Robert Delage

The Impact of UGT1A8, UGT1A9, and UGT2B7 Genetic Polymorphisms on the Pharmacokinetic Profile of Mycophenolic Acid After a Single Oral Dose in Healthy Volunteers

We studied whether polymorphisms in the UGT1A8, UGT1A9, and UGT2B7 genes, the enzymes producing the phenolic (MPAG) and acyl (AcMPAG) glucuronides of mycophenolic acid (MPA), could contribute to the interindividual variation observed in mycophenolate mofetil (MMF) pharmacokinetics (PKs). This study enrolled 17 healthy volunteers with no polymorphisms (controls) and 17 carriers of UGT1A9 —275/–2152 selected among 305 individuals genetically screened for UDP‐glucuronosyltransferase (UGT) polymorphisms. Additional investigative groups included carriers of UGT1A8*2 (A173G) (n=9), UGT1A8*3 (C277Y) (n=4), and UGT1A9*3 (M33T) (n=5). Genetic analysis also included UGT2B7 to detect UGT2B7*2 (His268Tyr) and the promoter haplotype −1248A>G, −1241T>C, −1054T>C, −842G>A, −268A>G, −102T>C. Kinetics were measured in plasma and urine after a single 1.5 g oral dose of MMF, by high‐performance liquid chromatography coupled with tandem mass spectrometry, over 12 h after drug intake. Compared to controls, MPA exposure was significantly lower for UGT1A9 −275/−2152 carriers, with no significant changes in MPAG. The estimates of enterohepatic (re)cycling (area under the concentration–time curve (AUC6–12 h/AUC0–12 h)) were significantly lower for MPA, MPAG, and AcMPAG in UGT1A9 −275/−2152 subjects. Compared with controls, UGT1A9*3 carriers had higher MPA and AcMPAG exposure, whereas homozygosity for the UGT1A8*2 allele and heterozygosity for UGT1A8*3 allele had no impact on MPA PKs. Compared with UGT2B7*1/*1 individuals (n=10), UGT2B7*2/*2 subjects (n=17) presented significantly higher free MPA Cmax values and elevated free and total MPA. Results indicate that after a single oral dose of MMF in healthy volunteers, specific UGT genotypes significantly alter MPA PKs and this clearly warrants additional studies with complete and detailed genetic profiling of UGT1A8, UGT1A9, and UGT2B7 genes.

Pharmacokinetics of mycophenolate mofetil and its glucuronide metabolites in healthy volunteers

UNLABELLED We previously reported that polymorphisms in the UGT2B7 and UGT1A9 genes are associated with significant alteration in the disposition of mycophenolic acid (MPA) in healthy volunteers. AIM This study further evaluates the impact of genetic polymorphisms at the UGT1A1, UGT1A7 and ABCC2 loci. METHODS Genetic analyses of five UGT candidate genes and ABCC2 were completed on 47 healthy subjects who received a single dose of 1.5 g mycophenolate mofetil and completed a 12-h pharmacokinetic profile. RESULTS Multivariate analyses indicate that the ABCC2 -24T promoter polymorphism is associated with a 25% increase in acyl mycophenolic acid phenolic glucuronide level. Subjects with combined ABCC2 -24T and UGT1A9*3 genotypes present a 169% increased exposure to AcMPAG. Homozygosity for UGT1A7 387G/391A (129Lys/131Lys) is associated with a modest but significant 7% reduction in MPA level. When these additional genetic factors are considered in the model, the effects of previously described UGT1A9 and UGT2B7 variations remain significant. No significant effect is observed for UGT1A1*28, UGT1A7 622T/C (Trp208Arg), UGT1A9 -440TC/-331CT, UGT1A9 -118 TA(9/10) and seven other ABCC2 SNPs. CONCLUSION We demonstrate that MPA disposition is a multigenic process, and that additional studies are required to ascertain the relationship between UGT, ABCC2 genotypes and MPA pharmacokinetics in transplant recipients.

All Patients with Persistent Polyclonal B Cell Lymphocytosis Present Bcl-2/Ig Gene Rearrangements

The bcl-2 gene belongs to a class of oncogenes involved in the inhibition of apoptosis. Most follicular lymphomas are associated with the t(14;18) translocation that juxtaposes the bcl-2 gene located on chromosome 18 to the immunoglobulin gene locus located on chromosome 14. Consequently, the bcl-2 gene is overly expressed and leads to an accumulation of mature clonal B cells. Prolonged survival of the B cell clone appears to be the early event in tumorigenesis, creating an increased risk of cumulative mutations. Interestingly, bcl-2/Ig gene rearrangements may be identified in nearly 50% of normal individuals but the outcome of normal individuals carrying high levels of t(14;18) is not well defined. Persistent polyclonal B cell lymphocytosis (PPBL) is a unique polyclonal lymphoproliferative disorder mostly restricted to women. We have recently demonstrated that PPBL is also associated with multiple bcl-2/Ig gene rearrangements. In this report, we have extended our analysis to additional patients and demonstrated that all patients presented multiple detectable t(14;18) translocated clones. In addition, Bcl-2 protein expression was increased. Our findings, along with the clinical features of PPBL, make this disorder an exceptional model for the study of B-cell homeostasis.

