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Maspin is a non-inhibitory member of the serpin family that affects cell behaviours related to migration and survival. We have previously shown that peptides of the isolated G α-helix (G-helix) domain of maspin show bioactivity. Migration, invasion, adhesion and proliferation of vascular smooth muscle cells (VSMC) are important processes that contribute to the build-up of atherosclerotic plaques. Here we report the use of functional assays of these behaviours to investigate whether other maspin-derived peptides impact directly on VSMC; focusing on potential anti-atherogenic properties. We designed 18 new peptides from the structural moieties of maspin above ten amino acid residues in length and considered them beside the existing G-helix peptides. Of the novel peptides screened those with the sequences of maspin strand 4 and 5 of beta sheet B (S4B and S5B) reduced VSMC migration, invasion and proliferation, as well as increasing cell adhesion. A longer peptide combining these consecutive sequences showed a potentiation of responses, and a 7-mer contained all essential elements for functionality. This is the first time that these parts of maspin have been highlighted as having key roles affecting cell function. We present evidence for a mechanism whereby S4B and S5B act through ERK1/2 and AMP-activated protein kinase (AMPK) to influence VSMC responses.

Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

T cells activate latent TGF-β by an LSKL peptide-sensitive mechanism, suggesting a role for thrombospondin-1 in T cell differentiation after kidney transplantation. 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.

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.

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.

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.

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 (NOTCH1(WT)FBXW7(WT)), 38% single NOTCH1 mutant (NOTCH1(Single)FBXW7(WT)), 3% just FBXW7 mutant (NOTCH1(WT)FBXW7(MUT)) and 24% either double NOTCH1 mutant (NOTCH1(Double)FBXW7(WT)) or mutant in both genes (NOTCH1(MUT)FBXW7(MUT)), hereafter called as NOTCH1±FBXW7(Double). There was no difference between groups in early response to therapy, but NOTCH1±FBXW7(Double) patients were more likely to be associated with negative minimal residual disease (MRD) post-induction than NOTCH1(WT)FBXW7(WT) 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 NOTCH1(WT)FBXW7(WT), NOTCH1(Single)FBXW7(WT) and NOTCH1±FBXW7(Double) patients, respectively (log-rank P for trend=0.005). Although 14 NOTCH1±FBXW7(Double) 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.

We investigated the benefit of adding all-trans retinoic acid (ATRA) to chemotherapy for younger patients with nonacute promyelocytic acute myeloid leukemia and high-risk myelodysplastic syndrome, and considered interactions between treatment and molecular markers. Overall, 1075 patients less than 60 years of age were randomized to receive or not receive ATRA in addition to daunorubicin/Ara-C/thioguanine chemotherapy with Ara-C at standard or double standard dose. There were data on FLT3 internal tandem duplications and NPM1 mutations (n = 592), CEBPA mutations (n = 423), and MN1 expression (n = 195). The complete remission rate was 68% with complete remission with incomplete count recovery in an additional 16%; 8-year overall survival was 32%. There was no significant treatment effect for any outcome, with no significant interactions between treatment and demographics, or cytarabine randomization. Importantly, there were no interactions by FLT3/internal tandem duplications, NPM1, or CEBPA mutation. There was a suggestion that ATRA reduced relapse in patients with lower MN1 levels, but no significant effect on overall survival. Results were consistent when restricted to patients with normal karyotype. ATRA has no overall effect on treatment outcomes in this group of patients. The study did not identify any subgroup of patients likely to derive a significant survival benefit from the addition of ATRA to chemotherapy. This study is registered at http://www.controlled-trials.com under ISRCTN17833622.

