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Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor–mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a–mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.

Relapse of acute lymphoblastic leukemia (ALL) remains a leading cause of childhood cancer-related death. Prior studies have shown clonal mutations at relapse often arise from relapse-fated subclones that exist at diagnosis. However, the genomic landscape, evolutionary trajectories, and mutational mechanisms driving relapse are incompletely understood. In an analysis of 92 cases of relapsed childhood ALL incorporating multimodal DNA and RNA sequencing, deep digital mutational tracking, and xenografting to formally define clonal structure, we identified 50 significant targets of mutation with distinct patterns of mutational acquisition or enrichment. CREBBP, NOTCH1, and RAS signaling mutations arose from diagnosis subclones, whereas variants in NCOR2, USH2A, and NT5C2 were exclusively observed at relapse. Evolutionary modeling and xenografting demonstrated that relapse-fated clones were minor (50%), major (27%), or multiclonal (18%) at diagnosis. Putative second leukemias, including those with lineage shift, were shown to most commonly represent relapse from an ancestral clone rather than a truly independent second primary leukemia. A subset of leukemias prone to repeated relapse exhibited hypermutation driven by at least three distinct mutational processes, resulting in heightened neoepitope burden and potential vulnerability to immunotherapy. Finally, relapse-driving sequence mutations were detected prior to relapse using droplet digital PCR at levels comparable with orthogonal approaches to monitor levels of measurable residual disease. These results provide a genomic framework to anticipate and circumvent relapse by earlier detection and targeting of relapse-fated clones. Significance: This study defines the landscape of mutations that preexist and arise after commencement of ALL therapy and shows that relapse may be propagated from ancestral, major, or minor clones at initial diagnosis. A subset of cases exhibits hypermutation that results in expression of neoepitopes that may be substrates for immunotherapeutic intervention. See related video: https://vimeo.com/442838617 See related commentary by Ogawa, p. 21. See related article by S. Dobson et al . This article is highlighted in the In This Issue feature, p. 5

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

AIM: To identify the effect of single nucleotide polymorphism (SNP) interactions on the risk of toxicity following radiotherapy (RT) for prostate cancer (PCa) and propose a new method for polygenic risk score incorporating SNP-SNP interactions (PRSi). MATERIALS AND METHODS: Analysis included the REQUITE PCa cohort that received external beam RT and was followed for 2 years. Late toxicity endpoints were: rectal bleeding, urinary frequency, haematuria, nocturia, decreased urinary stream. Among 43 literature-identified SNPs, the 30% most strongly associated with each toxicity were tested. SNP-SNP combinations (named SNP-allele sets) seen in ≥10% of the cohort were condensed into risk (RS) and protection (PS) scores, respectively indicating increased or decreased toxicity risk. Performance of RS and PS was evaluated by logistic regression. RS and PS were then combined into a single PRSi evaluated by area under the receiver operating characteristic curve (AUC). RESULTS: Among 1,387 analysed patients, toxicity rates were 11.7% (rectal bleeding), 4.0% (urinary frequency), 5.5% (haematuria), 7.8% (nocturia) and 17.1% (decreased urinary stream). RS and PS combined 8 to 15 different SNP-allele sets, depending on the toxicity endpoint. Distributions of PRSi differed significantly in patients with/without toxicity with AUCs ranging from 0.61 to 0.78. PRSi was better than the classical summed PRS, particularly for the urinary frequency, haematuria and decreased urinary stream endpoints. CONCLUSIONS: Our method incorporates SNP-SNP interactions when calculating PRS for radiotherapy toxicity. Our approach is better than classical summation in discriminating patients with toxicity and should enable incorporating genetic information to improve normal tissue complication probability models. ispartof: RADIOTHERAPY AND ONCOLOGY vol:159 pages:241-248 ispartof: location:Ireland status: published

This longitudinal cohort study compared ocular surface indicators in forty allogeneic hematopoietic stem cell transplant (HSCT) subjects with twenty healthy controls at baseline and identified changes in ocular graft-versus-host disease (oGVHD). Outcome measures included: Ocular Surface Disease Index (OSDI), tear osmolarity, Schirmer's test, Oxford corneal staining score, tear break-up time (TBUT), and tear and serum biomarkers (IFN-γ, IL-10, MMP-9, IL-12, IL-13, IL-17α, IL-1β, IL-2, IL-4, IL-6, IL-8, CXCL10, MCP-1, MIP-1α, RANTES, TNF-α). At baseline the HSCT group had higher median Oxford corneal staining score (1.7 vs. 0.0; P 0.0001), higher tear TNF-α (20.0 vs. 11.2 pg/mL; P 0.0001), lower tear RANTES (70.4 vs. 190.2 pg/mL; P 0.0001), higher serum IL-8 (10.2 vs. 4.5 pg/mL; P = 0.0008), and higher serum TNF-α (8.7 vs. 4.2 pg/mL; P 0.0001). The incidence of oGVHD was 62% and associated changes included increased Oxford corneal staining score (4.6 vs. 1.8, P = 0.0001), decreased Schirmer's test (3.0 vs. 10.0; P 0.0001), and decreased TBUT (4.7 vs. 9.0 s; P = 0.0004). Baseline differences in ocular surface indicators suggest a tendency toward ocular dryness in individuals with hematologic disorders preparing for HSCT. Individuals who developed oGVHD showed changes in corneal staining score, Schirmer's test, and TBUT.