Persistent polyclonal B-cell lymphocytosis : further evidence for a genetic disorder associated with B-cell abnormalities

Persistent polyclonal B‐cell lymphocytosis (PPBL) is an intriguing disorder diagnosed predominantly in women, usually cigarette smokers, characterized by an increase in the number of polyclonal B lymphocytes. Abnormality of the B‐cell population is also evidenced by the presence of multiple bcl‐2/Ig gene rearrangements and the finding of an additional long arm chromosome 3q+ (i3)(q10) within a significant proportion of B cells. The physiopathology of PPBL is unknown but its association with the HLA DR7 phenotype suggests a possible genetic disorder. To further determine whether PPBL has a genetic predisposition, we have undertaken an extensive study in a large family of a patient diagnosed with PPBL. Three individuals among the first‐degree relatives presented all the criteria for a diagnosis of PPBL. A slight increase in serum IgM without evidence of B‐cell proliferation was shown in two additional siblings. Multiple bcl‐2/Ig gene rearrangements, a typical feature of PPBL, were identified in 8/10 individuals among first‐degree relatives. A statistically significant association was found between the presence of these rearrangements and of a paternal HLA haplotype. We conclude that PPBL has a familial occurrence suggesting an underlying genetic defect. The development of the complete syndrome probably relies on unidentified additional co‐factors.

Multiple bcl‐2/Ig gene rearrangements in persistent polyclonal B‐cell lymphocytosis

Persistent polyclonal B‐cell lymphocytosis is a benign lymphoproliferative disorder of unknown aetiology occurring exclusively in women, characterized by typical binucleated lymphocytes, polyclonal expansion of B cells and elevated serum IgM. Owing to the role of Bcl‐2 oncogene in inhibition of apoptosis, we have investigated the presence of the bcl‐2/Ig gene rearrangement. Bcl‐2/Ig gene rearrangement was determined by polymerase chain reaction targeting the usual breakpoint regions of the t(14;18). Bcl‐2/Ig gene rearrangement was identified in all six patients and, more importantly, multiple rearrangements were present in five patients. The frequency of the bcl‐2/Ig gene rearrangement is estimated to be of one translocation in 1 × 102 to 1 × 103 peripheral blood mononuclear cells. We conclude that persistent polyclonal B‐cell lymphocytosis is associated with bcl‐2/Ig gene rearrangement. These findings are of clinical importance because these patients may be misdiagnosed as having a leukaemic expression of non‐Hodgkins lymphoma.

Lack of CD40‐dependent B‐cell proliferation in B lymphocytes isolated from patients with persistent polyclonal B‐cell lymphocytosis

Persistent B‐cell lymphocytosis (PPBL) is a haematological disorder diagnosed primarily in adult female smokers that is characterized by a polyclonal increase in peripheral blood B lymphocytes and a moderate elevation of serum IgM. B lymphocyte‐associated cellular abnormalities, such as the occurrence of multi‐lobed nuclei, increased bcl2/Ig gene rearrangements and the identification of an extra long‐arm chromosome (i3)(q10) in the B‐cell population, indicate that PPBL could be part of a multi‐step process leading to the emergence of a malignant B lymphoproliferation. However, the resulting impact on cellular functional properties remains to be elucidated. Our goal was to address that aspect via the study of B‐cell activity following stimulation through CD40, a key molecule of the tumour necrosis factor receptor superfamily involved in B lymphocyte development. In contrast to normal B cells, PPBL B lymphocytes were unable to respond to the proliferative signal delivered in vitro by CD40, indicating a defect in the CD40 activation pathway. Polymerase chain reaction amplification and sequencing of the receptor as well as FACScan analysis of patient B lymphocytes dismissed the possibility of a defect in either CD40 structure or expression. Moreover, Western blot analysis of tyrosine phosphorylation, an early event in the CD40‐signalling cascade, was similar in patients and controls, leading to the conclusion that the defect affecting B lymphocytes in PPBL patients is probably located downstream of that signalling cascade.