Purpose: To investigate the phytochemical uptake following human consumption of Montmorency tart cherry (L. Prunus Cerasus) and influence of selected phenolic acids on vascular smooth muscle cells in vitro. \ud Methods: In a randomized, double blinded, cross-over design, 12 healthy males consumed either 30 or 60 mL of Montmorency tart cherry concentrate. Following analysis of the juice composition, venous blood samples were taken before and 1, 2, 3, 5 and 8 h post consumption of the beverage. In addition to examining some aspects of the concentrate contents, plasma concentrations of protocatechuic (PCA), vanillic (VA) and chlorogenic acid (CHL) were analysed by reversed–phase high performance liquid chromatography (HPLC) with diode array for quantitation and mass spectrometry detection (LCMS) for qualitative purposes. Vascular smooth muscle cell migration and proliferation were also assessed in vitro. \ud Results Both the 30 mL and 60 mL doses of Montmorency cherry concentrate contained high amounts of total phenolics (71.37 ± 0.11; 142.73 ± 0.22 mg˙L¯1) and total anthoycanins (62.47 ± 0.31; 31.24 ± 0.16 mg˙L¯1), as well as large quantities of CHL (0.205 ± 0.24; 0.410 ± 0.48 mg˙L¯1) and VA (0.253 ± 0.84; 0.506 ± 1.68 mg˙L¯1). HPLC/LCMS identified two dihydroxybenzoic acids (PCA and VA) in plasma following MC concentrate consumption. Both compounds were most abundant 1-2 h post initial ingestion with traces detectable at 8 h post ingestion. Cell migration was significantly influenced by the combination of PCA and VA, but not in isolation. There was no effect of the compounds on cell proliferation. \ud Conclusions: These data show new information that phenolic compounds thought to exert vasoactive properties are bioavailable in vivo following MC consumption, and subsequently can influence cell behaviour. These data may be useful for the design and interpretation of intervention studies investigating the health effects of Montmorency cherries.

Summary: 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. © 2014 John Wiley and Sons Ltd.

Purpose Notch 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. Methods NOTCH1 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. Results NOTCH1 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). Conclusion NOTCH1 and FBXW7 mutant-positive patients do not fare sufficiently well to warrant an individualized treatment approach in future studies.

The molecular mechanisms involved in disease progression and relapse in T-cell acute lymphoblastic leukemia (T-ALL) are poorly understood. We used single nucleotide polymorphism array analysis to analyze paired diagnostic and relapsed T-ALL samples to identify recurrent genetic alterations in T-ALL. This analysis showed that diagnosis and relapsed cases have common genetic alterations, but also that relapsed samples frequently lose chromosomal markers present at diagnosis, suggesting that relapsed T-ALL emerges from an ancestral clone different from the major leukemic population at diagnosis. In addition, we identified deletions and associated mutations in the WT1 tumor suppressor gene in 2 of 9 samples. Subsequent analysis showed WT1 mutations in 28 of 211 (13.2%) of pediatric and 10 of 85 (11.7%) of adult T-ALL cases. WT1 mutations present in T-ALL are predominantly heterozygous frameshift mutations resulting in truncation of the C-terminal zinc finger domains of this transcription factor. WT1 mutations are most prominently found in T-ALL cases with aberrant rearrangements of the oncogenic TLX1, TLX3, and HOXA transcription factor oncogenes. Survival analysis demonstrated that WT1 mutations do not confer adverse prognosis in pediatric and adult T-ALL. Overall, these results identify the presence of WT1 mutations as a recurrent genetic alteration in T-ALL.