Objectives The R102G variant in complement 3 (C3) results in two allotypic variants: C3 fast (C3F) and C3 slow (C3S). C3F presents at increased frequency in patients with chronic kidney disease (CKD), our aim is to explore its role in CKD progression and mortality. Methods Delta (Δ) eGFR for 2038 patients in the Salford Kidney Study (SKS) was calculated by linear regression; those with ≤-3ml/min/1.73m2/yr were defined as rapid progressors (RP) and those with ΔeGFR between -0.5 and +1ml/min/1.73m2/yr, labelled stable CKD patients (SP).A group of 454 volunteers was used as a control group. In addition, all biopsy-proven glomerulonephritis (GN) patients were studied regardless of their ΔeGFR. R102G was analysed by real-time PCR, and genotypic and allelic frequencies were compared between RP and SP along with the healthy control group. Results There were 255 SP and 259 RP in the final cohort. Median ΔeGFR was 0.07 vs. -4.7 ml/min/ 1.73m2/yr in SP vs. RP. C3F allele frequency was found to be significantly higher in our CKD cohort (25.7%) compared with the healthy control group (20.6%); p = 0.008.However, there was no significant difference in C3F allele frequency between the RP and SP groups. In a subgroup analysis of 37 patients with IgA nephropathy in the CKD cohort (21 RP and 16 SP), there was a significantly higher frequency of C3F in RP 40.5% vs. 9.4% in SP; p = 0.003. In the GN group, Cox regression showed an association between C3F and progression only in those with IgA nephropathy (n = 114);HR = 1.9 (95% CI 1.1–3.1; p = 0.018) for individuals heterozygous for the C3F variant, increased further for individuals homozygous for the variant, HR = 2.8 (95% CI 1.2–6.2; p = 0.014). Conclusion The C3 variant R102G is associated with progression of CKD in patients with IgA nephropathy.

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. Significance: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse. See related video: https://vimeo.com/442838617 See related article by E. Waanders et al .

PURPOSE: REQUITE aimed to establish a resource for multi-national validation of models and biomarkers that predict risk of late toxicity following radiotherapy. The purpose of this article is to provide summary descriptive data. METHODS: An international, prospective cohort study recruited cancer patients in 26 hospitals in eight countries between April 2014 and March 2017. Target recruitment was 5300 patients. Eligible patients had breast, prostate or lung cancer and planned potentially curable radiotherapy. Radiotherapy was prescribed according to local regimens, but centres used standardised data collection forms. Pre-treatment blood samples were collected. Patients were followed for a minimum of 12 (lung) or 24 (breast/prostate) months and summary descriptive statistics were generated. RESULTS: The study recruited 2069 breast (99% of target), 1808 prostate (86%) and 561 lung (51%) cancer patients. The centralised, accessible database includes: physician- (47,025 forms) and patient- (54,901) reported outcomes; 11,563 breast photos; 17,107 DICOMs and 12,684 DVHs. Imputed genotype data are available for 4223 patients with European ancestry (1948 breast, 1728 prostate, 547 lung). Radiation-induced lymphocyte apoptosis (RILA) assay data are available for 1319 patients. DNA (n = 4409) and PAXgene tubes (n = 3039) are stored in the centralised biobank. Example prevalences of 2-year (1-year for lung) grade ≥2 CTCAE toxicities are 13% atrophy (breast), 3% rectal bleeding (prostate) and 27% dyspnoea (lung). CONCLUSION: The comprehensive centralised database and linked biobank is a valuable resource for the radiotherapy community for validating predictive models and biomarkers. PATIENT SUMMARY: Up to half of cancer patients undergo radiation therapy and irradiation of surrounding healthy tissue is unavoidable. Damage to healthy tissue can affect short- and long-term quality-of-life. Not all patients are equally sensitive to radiation "damage" but it is not possible at the moment to identify those who are. REQUITE was established with the aim of trying to understand more about how we could predict radiation sensitivity. The purpose of this paper is to provide an overview and summary of the data and material available. In the REQUITE study 4400 breast, prostate and lung cancer patients filled out questionnaires and donated blood. A large amount of data was collected in the same way. With all these data and samples a database and biobank were created that showed it is possible to collect this kind of information in a standardised way across countries. In the future, our database and linked biobank will be a resource for research and validation of clinical predictors and models of radiation sensitivity. REQUITE will also enable a better understanding of how many people suffer with radiotherapy toxicity. ispartof: RADIOTHERAPY AND ONCOLOGY vol:138 pages:59-67 ispartof: location:Ireland status: published

BackgroundAlthough several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear.ObjectiveWe sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients.MethodsAn IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS–specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens.ResultsActivation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS–high asthma with increased epithelial expression of IL-6TS–inducible genes in the absence of systemic inflammation. The IL-6TS–high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1β, IL-8, and IL-1β.ConclusionsLocal lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosisin the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis andTP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition.This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia. Refereed/Peer-reviewed

Recent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.

