1) or a profibrotic (M2) phenotype characterized by the expression of cell surface proteins such as CD206 and CD163 and soluble factors such as CC chemokine ligand 18 (CCL18). A key role for Mϕ in fibrosis has been observed in diverse organ settings. We studied the Mϕ population in a human model of peritoneal dialysis in which continuous stress due to dialysis fluids and recurrent peritonitis represent a risk for peritoneal membrane dysfunction reflected as ultrafiltration failure (UFF) and peritoneal fibrosis. METHODS We used flow cytometry and quantitative reverse transcription-polymerase chain reaction to analyse the phenotype of peritoneal effluent Mϕ and tested their ability to stimulate the proliferation of human fibroblasts. Mϕ from non-infected patients were compared with those from patients with active peritonitis. Cytokine production was evaluated by enzyme-linked immunosorbent assay (ELISA) in spent dialysates and cell culture supernatants. RESULTS CD206(+) and CD163(+) M2 were found within peritoneal effluents by flow cytometry analysis, with increased frequencies of CD163(+) cells during peritonitis (P = 0.003). TGFB1, MMP9 and CCL18 messenger RNA (mRNA) levels in peritoneal macrophages (pMϕ) were similar to those found in M2 cells differentiated in vitro. The ability of pMϕ to stimulate fibroblast proliferation correlated with CCL18 mRNA levels (r = 0.924, P = 0.016). CCL18 production by pMϕ was confirmed by immunostaining of cytospin samples and ELISA. Moreover, CCL18 effluent concentrations correlated with decreased peritoneal function, which was evaluated as dialysate to plasma ratio of creatinine (r = 0.724, P < 0.0001), and were significantly higher in patients with UFF (P = 0.0025) and in those who later developed sclerosing peritonitis (P = 0.024). CONCLUSIONS M2 may participate in human peritoneal fibrosis through the stimulation of fibroblast cell growth and CCL18 production as high concentrations of CCL18 are associated with functional deficiency and fibrosis of the peritoneal membrane.

Identification of new SNPs associated with severe toxicity to capecitabine

&NA; Predicting individual risk of chemotherapy‐induced severe adverse reaction is a critical issue when selecting the best treatment for cancer patients. SNPs have been identified in genes involved in the pharmacodynamics of fluoropyrimidines, and guidelines even recommend genotyping some DPYD variants in order to estimate the risk of toxicity. However, the predictive value of this approach remains insufficient, thus limiting its clinical implementation. The aim of the present study was to identify new genetic variants by selecting a group of tag SNPs in genes associated with the pharmacodynamics of fluoropyrimidines (CDA, DPYD, ENOSF1, CES1, TYMS, SLC22A7, TYMP, and UMPS). For this purpose, 23 selected SNPs were genotyped on an OpenArray™ platform in a cohort of 301 colorectal cancer patients receiving capecitabine‐based chemotherapy. Univariate and multivariate statistical analysis by logistic regression revealed 10 SNPs associated with severe adverse reactions to capecitabine (P < 0.05): rs1048977, rs12726436, and rs2072671 in CDA; rs12119882 in DPYD; rs2853741 in TYMS; rs699517 in TYMS/ENOSF1; rs2270860 and rs4149178 in SLC22A7; and rs2279199 and rs4678145 in UMPS. Except for rs2072671, no association had previously been reported between these SNPs and the risk of capecitabine‐induced toxicity. The use of tag SNPs to find new polymorphisms related to adverse reactions to capecitabine was successful. These new variants could increase the predictive power of currently available tests and thus prevent severe adverse reactions to capecitabine. Graphical abstract Figure. No caption available.

Continuation of bevacizumab and addition of hormone therapy following weekly paclitaxel therapy in HER2-negative metastatic breast cancer.