DNMT3A mutations (DNMT3AMUT) are recurrent in AML. They predominate in patients with intermediate-risk (IR) cytogenetics and are often co-incident with FLT3ITD and NPM1MUT. Their prognostic impact is unclear. Most reports suggest they are associated with a worse outcome, but a large study including 1060 younger adult IR patients found that DNMT3AMUT had no significant impact on survival endpoints. Variable results have also been reported for different FLT3/NPM1 subgroups. Missense mutations at R882 in exon 23 occur in ≈65% of patients, but other missense and truncation mutations occur throughout the gene, mainly in exons 13-23. There is limited information on the prognostic impact of the different mutations, although they may have differing functional consequences. We therefore screened exons 13-23 in DNA samples from 914 younger adult AML patients (median age 43 years) with IR cytogenetics treated on UK MRC trials and evaluated outcome according to type of DNMT3Amutation. Overall, 278 mutations were detected in 272 (30%) patients; 175 (64%) had R882 missense mutations, 59 (22%) other missense mutations, 35 (13%) truncations or in-frame deletions; 3 (1%) had 2 mutations of differing types. Median R882 mutant level in 172 mutated cases was 47% (range 15-85%), consistent with a heterozygous mutation in most cells. Patients with DNMT3AMUT were significantly older than those with DNMT3A wild-type (DNMT3AWT) (P Presence of DNMT3AMUT was associated with a poorer prognosis, but this difference was only seen if the results were analyzed separately according to NPM1 genotype, where DNMT3AMUT was associated with higher cumulative incidence of relapse (CIR) than DNMT3AWT in cases with NPM1MUT (49% vs 40%, P=.01) and NPM1WT (61% vs 58%, P=.5) genotype. Similarly, DNMT3AMUT patients had worse overall survival (OS) than DNMT3AWT patients with NPM1MUT (38% vs 50%, P=.008) and NPM1WT (15% vs 25%, P=.09) genotype. This statistical anomaly is an example of Simpson’s paradox. It results from the strong co-incidence of DNMT3A and NPM1 mutations with opposing prognostic associations that mask the effect seen separately when the groups are combined. Although the differences were smaller for NPM1WT cases, tests for heterogeneity showed that the impact of a DNMT3A mutation did not differ between NPM1MUT and NPM1WT for either CIR or OS, nor between the 4 genotypes defined by the combination of NPM1 and FLT3ITD genotypes. In multivariable analysis including age, WBC, NPM1 and FLT3ITD, DNMT3AMUT was a significant adverse risk factor for CIR (HR=1.27, CI=1.01-1.61; P=.04), and showed a trend for being adverse for OS (HR=1.19, CI=.98-1.45; P=.08). When outcome was considered according to the type of mutation (R882, other missense or truncations), for the NPM1MUT genotype cases CIR was highest in R882 and other missense cases (51%, 50%) and truncation cases were similar to DNMT3AWT (35%, 40%). For NPM1WT, CIR was highest in R882 cases (76%), similar in other missense and DNMT3AWT cases (55%, 58%) and lowest in truncation cases (40%). Consistent with this data, for NPM1MUT genotype, OS was lowest in R882 and other missense cases (35%, 38%), better in DNMT3AWT (50%) and highest in truncation cases (57%). For NPM1WT, OS was lowest in R882 cases (11%), and similar in DNMT3AWT, other missense and truncation cases (25%, 21%, 18% respectively). These data suggest that screening cannot be limited to the hotspot R882 mutations and that cases with missense mutations should be treated as poor risk, including those patients currently considered as favorable risk such as NPM1MUTFLT3WT. Conversely, truncation mutations have a different functional impact from missense mutations, more likely to result in haploinsufficiency than a dominant-negative effect, and these cases should be considered as equivalent to DNMT3AWT for prognostication and selection of therapy in 1st remission. Disclosures No relevant conflicts of interest to declare.