Recent genomic studies have identified chromosomal rearrangements defining new subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL), however many cases lack a known initiating genetic alteration. Using integrated genomic analysis of 1,988 childhood and adult cases, we describe a revised taxonomy of B-ALL, incorporating 23 subtypes defined by chromosomal rearrangements, sequence mutations, or heterogeneous genomic alterations, many of which show marked variation in prevalence according to age. Two subtypes have frequent alterations of the B lymphoid transcription factor gene PAX5. One, PAX5alt (7.4%), has diverse PAX5 alterations (rearrangements, intragenic amplifications or mutations), and a second subtype is defined by PAX5 p.Pro80Arg and biallelic PAX5 alterations. We show that p.Pro80Arg impairs B lymphoid development and promotes the development of B-ALL with biallelic Pax5 alteration in vivo. These results demonstrate the utility of transcriptome sequencing to classify B-ALL and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL; BCR-ABL1-like ALL) in children with National Cancer Institute (NCI) intermediate- or high-risk (HR) ALL is associated with poor outcome. Ph-like ALL is characterized by genetic alterations that activate cytokine receptor and kinase signaling and may be amenable to treatment with tyrosine kinase inhibitors. The prevalence, outcome, and potential for targeted therapy of Ph-like ALL in standard-risk (SR) ALL is less clear. We retrospectively analyzed a cohort of 1023 SR childhood B-ALL consecutively enrolled in the Children's Oncology Group AALL0331 clinical trial. The Ph-like ALL gene expression profile was identified in 206 patients, and 67 patients with either BCR-ABL1 (n = 6) or ETV6-RUNX1 (n = 61) were excluded from downstream analysis, leaving 139 of 1023 (13.6%) as Ph-like. Targeted reverse transcription polymerase chain reaction assays and RNA-sequencing identified kinase-activating alterations in 38.8% of SR Ph-like cases, including CRLF2 rearrangements (29.5% of Ph-like), ABL-class fusions (1.4%), JAK2 fusions (1.4%), an NTRK3 fusion (0.7%), and other sequence mutations (IL7R, KRAS, NRAS; 5.6%). Patients with Ph-like ALL had inferior 7-year event-free survival compared with non-Ph-like ALL (82.4 ± 3.6% vs 90.7 ± 1.0%, P = .0022), with no difference in overall survival (93.2 ± 2.4% vs 95.8 ± 0.7%, P = .14). These findings illustrate the significant differences in the spectrum of kinase alterations and clinical outcome of Ph-like ALL based on presenting clinical features and establish that genomic alterations potentially targetable with approved kinase inhibitors are less frequent in SR than in HR ALL.

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.

Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a high-risk subtype of ALL in children. There are conflicting data on the incidence and prognosis of Ph-like ALL in adults. Patients with newly diagnosed B-cell ALL (B-ALL) who received frontline chemotherapy at MD Anderson Cancer Center underwent gene expression profiling of leukemic cells. Of 148 patients, 33.1% had Ph-like, 31.1% had Ph+, and 35.8% had other B-ALL subtypes (B-other). Within the Ph-like ALL cohort, 61% had cytokine receptor-like factor 2 (CRLF2) overexpression. Patients with Ph-like ALL had significantly worse overall survival (OS), and event-free survival compared with B-other with a 5-year survival of 23% (vs 59% for B-other, P = .006). Sixty-eight percent of patients with Ph-like ALL were of Hispanic ethnicity. The following were associated with inferior OS on multivariable analysis: age (hazard ratio [HR], 3.299; P < .001), white blood cell count (HR, 1.910; P = .017), platelet count (HR, 7.437; P = .005), and Ph-like ALL (HR, 1.818; P = .03). Next-generation sequencing of the CRLF2+ group identified mutations in the JAK-STAT and Ras pathway in 85% of patients, and 20% had a CRLF2 mutation. Within the CRLF2+ group, JAK2 mutation was associated with inferior outcomes. Our findings show high frequency of Ph-like ALL in adults, an increased frequency of Ph-like ALL in adults of Hispanic ethnicity, significantly inferior outcomes of adult patients with Ph-like ALL, and significantly worse outcomes in the CRLF2+ subset of Ph-like ALL. Novel strategies are needed to improve the outcome of these patients.

Purpose BCR-ABL1–like acute lymphoblastic leukemia (ALL) is a recently identified B-cell ALL (B-ALL) subtype with poor outcome that exhibits a gene expression profile similar to BCR-ABL1-positive ALL but lacks the BCR-ABL1 fusion protein. We examined the outcome of children with BCR-ABL1–like ALL treated with risk-directed therapy based on minimal residual disease (MRD) levels during remission induction. Patients and Methods Among 422 patients with B-ALL enrolled onto the Total Therapy XV study between 2000 and 2007, 344 had adequate samples for gene expression profiling. Next-generation sequencing and/or analysis of genes known to be altered in B-ALL were performed in patients with BCR-ABL1–like ALL who had available material. Outcome was compared between patients with and those without BCR-ABL1–like ALL. Results Forty (11.6%) of the 344 patients had BCR-ABL1–like ALL. They were significantly more likely to be male, have Down syndrome, and have higher MRD levels on day 19 and at the end of induction than did other patients with B-ALL. Among 25 patients comprehensively studied for genetic abnormalities, 11 harbored a genomic rearrangement of CRLF2, six had fusion transcripts responsive to ABL tyrosine kinase inhibitors or JAK inhibitors, and seven had mutations involving the Ras signaling pathway. There were no significant differences in event-free survival (90.0% ± 4.7% [SE] v 88.4% ± 1.9% at 5 years; P = .41) or in overall survival (92.5% ± 4.2% v 95.1% ± 1.3% at 5 years; P = .41) between patients with and without BCR-ABL1–like ALL. Conclusion Patients who have BCR-ABL1–like ALL with poor initial treatment response can be salvaged with MRD-based risk-directed therapy and may benefit from identification of kinase-activating lesions for targeted therapies.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by genomic alterations that activate cytokine receptor and kinase signaling. We examined the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed patients with childhood B-lineage ALL with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of whom were excluded from additional analyses because of the presence of BCR-ABL1 (n = 46) or ETV6-RUNX1 (n = 11). Among the remaining 284 patients (20.4%), overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) identified in 63 patients (50.8% of those with CRLF2 rearrangement). Among the remaining patients, using reverse transcriptase polymerase chain reaction or transcriptome sequencing, we identified targetable ABL-class fusions (ABL1, ABL2, CSF1R, and PDGFRB) in 14.1%, EPOR rearrangements or JAK2 fusions in 8.8%, alterations activating other JAK-STAT signaling genes (IL7R, SH2B3, JAK1) in 6.3% or other kinases (FLT3, NTRK3, LYN) in 4.6%, and mutations involving the Ras pathway (KRAS, NRAS, NF1, PTPN11) in 6% of those with Ph-like ALL. We identified 8 new rearrangement partners for 4 kinase genes previously reported to be rearranged in Ph-like ALL. The current findings provide support for the precision-medicine testing and treatment approach for Ph-like ALL implemented in Children's Oncology Group ALL trials.