Background Bevacizumab plus taxane chemotherapy improves progression-free survival (PFS) versus taxane monotherapy in the first-line treatment of HER2-negative metastatic breast cancer (MBC) and appears promising in the second-line setting. This retrospective analysis evaluated the efficacy and safety of this combination in a real-world setting. Patients and methods Eligible patients received bevacizumab (10 mg/kg days 1 and 15, every 28 days) plus paclitaxel (80 mg/m2 days 1, 8, and 15, every 28 days) as first-line therapy for MBC, or as subsequent lines, including bevacizumab continuation therapy, at La Paz University Hospital between August 2007 and October 2012. End points included objective response rate (ORR), PFS, overall survival (OS), and safety. Results Seventy-eight patients were included. Median PFS was 12.8 months for patients receiving first-line treatment and 9.3 months for subsequent lines. Forty-five patients (57.7%) continued bevacizumab after stopping paclitaxel, and had significantly longer PFS and OS than those who did not (hazard ratio [HR] 0.40, 95% confidence interval [CI] 0.248–0.653, P<0.001; HR 0.39, 95% CI 0.218–0.710, P=0.002; respectively). In the continuation phase, estrogen receptor-positive patients had longer PFS and OS when receiving hormone therapy plus bevacizumab versus patients receiving only bevacizumab (HR 0.50, 95% CI 0.24–1.04, P=0.06; HR 0.43, 95% CI 0.16–1.16, P=0.09; respectively). Thirty-five patients (44.9%) reported grade 3–4 adverse events. Conclusion Bevacizumab plus paclitaxel was effective in HER2-negative MBC. Continuation of bevacizumab and addition of hormone therapy following paclitaxel therapy could be beneficial.

Use of exome sequencing to determine the full profile of genetic variants in the fluoropyrimidine pathway in colorectal cancer patients affected by severe toxicity

AIM To identify genetic variants associated with capecitabine toxicity in fluoropyrimidine pathway genes using exome sequencing. PATIENTS & METHODS Exomes from eight capecitabine-treated patients with severe adverse reactions (grade >2), among a population of 319, were sequenced (Ion Proton). SNPs in genes classified as potentially damaging (Sorting Intolerant from Tolerant and Polymorphism Phenotyping v2) were tested for association with toxicity in a validation cohort of 319 capecitabine-treated patients. RESULTS A total of 17 nonsynonymous genetic variants were identified. Of these, five putative damaging SNPs in DPYD, ABCC4 and MTHFR were genotyped in the validation cohort. DPYD rs1801160 was associated with the risk of toxicity (p = 0.029) and MTHFR rs1801133 with delayed administration of chemotherapy due to toxicity (p = 0.047). CONCLUSION Exome sequencing revealed two specific biomarkers of the risk of toxicity to capecitabine.

Sustained low peritoneal effluent CCL18 levels are associated with preservation of peritoneal membrane function in peritoneal dialysis

Peritoneal membrane failure (PMF) and, ultimately, encapsulating peritoneal sclerosis (EPS) are the most serious peritoneal dialysis (PD) complications. Combining clinical and peritoneal transport data with the measurement of molecular biomarkers, such as the chemokine CCL18, would improve the complex diagnosis and management of PMF. We measured CCL18 levels in 43 patients’ effluent and serum at baseline and after 1, 2, and 3 years of PD treatment by retrospective longitudinal study, and evaluated their association with PMF/EPS development and peritoneal risk factors. To confirm the trends observed in the longitudinal study, a cross-sectional study was performed on 61 isolated samples from long-term (more than 3 years) patients treated with PD. We observed that the patients with no membrane dysfunction showed sustained low CCL18 levels in peritoneal effluent over time. An increase in CCL18 levels at any time was predictive of PMF development (final CCL18 increase over baseline, p = .014; and maximum CCL18 increase, p = .039). At year 3 of PD, CCL18 values in effluent under 3.15 ng/ml showed an 89.5% negative predictive value, and higher levels were associated with later PMF (odds ratio 4.3; 95% CI 0.90–20.89; p = .067). Moreover, CCL18 levels in effluent at year 3 of PD were independently associated with a risk of PMF development, adjusted for the classical (water and creatinine) peritoneal transport parameters. These trends were confirmed in a cross-sectional study of 61 long-term patients treated with PD. In conclusion, our study shows the diagnostic capacity of chemokine CCL18 levels in peritoneal effluent to predict PMF and suggests CCL18 as a new marker and mediator of this serious condition as well as a new potential therapeutic target.