In AML, the favorable prognosis associated with mutations in the CEBPA gene is restricted to those cases with double CEBPA mutations (CEBPADM), consistent with the loss of normal CEBP/alpha activity from both alleles. Current recommendations are that CEBPADM-mutated patients should not receive a stem cell transplant in first remission. In general, these cases have 2 ‘classical’ mutations, an N-terminal out-of-frame insertion/deletion that leads to loss of the full-length p42 protein and increased levels of the p30 isoform translated from an internal start site, coupled with a C-terminal in-frame insertion/deletion in the DNA binding domain (DBD) or leucine zipper domain (LZD) that interferes with DNA binding or dimerization. However, in our study of 1427 younger adult patients, 26% of mutations did not fit this classical description due to either the location or type of mutation. Furthermore, of the CEBPADM cases, 20% had a classical plus a ‘non-classical’ mutation or a homozygous non-classical mutation. It will be important to understand the functional consequences of these atypical mutations if CEBPA genotype is to be used to determine patient management. As methylation profiling has shown that CEBPADM cases form a distinct hypermethylated cluster, we investigated whether this can provide information about non-classical cases. A test set of 40 diagnostic samples were analyzed on the Illumina Infinium 27K Human Methylation Array, all normal karyotype with wild type (WT) NPM1, FLT3ITD and FLT3TKD; 10 were CEBPADM, 30 CEBPAWT. Unsupervised cluster analysis showed that the 10 CEBPADM cases clustered within a group of 16 hypermethylated cases that separated from 24 hypomethylated cases. A methylation signature was created from the 25 most-differentially methylated CpG sites between the CEBPADM and CEBPAWT cases and used to examine a validation set of 95 samples analyzed on the Illumina Infinium 450K Human Methylation Array (31 CEBPADM, 38 single-mutated CEBPA [CEBPASM], 26 CEBPAWT). This included 38 cases with non-classical mutations, 14 of them CEBPADM. On unsupervised cluster analysis, most CEBPADM cases (81%) fell in a hypermethylated group that was distinct from CEBPASM and CEBPAWT cases, with no segregation between the latter. We derived a genotype predictor by comparing the % methylation in a sample at each of the 25 CpG sites with that in the CEBPADM and CEBPAWT signatures to determine which signature the sample data most approximated. This correctly predicted 25/31 (81%) of the CEBPADM cases, including 2 with missense DBD/LZD mutations (A295P, N321S) coupled with a classic N or C mutation, 2 with homozygous classic C mutations, indicating that presence of the p30 isoform is not required for the methylation profile, and 5 with a classic N mutation coupled with a truncating mutation in the middle of the gene, consistent with the presence of the p30 isoform alone. This data was supported by functional evaluation of mutant constructs in a luciferase reporter assay to assess DNA binding and transactivation activity (TA). Classic CEBPADM constructs all had significantly lower TA than CEBPAWT (mean 12%, 27%, 15% of CEBPAWT for homozygous N, homozygous C and N+C constructs). Combination of a classic N mutation with missense DBD mutations (A295P, R297P, R300P), a LZD truncation (K313fs) or a middle region truncation (Q209fs, A238fs), all led to ≤15% activity consistent with almost complete loss of CEBP/alpha activity. Of the 6 CEBPADM cases that did not cluster as expected, 1 with classic N+C mutations had a lower mutant level (mean 28% for the 2 mutations compared to 45% for 9 other pairs with available data) and 1 had a homozygous missense LZD mutation that did not show reduced TA that could explain the discrepancy. The other 4 all had high mutant level (mean level ≥39%) and biallelic mutations as assessed by cloning, and relevant constructs showed low TA (≤19%). The reason for their misclassification is therefore not apparent, although we cannot exclude the possibility of other coincident mutations influencing methylation. These data indicate that the hypermethylated profile associated with CEBPADM cases holds true for most of the CEBPA mutations identified in patients and can be used to support predicted functional consequence of the mutations. This may be particularly useful in determining management in CEBPADM cases with non-classical mutations. Disclosures No relevant conflicts of interest to declare.