Purpose Philadelphia chromosome (Ph) –like acute lymphoblastic leukemia (ALL) is a high-risk subtype of childhood ALL characterized by kinase-activating alterations that are amenable to treatment with tyrosine kinase inhibitors. We sought to define the prevalence and genomic landscape of Ph-like ALL in adults and assess response to conventional chemotherapy. Patients and Methods The frequency of Ph-like ALL was assessed by gene expression profiling of 798 patients with B-cell ALL age 21 to 86 years. Event-free survival and overall survival were determined for Ph-like ALL versus non–Ph-like ALL patients. Detailed genomic analysis was performed on 180 of 194 patients with Ph-like ALL. Results Patients with Ph-like ALL accounted for more than 20% of adults with ALL, including 27.9% of young adults (age 21 to 39 years), 20.4% of adults (age 40 to 59 years), and 24.0% of older adults (age 60 to 86 years). Overall, patients with Ph-like ALL had an inferior 5-year event-free survival compared with patients with non–Ph-like ALL (22.5% [95% CI, 14.9% to 29.3%; n = 155] v 49.3% [95% CI, 42.8% to 56.2%; n = 247], respectively; P < .001). We identified kinase-activating alterations in 88% of patients with Ph-like ALL, including CRLF2 rearrangements (51%), ABL class fusions (9.8%), JAK2 or EPOR rearrangements (12.4%), other JAK-STAT sequence mutations (7.2%), other kinase alterations (4.1%), and Ras pathway mutations (3.6%). Eleven new kinase rearrangements were identified, including four involving new kinase or cytokine receptor genes and seven involving new partners for previously identified genes. Conclusion Ph-like ALL is a highly prevalent subtype of ALL in adults and is associated with poor outcome. The diverse range of kinase-activating alterations in Ph-like ALL has important therapeutic implications. Trials comparing the addition of tyrosine kinase inhibitors to conventional therapy are required to evaluate the clinical utility of these agents in the treatment of Ph-like ALL.

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2D-BCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered., Acute lymphoblastic leukaemia is characterized by chromosomal rearrangements. Here, the authors carry out RNA-sequencing on a large cohort of patients and identify recurrent rearrangements of MEF2D, which lead to increased transcriptional activity of the gene, and cellular transformation in vitro.

The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24-31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32-39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.

One recently identified subtype of pediatric B-precursor acute lymphoblastic leukemia (ALL) has been termed BCR-ABL1–like or Ph-like because of similarity of the gene expression profile to BCR-ABL1 positive ALL suggesting the presence of lesions activating tyrosine kinases, frequent alteration of IKZF1, and poor outcome. Prior studies demonstrated that approximately half of these patients had genomic lesions leading to CRLF2 overexpression, with half of such cases harboring somatic mutations in the Janus kinases JAK1 and JAK2. To determine whether mutations in other tyrosine kinases might also occur in ALL, we sequenced the tyrosine kinome and downstream signaling genes in 45 high-risk pediatric ALL cases with either a Ph-like gene expression profile or other alterations suggestive of activated kinase signaling. Aside from JAK mutations and 1 FLT3 mutation, no somatic mutations were found in any other tyrosine kinases, suggesting that alternative mechanisms are responsible for activated kinase signaling in high-risk ALL.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL invivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function.

SummaryGenomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.

