Sarah Jenkinson

Outcome for children and young people with Early T‐cell precursor acute lymphoblastic leukaemia treated on a contemporary protocol, UKALL 2003

We investigated the outcome for children and young people with Early T‐precursor acute lymphoblastic leukaemia (ETP‐ALL), a recently described poor prognosis sub‐group of T‐ALL, treated on a contemporary protocol, UKALL 2003. After a median follow‐up of 4 years and 10 months, the ETP sub‐group, representing 16% of T‐ALL patients, had non‐significantly inferior 5‐year event‐free survival (76·7% vs. 84·6%, P = 0·2) and overall survival (82·4% vs. 90·9%, P = 0·1), and a higher relapse rate (18·6% vs. 9·6%, P = 0·1) compared to typical T‐ALL. ETP‐ALL has an intermediate risk outcome, which does not warrant experimental treatment or first remission allogeneic transplant for the group universally.

Prognostic Implications of NOTCH1 and FBXW7 Mutations in Adults With T-Cell Acute Lymphoblastic Leukemia Treated on the MRC UKALLXII/ECOG E2993 Protocol

PURPOSEnNotch pathway activation by mutations in either NOTCH1 and/or FBXW7 is one of the most common molecular events in T-cell acute lymphoblastic leukemia (T-ALL) and, in pediatric disease, predicts for favorable outcome. Their prognostic significance in adult T-ALL is unclear. We sought to evaluate the outcome according to mutation status of patients with adult T-ALL treated on the United Kingdom Acute Lymphoblastic Leukaemia XII (UKALLXII)/Eastern Cooperative Oncology Group (ECOG) E2993 protocol.nnnMETHODSnNOTCH1 and FBXW7 were screened by a combination of denaturing high-performance liquid chromatography and sequencing in 88 adult patients with T-ALL treated on the UKALLXII/ECOG E2993 protocol and compared with clinical characteristics and outcome.nnnRESULTSnNOTCH1 and FBXW7 mutations were common (60% and 18%, respectively) and were not associated with age or WBC count. NOTCH1 heterodimerization domain mutations were associated with FBXW7 mutations (P = .02), and NOTCH1 proline, glutamic acid, serine, threonine (PEST) rich domain and FBXW7 mutations were mutually exclusive. There were an equal number of high- and standard-risk patients in the NOTCH1 and FBXW7 mutated (MUT) groups. Patients wild type (WT) for both markers trended toward poorer event-free survival (EFS; MUT v WT, 51% v 27%, P = .10; hazard ratio, 0.6). Analysis by each marker individually was not significantly predictive of outcome (NOTCH1 MUT v WT, EFS 49% v 34%, P = .20; FBXW7 MUT v WT, EFS 53% v 41%, P.72).nnnCONCLUSIONnNOTCH1 and FBXW7 mutant-positive patients do not fare sufficiently well to warrant an individualized treatment approach in future studies.

The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule

Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates dl-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.

Impact of NOTCH1/FBXW7 mutations on outcome in pediatric T-cell acute lymphoblastic leukemia patients treated on the MRC UKALL 2003 trial

Activating mutations in the NOTCH1 pathway are frequent in pediatric T-cell acute lymphoblastic leukemia (T-ALL) but their role in refining risk stratification is unclear. We screened 162 pediatric T-ALL patients treated on the MRC UKALL2003 trial for NOTCH1/FBXW7 gene mutations and related genotype to response to therapy and long-term outcome. Overall, 35% were wild-type (WT) for both genes (NOTCH1WTFBXW7WT), 38% single NOTCH1 mutant (NOTCH1SingleFBXW7WT), 3% just FBXW7 mutant (NOTCH1WTFBXW7MUT) and 24% either double NOTCH1 mutant (NOTCH1DoubleFBXW7WT) or mutant in both genes (NOTCH1MUTFBXW7MUT), hereafter called as NOTCH1±FBXW7Double. There was no difference between groups in early response to therapy, but NOTCH1±FBXW7Double patients were more likely to be associated with negative minimal residual disease (MRD) post-induction than NOTCH1WTFBXW7WT patients (71% versus 40%, P=0.004). Outcome improved according to the number of mutations, overall survival at 5 years 82%, 88% and 100% for NOTCH1WTFBXW7WT, NOTCH1SingleFBXW7WT and NOTCH1±FBXW7Double patients, respectively (log-rank P for trend=0.005). Although 14 NOTCH1±FBXW7Double patients were classified as high risk (slow response and/or MRD positive), only two had disease progression and all remain alive. Patients with double NOTCH1 and/or FBXW7 mutations have a very good outcome and should not be considered for more intensive therapy in first remission, even if slow early responders or MRD positive after induction therapy.