Although outcome has improved for pediatric patients with T-ALL, ≈25% of cases relapse and prognosis post-relapse remains poor. Molecular characterisation at diagnosis can provide additional information for risk-stratification. We previously reported that patients with double NOTCH1 or NOTCH1+FBXW7 mutations (NOTCH1±FBXW7Double) have a very good outcome and should not be considered for more intensive therapy in first remission, even if slow responders or MRD-positive after induction therapy. However, recent studies have suggested that this may be modulated by the presence of coincident abnormalities such as in the PTEN gene. Truncating mutations and genomic loss of this gene have been described in T-ALL, but their prognostic impact in patients is unclear, with reports of either no effect or reduced event-free survival (EFS). Furthermore, subgroup analysis has shown that the adverse impact of a PTEN mutation is either not seen in the presence of a NOTCH1 mutation or, conversely, that it ablates the benefit of a NOTCH1 mutation. In order to determine whether these abnormalities impact on the good outcome seen in NOTCH1±FBXW7Double cases in our cohort, and whether they can refine stratification of cases with single NOTCH1 mutations (NOTCH1Single) or wild-type NOTCH1 (NOTCH1WT), we investigated PTEN genotype in samples from 145 patients treated on the MRC UKALL2003 trial and correlated this with outcome in the different subgroups. The entire coding region (exons 1-9) of the PTEN gene was screened for mutations using heteroduplex analysis. Samples with abnormal chromatograms were further investigated. Mutations were detected in 21 patients (14%); 17 (81%) had exon 7 mutations, 2 exon 6+7, and 2 exon 5 mutations. All were small insertions, deletions or indels; 89% were predicted to lead to C-terminal truncation and loss of protein function, 11% were in-frame size changes. Exon 7 mutant levels were quantified by size analysis in 19 patients; median total mutant level was 48% of all PTEN alleles (range, 10%-96%). Of note, in the 21 mutated cases, only 7 (33%) had a single mutation; 8 had 2, 3 had 3, and 3 had 4 mutations. Based on total mutant level, 11 cases were considered to have monoallelic (heterozygous) mutations and 10 cases biallelic (homozygous/hemizygous or compound heterozygous) mutations. To investigate loss of genomic material, Illumina CytoSNP-850k SNP array analysis was performed on all samples. Partial or complete loss of the PTEN gene was detected in 15 patients (10%), 12 with heterozygous and 3 homozygous loss. This data was consistent with quantitative analysis of the relative allele levels of two common intronic SNPs (rs1903858 and rs555895) studied in 76 informative patients, which indicated that in 2 of 6 informative cases with heterozygous loss, only the 3’ end of the gene was deleted. Putting together the mutation and SNP data, 32 patients (22%) had abnormalities in the PTEN gene (PTENABN), 19 (59%) scored as monoallelic (PTENMONO), and 13 (41%) biallelic (PTENBI). There was no significant difference according to overall PTEN genotype in either early response to therapy (P>.99) or MRD status at day 29 of induction therapy (P=.28). Long-term outcome also did not significantly differ, EFS at 5 years was 78% in PTENABN and 85% in PTENWT patients (P=.37), overall survival (OS) 81% and 91% respectively (P=.1). These results did not change if grouped according to PTEN type, EFS 74% in PTENMONO and 85% in PTENBI patients (P=.46), although the number of patients in these groups was very small. The incidence of PTEN abnormalities did not differ according to NOTCH1/FBXW7 genotype, 59% PTENABN patients had a NOTCH1/FBXW7 mutation compared to 67% PTENWT patients (P=.7). There was no evidence that PTEN genotype impacted on the favorable outcome of the NOTCH1±FBXW7Double group, none of the 5 PTENABNNOTCH1±FBXW7Double patients relapsed and all remain alive. Similarly, no significant difference was observed in the NOTCH1Single and NOTCH1WT patients. In conclusion, although we found that loss of PTEN through either gene mutation or genomic deletion was relatively common in pediatric T-ALL patients, including total loss of PTEN in nearly one-half of mutated patients, this appeared to have no effect on either response to treatment or long-term outcome with current therapies, and therefore screening of PTEN is not warranted in pediatric T-ALL for potential use in risk-adapted therapy. Disclosures No relevant conflicts of interest to declare.