As controversy exists regarding the prognostic significance of genomic rearrangements of CRLF2 in pediatric B-precursor acute lymphoblastic leukemia (ALL) classified as standard/intermediate-risk (SR) or high-risk (HR), we assessed the prognostic significance of CRLF2 mRNA expression, CRLF2 genomic lesions (IGH@-CRLF2, P2RY8-CRLF2, CRLF2 F232C), deletion/mutation in genes frequently associated with high CRLF2 expression (IKZF1, JAK, IL7R), and minimal residual disease (MRD) in 1061 pediatric ALL patients (499 HR and 562 SR) on COG Trials P9905/P9906. Whereas very high CRLF2 expression was found in 17.5% of cases, only 51.4% of high CRLF2 expressors had CRLF2 genomic lesions. The mechanism underlying elevated CRLF2 expression in cases lacking known genomic lesions remains to be determined. All CRLF2 genomic lesions and virtually all JAK mutations were found in high CRLF2 expressors, whereas IKZF1 deletions/mutations were distributed across the full cohort. In multivariate analyses, NCI risk group, MRD, high CRLF2 expression, and IKZF1 lesions were associated with relapse-free survival. Within HR ALL, only MRD and CRLF2 expression predicted a poorer relapse-free survival; no difference was seen between cases with or without CRLF2 genomic lesions. Thus, high CRLF2 expression is associated with a very poor outcome in high-risk, but not standard-risk, ALL. This study is registered at www.clinicaltrials.gov as NCT00005596 and NCT00005603.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Genes involved in sex hormone pathways are candidates for influencing bone strength. Polymorphisms in these genes were tested for association with heel quantitative ultrasound (QUS) parameters in middle-aged and elderly European men. Men 40-79 yr of age were recruited from population registers in eight European centers for the European Male Aging Study (EMAS). Polymorphisms were genotyped in AR, ESR1, ESR2, CYP19A1, CYP17A1, SHBG, SRD5A2, LHB, and LHCGR. QUS parameters broadband ultrasound attenuation (BUA) and speed of sound (SOS) were measured in the heel and used to derive BMD. The relationships between QUS parameters and polymorphisms were assessed using linear regression adjusting for age and center. A total of 2693 men, with a mean age of 60.1 +/- 11.1 (SD) yr were included in the analysis. Their mean BUA was 80.0 +/- 18.9 dB/Mhz, SOS was 1550.2 +/- 34.1 m/s, and BMD was 0.542 +/- 0.141 g/cm(2). Significant associations were observed between multiple SNPs in a linkage disequilibrium (LD) block within CYP19A1, peaking at the TCT indel with the deletion allele associating with reduced ultrasound BMD in heterozygotes (beta =-0.016, p = -0.005) and homozygotes (beta = -0.029, p = 0.001). The results for BUA and SOS were similar. Significant associations with QUS parameters were also observed for the CAG repeat in AR and SNPs in CYP17A1, LHCGR, and ESR1. Our data confirm evidence of association between bone QUS parameters and polymorphisms in CYP19A1, as well as modest associations with polymorphisms in CYP17A1, ESR1, LHCGR, and AR in a population sample of European men; this supports a role for genetically determined sex hormone actions in influencing male bone health.

The individual variability in the waning androgenic-anabolic functions of aging men may be influenced by the CAG repeat polymorphism in exon 1 of the androgen receptor (AR), affecting androgen sensitivity. However, findings on its phenotypic effects are inconclusive.The aim was to investigate the relationships between health status, various reproductive hormones, and the AR CAG repeat length.We conducted a multinational prospective cohort observational study with cross-sectional baseline data.This was a population survey of community-dwelling men.Men (40-79 yr old; n = 3369) were randomly recruited from centers in eight European countries; CAG repeat analysis was performed in 2878 men.We measured the correlations of the CAG repeat length with selected endocrine, metabolic, and phenotypic parameters related to aging and sex hormone action.Only minor differences were found in CAG repeat lengths between the eight European countries. They showed significant positive association with total, free, and bioavailable levels of testosterone (T) and estradiol. FSH but not LH correlated inversely with CAG repeat length. Significant associations were found with bone ultrasound parameters at the calcaneus. Negative correlation was found with triglycerides, but not with other blood lipids or with anthropometry, blood pressure, hemoglobin, insulin sensitivity, or sexual and prostatic functions.The AR CAG repeat length correlates significantly with serum T and estradiol of aging men. Weaker transcriptional activity of the AR with longer CAG-encoded polyglutamine repeats appears to be totally or nearly totally compensated for by higher T levels. The residual phenotypic correlations may reflect differences in estrogen levels/actions after aromatization of the higher T levels.

Summary Background A defect in hypothalamic–pituitary–adrenal (HPA) axis function has been suggested to contribute to susceptibility to rheumatoid arthritis (RA). Objective To investigate polymorphisms of the glucocorticoid receptor (GR) gene and determine any associations with RA. Methods Three GR polymorphisms that tag 95% of all haplotypes across the GR gene were genotyped. These are an intron B Bcl1 polymorphism, a ttg insertion/deletion within intron F (rs2307674) and the single nucleotide polymorphism (SNP) lying in the 3′ untranslated region of exon 9b (rs6198). The dye terminator-based SNaPshot method or size resolution by capillary electrophoresis was performed. The study population comprised 198 UK Caucasian RA cases and 393 ethnically matched controls. Results No significant single point or haplotypic associations were found for GR polymorphisms with RA susceptibility. Furthermore, no evidence for GR polymorphisms with aspects of RA severity was seen. Conclusion In this study of the most comprehensive coverage of GR polymorphisms with RA, no significant contributing role for GR polymorphisms with RA was found.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