Simpson's Paradox and the Impact of Different DNMT3A Mutations on Outcome in Younger Adults With Acute Myeloid Leukemia

PURPOSEnTo evaluate the impact of DNMT3A mutations on outcome in younger patients with cytogenetic intermediate-risk acute myeloid leukemia.nnnPATIENTS AND METHODSnDiagnostic samples from 914 patients (97% < 60 years old) were screened for mutations in DNMT3A exons 13 to 23. Clinical outcome was evaluated according to presence or absence of a mutation and stratified according to type of mutation (R882, non-R882 missense, or truncation).nnnRESULTSnDNMT3A mutations (DNMT3A(MUT)) were identified in 272 patients (30%) and associated with a poorer prognosis than wild-type DNMT3A, but the difference was only seen when the results were stratified according to NPM1 genotype. This example of Simpsons paradox results from the high coincidence of DNMT3A and NPM1 mutations (80% of patients with DNMT3A(MUT) had NPM1 mutations), where the two mutations have opposing prognostic impact. In the stratified analyses, relapse in patients with DNMT3A(MUT) was higher (hazard ratio, 1.35; 95% CI, 1.07 to 1.72; P = .01), and overall survival was lower (hazard ratio, 1.37; 95% CI, 1.12 to 1.87; P = .002). The impact of DNMT3A(MUT) did not differ according to NPM1 genotype (test for heterogeneity: relapse, P = .4; overall survival, P = .9). Further analysis according to the type of DNMT3A mutation indicated that outcome was comparable in patients with R882 and non-R882 missense mutants, whereas in those with truncation mutants, it was comparable to wild-type DNMT3A.nnnCONCLUSIONnThese data confirm that presence of a DNMT3A mutation should be considered as a poor-risk prognostic factor, irrespective of the NPM1 genotype, and suggest that further consideration should be given to the type of DNMT3A mutation.

Impact of PTEN abnormalities on outcome in pediatric patients with T-cell acute lymphoblastic leukemia treated on the MRC UKALL2003 trial

PTEN gene inactivation by mutation or deletion is common in pediatric T-cell acute lymphoblastic leukemia (T-ALL), but the impact on outcome is unclear, particularly in patients with NOTCH1/FBXW7 mutations. We screened samples from 145 patients treated on the MRC UKALL2003 trial for PTEN mutations using heteroduplex analysis and gene deletions using single nucleotide polymorphism arrays, and related genotype to response to therapy and long-term outcome. PTEN loss-of-function mutations/gene deletions were detected in 22% (PTENABN). Quantification of mutant level indicated that 67% of mutated cases harbored more than one mutant, with up to four mutants detected, consistent with the presence of multiple leukemic sub-clones. Overall, 41% of PTENABN cases were considered to have biallelic abnormalities (mutation and/or deletion) with complete loss of PTEN in a proportion of cells. In addition, 9% of cases had N- or K-RAS mutations. Neither PTEN nor RAS genotype significantly impacted on response to therapy or long-term outcome, irrespective of mutant level, and there was no evidence that they changed the highly favorable outcome of patients with double NOTCH1/FBXW7 mutations. These results indicate that, for pediatric patients treated according to current protocols, routine screening for PTEN or RAS abnormalities at diagnosis is not warranted to further refine risk stratification.