Mechanisms Involved in Hypersensitivity Reactions to Polysulfone Hemodialysis Membranes

Several cases of patients with anaphylactic or systemic hypersensitivity reactions to polysulfone (PS) hemodialysis (HD) membranes and tolerance to cellulose triacetate (CTA) membranes have recently been reported. To investigate the mechanisms involved in PS hypersensitivity, basophil, T cell, and complement activation were analyzed in acute-phase samples from two patients with systemic reactions to PS-based membranes. Basophil and T cell activation, as well as higher serum tryptase levels were detected in acute-phase samples compared with basal levels. Complement levels (C3 and C4) were decreased in acute-phase samples from PS-allergic patients to a higher extent than in samples from control donors taken at the same time points, indicating complement activation during the acute reactions. An experimental external circuit was established on pediatric membranes after rinsing with low or high priming volumes of saline solution, to analyze basophils, T cells, and complement activation in blood samples from 10 PS-allergic and 8 nonallergic HD patients upon contact with PS-based or CTA membranes. Predialysis and postdialysis samples were collected. Basophils from PS-allergic patients exhibited increased degranulation, and T cells showed significantly increased activation after contact with PS-based membranes primed with low volumes of saline. No activation was detected in leukocytes from nonallergic patients under the same experimental conditions. Membrane priming with high volumes of saline abrogated activation of basophils and T cells. However, basophils from allergic donors showed significantly higher responses to Fcεc stimulation after contact with PS membranes. Basophil degranulation and elevated serum tryptase levels in allergic patients during acute reactions support the systemic activation of mast cells and basophils during hypersensitivity reactions to PS-based membranes. A leachable component of the membranes might be responsible for cell activation in some patients.

High Frequencies of CMV-specific CD8 and CD4 T Cell Subsets are Needed Pretransplant to Protect from CMV Replication in Kidney Transplant Recipients Treated with Thymoglobulin

Background Diagnostic of CMV-specific T cell immunity in solid organ transplant patients is showing to be a useful tool in predicting CMV infection after transplantation. Induction treatment with anti-lymphocyte polyclonal antibodies (thymoglobulin) causes lymphocyte depletion from the first week to beyond one-year post-trasplantation. Pretransplant immunological analysis in patients treated with thymoglobulin could guide the most appropriate prevention strategy. Methods We performed a prospective study of patients with kidney transplant who were treated with thymoglobulin as induction treatment and with universal prophylaxis for CMV infection. CD4 and CD8 T-cell responses to pp65 and IE-1 CMV antigens in 37 CMV-seropositive and 9 CMV-seronegative patients were investigated. Intracellular flow cytometry was employed to determine IFN-&ggr; production as a functional readout. The response was analyzed in pretransplant samples and at 1, 6, 12 and 24 months post-transplant. Results In CMV-seropositive patients who did not develop CMV infection post-transplant, pretransplant CD8 T-cell response to IE-1 (p= .004) and CD8 and CD4 T-cell responses to pp65 (p=.04 and p=.002, respectively) were significantly higher than it in patients with CMV replication (Figure 1) ROC curve analysis showed that low frequencies of IE-1-specific (< .13%) or pp65-specific (< .88%) CD8 T cells or pp65-specific (< .13%) CD4 T cells predicted the development of CMV infection (Figure 2). Figure. No caption available. Figure. No caption available. Conclusions Functional assessment of pretransplant CMV-specific CD4 and CD8 T-cell immunity in seropositive patients who are going to receive thymoglobulin allows the identification of patients at risk of developing CMV infection post-transplantation.

Clinical utility of ABCB1 genotyping for preventing toxicity in treatment with irinotecan

ABSTRACT Preventing severe irinotecan‐induced adverse reactions would allow us to offer better treatment and improve patients’ quality of life. Transporters, metabolizing enzymes, and genes involved in the folate pathway have been associated with irinotecan‐induced toxicity. We analyzed 12 polymorphisms in UGT1A1, ABCB1, ABCG2, ABCC4, ABCC5, and MTHFR in 158 patients with metastatic colorectal cancer treated with irinotecan and studied the association with grade >2 adverse reactions (CTCAE). Among the most frequent ADRs, the SNPs rs1128503, rs2032582, and rs1045642 in ABCB1 and rs1801133 in MTHFR were associated with hematological toxicity and overall toxicity. The SNP rs11568678 in ABCC4 was also associated with overall toxicity. After correction of P values using a false discovery rate, only ABCB1 variants remained statistically significant. Haplotype analysis in ABCB1 showed an 11.3‐fold and 4.6‐fold increased risk of hematological toxicity (95% CI, 1.459–88.622) and overall toxicity (95% CI, 2.283–9.386), respectively. Consequently, genotyping of the three SNPs in ABCB1 can predict overall toxicity and hematological toxicity with a diagnostic odds ratio of 4.40 and 9.94, respectively. Genotyping of ABCB1 variants can help to prevent severe adverse reactions to irinotecan‐based treatments in colorectal cancer.