To the editor: Hypodiploid acute lymphoblastic leukemia (ALL) with

Background The Pediatric Preclinical Testing Program (PPTP) is an initiative supported by the National Cancer Institute (NCI) to identify novel therapeutic agents that may have significant activity against childhood cancers. The PPTP has established panels of childhood cancer xenografts and cell lines to be used for in vivo and in vitro testing. These include panels for Wilms tumor, sarcomas (rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma), neuroblastoma, brain tumors (glioblastoma, ependymoma, and medulloblastoma), rhabdoid tumors (CNS and renal), and acute lymphoblastic leukemia (ALL). Here, we describe the characteristics of the in vivo tumor panels and report results for the in vivo evaluation of two standard agents, vincristine and cyclophosphamide. Procedures Solid tumors were grown subcutaneously in immune-deficient mice and tumor dimensions were measured weekly. ALL xenografts were inoculated intravenously and human CD45-positive cells were enumerated weekly. Results Vincristine-induced objective responses in 6 of 24 (25%) and cyclophosphamide-induced objective responses in 18 of 28 (64%) solid tumor models. Comparable assessments of high levels of activity for these two agents were obtained using a tumor growth delay (TGD) measure. Both agents induced regressions in each of the ALL models evaluated. Conclusions We have established 51 solid tumor and 10 ALL in vivo models. The models identify vincristine and cyclophosphamide as having broad-spectrum activity. The PPTP tumor panels appear to generally recapitulate the activity of these agents against specific childhood cancers and to have the potential for identifying novel agents having significant clinical activity.

Acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA) is characterized by distinct presenting features and inferior prognosis compared with pediatric ALL. We performed a genome-wide association study (GWAS) to comprehensively identify inherited genetic variants associated with susceptibility to AYA ALL. In the discovery GWAS, we compared genotype frequency at 635 297 single nucleotide polymorphisms (SNPs) in 308 AYA ALL cases and 6,661 non-ALL controls by using a logistic regression model with genetic ancestry as a covariate. SNPs that reached P ≤ 5 × 10(-8) in GWAS were tested in an independent cohort of 162 AYA ALL cases and 5,755 non-ALL controls. We identified a single genome-wide significant susceptibility locus in GATA3: rs3824662, odds ratio (OR), 1.77 (P = 2.8 × 10(-10)) and rs3781093, OR, 1.73 (P = 3.2 × 10(-9)). These findings were validated in the replication cohort. The risk allele at rs3824662 was most frequent in Philadelphia chromosome (Ph)-like ALL but also conferred susceptibility to non-Ph-like ALL in AYAs. In 1,827 non-selected ALL cases, the risk allele frequency at this SNP was positively correlated with age at diagnosis (P = 6.29 × 10(-11)). Our results from this first GWAS of AYA ALL susceptibility point to unique biology underlying leukemogenesis and potentially distinct disease etiology by age group.

SummaryAlterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.

BACKGROUND Philadelphia chromosome–like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR–ABL1–positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6–NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.)

We developed Copy Number Segmentation by Regression Tree in Next Generation Sequencing (CONSERTING), an algorithm for detecting somatic copy-number alteration (CNA) using whole-genome sequencing (WGS) data. CONSERTING performs iterative analysis of segmentation on the basis of changes in read depth and the detection of localized structural variations, with high accuracy and sensitivity. Analysis of 43 cancer genomes from both pediatric and adult patients revealed novel oncogenic CNAs, complex rearrangements and subclonal CNAs missed by alternative approaches.

Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biological determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways, and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse. However, DNA sequence mutations in ALL have not been analysed in detail. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis-relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CREBBP, also known as CBP). The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the histone acetyltransferase domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL.

Treatment with the nucleoside analog cytarabine has been shown to mimic changes in gene expression associated with downregulation of the EWS-FLI1 oncogene in Ewing sarcoma cell lines, selectively inhibit their growth in vitro, and cause tumor regression in athymic nude mice. For this report cytarabine was studied in vitro against a panel of 23 pediatric cancer cell lines and in vivo against 6 Ewing sarcoma xenografts. Acute lymphoblastic leukemia cell lines were the most sensitive to cytarabine in vitro (median IC(50) 9 nM), while Ewing sarcoma cell lines showed intermediate sensitivity (median IC(50) 232 nM). Cytarabine at a dose of 150 mg/kg administered daily 5× failed to significantly inhibit growth of five xenograft models, but reduced growth rate of the A673 xenograft by 50%. Cytarabine shows no differential in vitro activity against Ewing sarcoma cell lines and is ineffective in vivo against Ewing sarcoma xenografts at the dose and schedule studied.

BACKGROUND/OBJECTIVE: Variants in the interleukin-23 receptor (IL23R) and the autophagy-related 16-like 1 (ATG16L1) genes have been associated with an increased risk of Crohn’s disease (CD). Both genes were identified through genome-wide association scans and subsequent studies have validated these associations. To assess the effect size of these variants, an independent case-control association study and meta-analysis were performed.METHODS: British Caucasian subjects with inflammatory bowel disease (n=500) and 877 ethnically matched controls were genotyped for the disease-associated variants inIL23RandATG16L1. In addition, meta-analyses of 12,991 patients and 14,598 controls, and 11,909 patients and 15,798 controls, were conducted on independently published data for the associations betweenIL23RandATG16L1variants and CD, respectively.RESULTS: In the present cohort, both susceptibility variants showed highly significant associations, includingIL23R(rs11209026, P=0.0006; OR 0.37; 95% CI 0.21 to 0.67) andATG16L1(rs2241880, P=0.0017; OR 1.36; 95% CI 1.12 to 1.66). The meta-analysis based on the random effects model showed similar combined effects for rs11209026 (n=26, OR 0.41; 95% CI 0.37 to 0.46) and rs2241880 (n=25, OR 1.33; 95% CI 1.28 to 1.39). There was no statistically significant gene-gene interaction between caspase recruitment domain (CARD15) variants and theIL23RorATG16L1polymorphisms (P=0.44 and P=0.24, respectively).CONCLUSION: The present cohort and meta-analysis provides strong evidence that, in addition toCARD15, polymorphisms in bothIL23RandATG16L1alter susceptibility to CD and that these effects are consistent across all populations of European ancestry; however, onlyATG16L1is relevant to inflammatory bowel disease in the Asian population.