Renal allograft rejection: Examination of delayed differentiation of Treg and Th17 effector T cells

Antigen presentation after kidney transplantation occurs in lymphoid tissues remote from the allograft, with activated T cells then migrating towards the graft. This study examined the possibility that these activated T cells can differentiate to acquire Th17 or Treg phenotypes after a time consistent with their arrival within renal allograft tissues. An immunocytochemical study was performed to demonstrate the response to intragraft TGF-β and the phenotype of lymphoid cells within rejecting human renal allograft tissue. A series of in vitro experiments was then performed to determine the potential to induce these phenotypes by addition of appropriate cytokines 3days after initial T cell activation. During renal allograft rejection there was a strong response to TGF-β, and both FOXP3 and IL-17A were expressed by separate lymphoid cells in the graft infiltrate. FOXP3 could be induced to high levels by the addition of TGF-β1 3days after the initiation of allogeneic mixed leukocyte culture. This Treg marker was enriched in the sub-population of T cells expressing the cell-surface αE(CD103)β7 integrin. The RORγt transcription factor and IL-17A were induced 3days after T cell activation by the addition of TGF-β1, IL-1β, IL-6 and IL-23; many of these Th17 cells also co-expressed CD103. T cells can develop an effector phenotype following cytokine stimulation 3days after initial activation. This suggests that the intragraft T cell phenotype may be indicative of the prevailing cytokine microenvironment.

Kidney transplantation: analysis of the expression and T cell-mediated activation of latent TGF-β

Activated T cells infiltrate a renal allograft during rejection and can respond to TGF‐β within the tubules, causing local differentiation and expression of the αE(CD103)β7 integrin. This study was performed to examine the expression of latent TGF‐β within renal allograft tissues and to define a mechanism by which T cells can activate and respond to this latent factor. Rejecting renal allograft biopsy tissues showed increased expression of the latent TGF‐β complex, which was localized around the tubules by a mechanism that might involve interaction with heparan sulfate in the basement membrane. A cultured renal TEC line also expressed the latent complex, but these cells did not respond to this form of TGF‐β by pSmad 3. However, coculture of these cells with activated T cells induced the expression of CD103, suggesting that T cells can activate and respond to the latent TGF‐β associated with TEC. Although activated T cells expressed little cell‐surface TSP‐1, this was increased by culture with fibronectin or fibronectin‐expressing renal TEC. Blockade of TSP‐1 using LSKL peptides reduced the potential of activated T cells to differentiate in response to latent TGF‐β. This study suggests that penetration of renal tubules by activated T cells leads to increased expression of T cell‐surface TSP‐1, allowing activation of latent TGF‐β sequestered on heparan sulfate within the microenvironment. This mechanism may be important for localized phenotypic maturation of T cells that have infiltrated the kidney during allograft rejection.

Prognostic impact of the absence of biallelic deletion at the TRG locus for pediatric patients with T-cell acute lymphoblastic leukemia treated on the Medical Research Council UK Acute Lymphoblastic Leukemia 2003 trial

Risk-stratification and treatment intensification according to minimal residual disease (MRD) analysis has improved outcomes of patients with acute lymphoblastic leukemia (ALL).[1][1],[2][2] However, a significant proportion of patients with T-cell ALL (T-ALL) still experience early relapse or

Chapter 4:Drug Transporters in the Kidney

With a high expression of both uptake and efflux transporters, together with metabolic enzymes, the proximal tubule in the kidney plays a major role in determining the absorption, distribution, metabolism and elimination of a wide range of molecules. Since most members of the solute carrier and ATPase binding cassette families that transport drug molecules in the kidney have broad substrate specificity, there is a need to identify clinically important transporter mediated drug–drug interactions that may result in nephrotoxicity. To address this, efforts have been made to elucidate the mechanisms of drug–drug interactions and toxicity and better understand renal drug transport. The importance of transporters in the kidney has led regulatory agencies around the world to mandate drug–drug interaction and nephrotoxicity safety studies for new molecular entities that have substantial renal elimination. This review summarises the key data on the identification and characterisation of transporters found in the proximal tubule of the kidney. Differences and similarities in transporter expression and function between human and rodent species are also discussed. In addition, current renal in vitro models are explored, along with recent developments in this area.