Prominent Levels of the Profibrotic Chemokine CCL18 during Peritonitis: In Vitro Downregulation by Vitamin D Receptor Agonists

Peritoneal dialysis (PD) is used as a renal replacement therapy, which can be limited by peritoneal membrane ultrafiltration failure (UFF) secondary to fibrotic processes. Peritonitis, a frequent complication of PD, is a major risk factor for peritoneal membrane fibrosis and UFF. Low peritoneal levels of the chemokine CCL18 are associated with preservation of peritoneal membrane function in PD. Given that CCL18 is involved in fibrotic processes and recurrent peritonitis, it is a risk factor for peritoneal membrane failure; thus, we evaluated CCL18 concentrations in peritoneal effluents from patients undergoing peritonitis episodes. Pharmacological interventions aimed at diminishing the production of CCL18 were also explored. Fivefold higher CCL18 peritoneal concentrations were found during acute bacterial peritonitis, in parallel with the increased infiltration of macrophages. Unexpectedly, CCL18 was also highly (50-fold) increased during sterile eosinophilic peritonitis, and peritoneal eosinophils were found to express CCL18. In vitro treatment of peritoneal macrophages with the vitamin D receptor agonist paricalcitol was able to reduce the secretion and the expression of CCL18 in isolated peritoneal macrophages. In conclusion, our study suggests that the chemokine CCL18 can be a mediator of peritoneal membrane failure associated with peritonitis episodes as well as providing a new potential therapeutic target.

PKP-007 Dpyd snps and disease free survival after capecitabine based adjuvant treatment in colorectal cancer

Background DPYD has a key role in fluoropyrimidines metabolism. The relationship between enzyme activity and drug efficacy and toxicity has been widely studied, but the predictive value of the most accepted variants is still poor. Hence the search for new biomarkers is needed. Purpose To analyse if single nucleotide polymorphisms (SNPs) in the DPYD exon regions have an influence on disease free survival (DFS) in colorectal cancer patients treated with capecitabine based adjuvant chemotherapy. Material and methods The study design was observational, ambispective and multicentric. The study population included 138 adult patients with stages II and III colorectal cancer that received capecitabine based adjuvant chemotherapy. DNA was isolated from peripheral blood samples and seven polymorphisms (rs12119882, rs1801158, rs1801159, rs291592, rs291593, rs44221623, rs6668296) in the DPYD exon regions were genotyped through OpenArray technology. DFS was estimated by the Kaplan–Meier method. The relationship between polymorphisms and DFS was explored using the Cox regression model with tumour stage, treatment and hospital as co-variables. Results 76.8% of patients received capecitabine in combination with oxaliplatin (XELOX regimen), and the remaining 23.2% monotherapy. Median follow-up time was 30.1 months (range 7–171.9). At the cut-off date, 35 patients had relapsed (25.4%). Patients harbouring the DPYD rs291593 GG genotype had a worse DFS than those carriers of the GA/AA variants (HR 2.15; 95% CI 1.10–4.23; p=0.026). Patients with the TT homozygous genotype in DPYD rs1801159 also showed a trend to shorter DFS than heterozygous or homozygous carriers of the C allele when analysed by Kaplan–Meier (p=0.019). In multivariate analysis, differences remained in the limit of the statistical significance (HR 2.16; 95% CI 1.00–4.67; p=0.051). No statistically significant association was found between DFS and the other polymorphisms that were studied. Conclusion Genotyping of exonic genetic variants in DPYD are related to DFS after capecitabine based adjuvant chemotherapy in CRC patients and could be a successful approach to find new pharmacogenetic predictors of tumour relapse. However, more studies in larger cohorts with a longer follow-up are needed. No conflict of interest