Introduction The STIL-TAL1 fusion is found in 16% cases of paediatric and adolescent T-ALL, making it one of the most common T-ALL subgroups. Our study considers this leukaemia subtype in the context of a complex ecosystem that is diverse, evolving and subject to selective pressures. We used single cell methods to understand the order of co-operating mutational events and the clonal evolution of mutations in genes that are re-iteratively targeted, such as PTEN. Methods Diagnostic DNA from five STIL-TAL1 positive T-ALL cases was exome sequenced using Agilent SureSelect Human all Exon kit plus Illumina paired end sequencing. Driver copy number alterations and NOTCH1/PTEN exon 7 mutation status had been identified in a previous study and candidate driver mutations for inclusion in single cell experiments were validated by sequencing or Q-PCR using custom assays. Where more than one mutation was present within the same exon of a candidate driver gene, cloning experiments were carried out to verify the independent mutation sequences. Material from xenograft transplants was available in three of the five cases to assess their clonal heterogeneity in the leukaemia initiating cell compartment. Single cell multiplex Q-PCR was used to examine the single cell genetics of the pre-defined mutation events. Briefly, single cells were sorted and lysed prior to multiplex specific (DNA) target amplification and Q-PCR using the 96.96 dynamic microfluidic array and the BioMark HD (Fluidigm, UK). Copy number assays for the 1p33 deletion and custom assays for the patient specific STIL-TAL1 fusion breakpoints were used to confirm that the 1p33 deletion leading to this gene fusion was a clonal event. Results The only aberrant events common to all five samples were CKDN2A copy number loss and the 1p33 deletion that results in the STIL-TAL1 fusion. Exome sequencing revealed further mutations in known T-ALL drivers including NOTCH1, PTEN and PHF6 as well as candidate driver mutations in FREM2, PIK3CD, RPL14, BMPR1A and CDH18. Both NOTCH1 and PTEN demonstrated re-iterative inactivation and this was investigated in detail for PTEN. Case 1 had multiple PTEN exon 7 mutations and sub-clonal copy number loss. Case 2 had parallel frameshift mutations in PTEN exons 5 and 7. Case 3 contained an exon 8 mutation and multiple PTEN exon 7 mutations. In this case the three most frequent PTEN exon 7 indels were validated and tracked in a single cell multiplex Q-PCR experiment. This revealed a branching sub-clonal genetic architecture (see figure 1) in which all malignant cells at the proposed apex of the branching architecture harboured the STIL-TAL1 fusion and CDKN2A deletion with copy number losses of 4p, 6q and FREM2 and PTEN mutations occurring as sub-clonal events. PTEN indels 2 and 3 were found co-localised in the same sub-clone. Preliminary analysis of the paired mouse xenograft bone marrow did not detect PTEN exon 7 indels 1 – 3 in 84 single cells. However, bulk Sanger Sequencing analysis did identify the PTEN exon 8 mutation in the mouse. Ongoing work is in progress to determine whether single cells of the xenograft carry alternative PTEN exon 7 mutations detected in the diagnostic sample exome data and to characterise in which diagnostic sub-clone the PTEN exon 8 mutation resides. Conclusions This study demonstrates how exome sequencing and single cell multiplex Q-PCR can be used as complementary tools to understand the sub-clonal complexity of STIL-TAL1 T-ALL. PTEN inactivation is sub-clonal by single cell analysis, demonstrating the parallel evolution of multiple independent PTEN inactivated sub-clones, highlighting PTEN inactivation as a key event in this T-ALL subgroup. In a wider cohort of 20 patients collected by our group at least 50% had PTEN inactivation as assessed by sequencing of exon 7 and copy number data alone. Results indicate a strong evolutionary pressure selecting for mutational events that result in inactivation of the PTEN-PI3Kinase pathway. These events occur via multiple mechanisms, including copy number loss and truncating mutations, which are not limited to the known T-ALL hotspot in exon 7. Current work is focussing on using a similar approach to examine the clonal evolution of NOTCH1 mutations in STIL-TAL1 T-ALL samples in diagnostic and xenograft samples of cases 4 and 5. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract 1358 The PI3K/mTOR and NOTCH pathways are promising therapeutic targets for the treatment of T-ALL. Hyperactivation of the PI3K/mTOR pathway occurs frequently, predominantly due to loss of PTEN function through deletion, mutation, microRNA induced downregulation or post-translational modification. NOTCH signalling is aberrantly activated in the majority of patients, most commonly due to mutation of Notch-1. Activation of NOTCH signalling can also positively regulate mTOR activity and increase PI3K/Akt signalling via downregulation of PTEN expression. We examined the effects of PI3K/mTOR blockade, using the dual inhibitor PI-103, on the proliferation and survival of T-ALL cell lines with various combinations of NOTCH and PTEN abnormalities. There was marked reduction in the proliferation of all T-ALL cell lines tested, regardless of their PTEN status or level of activated Akt. However, using Annexin-V/PI staining, we observed significant induction of cell death ( Blockade of NOTCH signalling, using a gamma secretase inhibitor (GSI), had no effect on cell survival and only a modest effect on cell proliferation: only 5/13 NOTCH deregulated cell lines, all of which expressed wild-type PTEN, showed a clear reduction in cell number. By contrast only 1 of the 8 GSI-resistant cell lines expressed wild-type PTEN. We tested the effect of combined blockade of PI3K/mTOR and NOTCH pathways using PI-103 + GSI to determine the extent of any non-overlapping effects. In NOTCH-mutant/PTEN-WT cells, combined blockade led to a reduction in cell size and number with a more rapid and marked increase in cell cycle arrest and reduced levels of Cdk4 and Cyclin D3, than achieved with either agent alone. Further, 8/13 cell lines with deregulated Notch showed a significant reduction in cell survival with PI-103+GSI compared with PI-103 alone. In 5/6 NOTCH-mutant/PTEN-WT cell lines survival fell to ≤50% below control, with rapid commitment to cell death (48–72 hours). This mutational context represents the majority of primary T-ALL samples at presentation - confirmation of the enhanced effect of dual PI3K/mTOR and NOTCH blockade was obtained in primary T-ALLs cultured in suspension or on stromal support. Utilizing selective inhibitors of the PI3K (PIK90) or mTOR (rapamycin) enzymes in combination with GSI we found that blockade of both enzymes was required to achieve maximal levels of cell death. c-MYC is an important oncogene in T-ALL - it is a direct transcriptional target of Notch signaling and protein stability is modulated via the PI3K/Akt/GSK3 module. Therefore, we examined the effects of PI3K/mTOR and NOTCH blockade on levels of nuclear c-MYC in cells sensitive to inhibition of both pathways. c-MYC was downregulated by GSI but, surprisingly, increased after 48h of PI-103 treatment. This followed increased levels of nuclear Notch intracellular domain and could be abolished by the addition of GSI, indicating a NOTCH-dependent mechanism. Gene expression microarray analysis confirmed global upregulation of NOTCH signalling in PI-103 treated cells and the suppression of this effect by addition of GSI. These findings were confirmed for Notch target genes (Deltex-1, c-MYC, Hes-1, CD21 and GIMAP5) by qPCR and flow cytometry (CD21). Upregulation of Notch-MYC proliferation and survival signals could explain why T-ALL cells survive after PI3K/mTOR blockade. We found that maintenance of c-MYC levels by retroviral expression counteracted the effects of PI3K/mTOR/NOTCH blockade and comparable levels of cell death, to those seen with PI-103 plus GSI, were seen in cells treated with PI-103 + the c-MYC inhibitor, 10058-F4. Our data show that targeting PI3K/mTOR can upregulate NOTCH-MYC activity in T-ALL cells with aberrant NOTCH signalling. These finding have implications for the use of PI3K inhibitors for the treatment of T-ALL, and other malignancies with activated NOTCH signaling, and provide a rational basis for the use of drug combinations that target both pathways. Disclosures: No relevant conflicts of interest to declare.