ObjectiveData remain divergent regarding the activational effects of endogenous hormones on adult cognitive function. We examined the association between cognition, hormones and androgen receptor (AR) CAG repeat length in a large cohort of men.DesignCommunity-based, cross-sectional study of 3369 men aged 40–79 years.MethodsCognition tests were the Rey-Osterrieth Complex Figure, Camden Topographical Recognition Memory and Digit-Symbol Substitution. A fluid cognition (FC) z-score was computed from the individual tests. Testosterone, oestradiol (OE2) and 5α-dihydrotestosterone were measured by gas chromatography–mass spectrometry; DHEAS, LH, FSH and sex hormone-binding globulin (SHBG) by electrochemiluminescence. Free testosterone and OE2 were calculated from total hormone, SHBG and albumin. CAG repeat lengths were assayed by PCR genotyping.ResultsTotal testosterone and free testosterone were associated with higher FC z-scores, LH and FSH with lower FC z-scores in age-adjusted linear regressions. After adjusting for health, lifestyle and centre, a modest association was only observed between DHEAS and a lower FC z-score (β=−0.011, P=0.02), although this was driven by subjects with DHEAS levels >10 μmol/l. Locally weighted plots revealed no threshold effects between hormones and FC. There was no association between CAG repeat length and FC z-score after adjustment for age and centre (β=−0.007, P=0.06), nor any interaction effect between CAG repeat length and hormones.ConclusionOur results suggest that endogenous hormones are not associated with a vision-based measure of FC among healthy, community-dwelling men. Further studies are warranted to determine whether ‘high’ DHEAS levels are associated with poorer performance on a broader range of neuropsychological tests.

Context: Polymorphisms in genes involved in regulation, biosynthesis, metabolism, and actions of testicular sex hormones may influence hormone balance and phenotype of aging men. Objective: We investigated the relationships between polymorphisms in genes related to pituitary-testicular endocrine function and health status. Design and Setting: Using cross-sectional baseline data, we conducted a multinational prospective cohort observational study consisting of a population survey of community-dwelling men. Participants: A total of 2748 men, aged 40-79 (mean +/- SD, 60.2 + 11.2) yr, were randomly recruited from eight European centers. Forty-three polymorphisms were genotyped in the following genes: androgen receptor (AR), estrogen receptor-alpha and -beta (ESR1 and ESR2), steroid 5alpha-reductase type II (SRD5A2), 17alpha-hydroxylase/17,20-lyase (CYP17A1), aromatase (CYP19A1), sex hormone-binding globulin (SHBG), LH beta-subunit (LHB), and LH receptor (LHCGR). Main Outcome Measures: We measured the associations between gene polymorphisms and endocrine, metabolic, and phenotypic parameters related to aging and sex hormone action. Results: Several polymorphisms in SHBG, ESR2, AR, CYP19A1, and LHB were significantly associated with circulating levels of SHBG, LH, total, free, and bioavailable testosterone and estradiol, the LH x testosterone product, and indices of insulin sensitivity. Apart from several previously reported associations between genes affecting estrogen levels and heel ultrasound parameters, no associations existed between polymorphisms and nonhormonal variables (anthropometry, blood lipids, blood pressure, hemoglobin, prostate symptoms, prostate-specific antigen, sexual dysfunction, cognition). Conclusion: In aging men, polymorphisms in genes related to the pituitary-testicular endocrine function significantly influence circulating LH, testosterone, and estradiol levels, but the downstream effects may be too small to influence secondary phenotypic parameters.

This study provides the first analysis of the TPMT mutant allele frequency in a sample of the Jordanian population and indicates that TPMT*3A is the most common allele in Jordanian subjects. Thiopurine methyltransferase TPMT catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Thiopurine methyltransferase (TPMT) polymorphisms are the major determinants of interindividual differences in the severe haematological toxicity of 6-mercaptopurine. Several variants in the TPMT gene have been identified that correlate with a low activity phenotype. Four variant alleles, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C, are responsible for over 80% of the low or undetectable enzyme activity. The allelic frequency of TPMT variants has been established in many populations. In this study, the frequencies of four (TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C) variants were investigated in 169 healthy Jordanian men (18–45 years of age). Single nucleotide polymorphisms (SNPs) were genotyped using the Sequenom® MassARRAY technology (Sequenom®; San Diego, CA, USA). TPMT*3A and TPMT*3C were the only deficiency alleles detected in the Jordanian population with an allele frequency of 0.59% and 0.30% respectively. The TPMT*3A allele frequency is found to be lower than in the European Caucasian population. TPMT*3A and TPMT*3C were the only deficiency alleles detected in the Jordanian population with an allele frequency of 0.59% and 0.30% respectively. The TPMT*3A allele frequency is found to be lower than in the European Caucasian population.