Autologous Hematopoietic Stem Cell Transplantation for Refractory Lymphomatoid Granulomatosis

Abstract Lymphomatoid granulomatosis (LG) is a rare lymphoproliferative disorder. There is no standard therapy for refractory patient. Here we present the case of a patient with LG of the lung and the brain who was refractory to polychemotherapy. An autologous hematopoietic stem cell transplantation was done and the patient achieved a complete remission. This represents the first case of high-dose chemotherapy with hematopoietic stem cell support in this disease.

Analysis of expressed VH genes in persistent polyclonal B cell lymphocytosis reveals absence of selection in CD27+IgM+IgD+ memory B cells

Persistent polyclonal B cell lymphocytosis (PPBL) is a hematological disorder diagnosed predominantly in women, characterized by a polyclonal increase in the number of peripheral blood B lymphocytes. Abnormality of the B cell population was evidenced by the finding of multiple bcl‐2/Ig gene rearrangements and an additional long‐arm chromosome within a significant proportion of B cells. To gain further insight about the developmental status of B lymphocytes in PPBL, analysis of cell surface Ig receptors was undertaken. An important expansion of the CD27+IgM+IgD+ B cell population was noted in PPBL patients (n=4). When investigated by PCR, pattern of heavy chain variable region (VH) genes usage in patients (n=6) was shown tobe similar to that observed in healthy individuals (n=3). In‐depth investigation was then conducted through cloning and sequencing of individual VH genes in three of those patients. They were mostly found to be mutated (21/29), correlating with the observed increase in CD27 expression, a marker of memory B cells. Altogether, these data clearly point out to the exact nature of the expanding B cell subset in patients. Finally, analysis of the repartition of recombinant versus silent mutations in framework regions (FR) of Ig genes showed no evidence of positive antigenic selection following somatic hypermutation. Thus, we suggest that a lack of response to physiological signals responsible for the elimination of low affinity memory IgM+IgD+ B cells in germinal centers could play an important role in the development of PPBL.

Overall survival in lower IPSS risk MDS by receipt of iron chelation therapy, adjusting for patient‐related factors and measuring from time of first red blood cell transfusion dependence: an MDS‐CAN analysis

Analyses suggest iron overload in red blood cell (RBC) transfusion‐dependent (TD) patients with myleodysplastic syndrome (MDS) portends inferior overall survival (OS) that is attenuated by iron chelation therapy (ICT) but may be biassed by unbalanced patient‐related factors. The Canadian MDS Registry prospectively measures frailty, comorbidity and disability. We analysed OS by receipt of ICT, adjusting for these patient‐related factors. TD International Prognostic Scoring System (IPSS) low and intermediate‐1 risk MDS, at RBC TD, were included. Predictive factors for OS were determined. A matched pair analysis considering age, revised IPSS, TD severity, time from MDS diagnosis to TD, and receipt of disease‐modifying agents was conducted. Of 239 patients, 83 received ICT; frailty, comorbidity and disability did not differ from non‐ICT patients. Median OS from TD was superior in ICT patients (5·2 vs. 2·1 years; P < 0·0001). By multivariate analysis, not receiving ICT independently predicted inferior OS, (hazard ratio for death 2·0, P = 0·03). In matched pair analysis, OS remained superior for ICT patients (P = 0·02). In this prospective, non‐randomized analysis, receiving ICT was associated with superior OS in lower IPSS risk MDS, adjusting for age, frailty, comorbidity, disability, revised IPSS, TD severity, time to TD and receiving disease‐modifying agents. This provides additional evidence that ICT may confer clinical benefit.

Hematopoietic stem cell transplantation in patients with lymphomatoid granulomatosis: a European group for blood and marrow transplantation report.

Lymphomatoid granulomatosis (LG) is a very rare, Epstein-Barr virus-associated lymphoproliferative disorder of B cells. Prognosis is poor, particularly after relapse and no curative treatment exists. We report the results of high-dose therapy and autologous stem cell transplantation (ASCT) or reduced-intensity conditioning and allogeneic stem cell transplantation (alloSCT) in patients with multiply relapsed LG. A European Group for Blood and Marrow Transplantation survey identified 10 patients who had received 9 ASCT and 4 alloSCT. All patients had active disease at the time of transplantation. With a median follow-up of 5.1 (range, 1.4 to 6.3) years, 6 patients are alive and disease-free. Two ASCT patients died of septicemia early after transplantation, and 1 committed suicide after being in continuous complete remission 19 months after ASCT. Another patient allografted 4 years after ASCT remained disease-free but died of severe graft-versus-host disease 3 months after alloSCT. High-dose therapy followed by ASCT and alloSCT are effective therapeutic options and should be considered in all patients with refractory and multiply relapsed LG.