A risk-adapted approach to the treatment of patients with acute lymphoblastic leukemia (ALL) has the potential of improving survival in high-risk patients and reducing therapy-related long-term sequelae in those at low-risk. The use of molecular markers that can further define risk are needed in this disease. Activating mutations of Notch-1 have been reported in over 50% of patients with T-ALL. Mutations in the heterodimerization domain (HD) lead to ligand-independent cleavage, whilst truncating mutations of the C-terminal PEST domain disrupt binding to the negative regulator FBXW7, leading to prolonged half-life of intracellular Notch (ICN). More recently, Notch-activating mutations in FBXW7 that prevent ICN degradation have been reported in 20% of patients. In pediatric patients treated on the ALL-BFM 2000 protocol, patients with Notch-1 mutations (MUT) had an excellent outcome with an event free survival (EFS) of 90% compared to 71% in wild type (WT) patients. The impact of FBXW7 mutations was not evaluated. We sought to address whether adults with Notch-1 and/or FBXW7 mutations also faired sufficiently well that they might avoid treatment intensification/transplantation. Notch-1 HD-N, HD-C, TAD and PEST domains, and FBXW7 WD40 domain were screened by denaturing high performance liquid chromatography and/or bidirectional sequencing in a cohort of 88 adult T-ALL patients treated on the MRC UKALLXII/ECOG2993 trial (54 UKALLXII, 34 ECOG2993). Overall, 51 patients (58%) had at least one Notch-1 mutation; 37 in the HD only, 8 in the PEST domain only and 6 in both HD and PEST domains. Sixteen patients (18%) had an FBXW7 mutation (7 R465C, 3 R505C, 2 R479Q, 2 R479L, 1 R465H, and 1 G423V) and, of note, 15 of these mutations altered conserved arginine residues in the WD40 domain thought to be responsible for binding to the Notch-1 PEST domain. Five FBXW7 MUT patients were Notch-1 WT. There was a positive association between having an FBXW7 mutation and a Notch-1 mutation in the HD only (Fishers exact test 2P.02). Furthermore, no patient had both an FBXW7 and a PEST mutation. This is consistent with the hypothesis that Notch-1 HD and FBXW7 mutations act in concert similar to dual HD and PEST mutations. Complete remission was achieved in 96% of patients. Median follow-up was 3.5 years. The Notch-1 WT and MUT groups received similar treatment (8 Vs 15 sibling allograft; 3 Vs 7 autograft; 3 Vs 3 matched unrelated donor allograft; 19 Vs 24 chemotherapy with maintenance alone; P.84). There was no significant association with white cell count (WCC) or age according to either Notch-1 or FBXW7 mutational status (Mantel Haenszel test for trend P>0.1 for each case). Outcome is shown in table 1. In contrast to the data in pediatric T-ALL, in this adult cohort Notch-1 mutational status was not associated with improved EFS or overall survival (OS); nor was there an association according to the type of Notch-1 mutation. Similarly, FBXW7 mutational status alone was not predictive of outcome. There was a suggestion that patients with either a Notch and/or an FBXW7 mutation had better EFS, although this did not reach statistical significance. Adjusting for treatment received in first remission did not affect the results. In conclusion, the outcome of adult patients remains poor irrespective of their Notch-1 and/or FBXW7 mutation status, and there is no evidence that these markers should influence therapy in this age group. 5 yr EFS (WT v MUT) OR (CI) log rank 2p Notch 37% v 47% 0.79 (0.44–1.42) 0.4 FBXW7 41% v 53% 0.85 (0.40–1.82) 0.7 NOTCH +/− FBXW7 31% v 49% 0.70 (0.38–1.29) 0.3 5 yr OS WT v MUT) OR (CI) log rank 2p Notch 47% v 51% 0.87 (0.47–1.62) 0.7 FBXW7 46% v 62% 0.76 (0.35–1.66) 0.5 NOTCH +/− FBXW7 44% v 52% 0.82 (0.43–1.56) 0.5