OBJECTIVE: The HLA 8.1 ancestral haplotype (HLA-B*08/DRB1*03/DQA1*05/DQB1*02) is associated with adult/juvenile idiopathic inflammatory myopathy (IIM), but confers a greater strength of association in patients possessing anti-Jo-1 or anti-PM-Scl antibodies. The HLA-DPB1 gene is centromeric to other HLA class II loci and separated by a recombination hotspot. We investigated whether HLA-DPB1 associations differ between anti-Jo-1 and anti-PM-Scl antibody-positive IIM cases. METHODS: Two hundred and thirty-three adult IIM patients (73% females, 49.4 +/- 13.6 years) with PM (n = 89), DM (n = 88) and myositis associated with another CTD (n = 55) and 85 juvenile DM patients (75% females, 6.2 +/- 3.6 years) were compared with 678 UK Caucasian controls. Patients/controls were genotyped for HLA-DPB1 and DRB1 alleles. Myositis-specific and associated antibodies were identified in cases using immunoprecipitation. RESULTS: HLA-DPB1*0101 was associated with IIM overall [22 vs 13% controls, corrected probability (P(corr)) = 2 x 10(-03); odds ratio (OR) 2.0; 95% CI 1.4, 2.9], PM (P(corr) = 7 x 10(-03); OR 2.5; 95% CI 1.5, 4.4) and anti-Jo-1 (P(corr) = 3 x 10(-5); OR 4.1; 95% CI 2.1, 7.8). No significant DPB1*0101 difference was present between anti-PM-Scl cases and controls. The HLA-DPB1*0101 association in IIM overall cases was dependent on the presence of DRB1*03. A number of HLA-DRB1*03/DPB1 haplotypes were identified, but only DRB1*03/DPB1*0101 was associated with anti-Jo-1 antibody-positive cases. CONCLUSIONS: The HLA-DRB1*03/DPB1*0101 haplotype is a risk factor for anti-Jo-1 antibody-positive IIM. Thus, although DRB1*03 is strongly associated with possession of either anti-Jo-1 or anti-PM-Scl, differing antibody associations are observed at the HLA-DPB1 locus.

Background Streptococcus pneumoniae (pneumococcus) is responsible for over one million deaths per year, with young children, the elderly and immunocompromised individuals being most at risk. Approximately half of East African children have been reported to be asymptomatic carriers of pneumococcus with invasive infection occurring after the disruption of the respiratory membrane which is believed to be caused by the host immune response. Racial incidence of invasive pneumococcal disease (IPD) is higher in certain populations even after adjusting for environmental factors suggesting a genetic component to disease susceptibility. The nitric oxide synthase 2A (NOS2A) gene is responsible for the production of nitric oxide under pathological conditions including host defence against bacterial infection. Nitric oxide is a modulator of apoptotic and inflammatory cascades and endothelial permeability. We hypothesised that genetic variants within this gene may predispose to disease risk and survival. Methods A cohort of 299 children with IPD (221 meningitis, 41 pneumonia and 37 with bacteraemia) and 931 age matched controls from Malawi were used in this study. We investigated nine haplotype tagging single nucleotide polymorphisms within the NOS2A gene and compared the presence or absence of the minor alleles in cases and controls and survivors and non-survivors within the cases. Results We observed no significant associations between cases and controls or with survival in either all IPD cases or in the separate analysis of meningitis cases. A near significant association was obtained for the comparison of rs8078340 in cases and controls (p-value, 0.078). However, results were unadjusted for multiple testing. Conclusion Our results suggest that polymorphic variation within the NOS2A gene does not influence invasive pneumococcal disease susceptibility or survival.

Objective: To study whether there is an association between the frequency of functional polymorphisms in the toll-like receptor 4 (TLR4) and cluster differentiation 14 (CD14) genes and periodontitis. Methodology: Genotyping for the TLR4 single-nucleotide polymorphisms (SNPs) Asp299Gly, Thr399Ile and the CD14 SNPs −159 and −1359 was completed for subjects with periodontal disease compared with control subjects. Two disease populations were investigated: 73 subjects with aggressive periodontitis (AgP; 28 males, 45 females) and 95 males with chronic periodontitis (CP). The TLR4 and CD14 polymorphisms were determined using SNaPshot primer extension with capillary electrophoresis. Comparison of allele and genotype frequencies for each polymorphism was by Fisher's exact test or χ2 analysis. Results: The TLR4 Asp299Gly genotype was present in a significantly (p=0.026) lower proportion of AgP subjects (5.5%) compared with control subjects (16.3%). The unadjusted odds ratio for the Asp299Gly genotype to be associated with AgP was 0.30, 95% confidence interval 0.10–0.91. No differences were found in the prevalence of the TLR4 Asp299Gly genotype in men with CP (18.9%) compared with an age-matched control group with no evidence of periodontitis (17%). In addition, there was no difference in the distribution of the CD14 polymorphisms in either the AgP or CP populations studied compared with controls. Conclusion: It is concluded that in West European Caucasians, the Asp299Gly TLR4 gene polymorphism is associated with a decreased risk of AgP but not CP. Promoter polymorphisms of the CD14 gene, however, did not influence susceptibility to inflammatory periodontitis in the population cohorts studied.