Clonal Events Research Articles

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study.

BackgroundAntimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei.Methods and FindingsWe performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei.ConclusionsThis study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.

Analyzing tumor heterogeneity and driver genes in single myeloid leukemia cells with SBCapSeq.

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor's major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.

Whole-exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole-exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence.

Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.

Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and are disrupted at the genomic level in a spectrum of human tumours including haematological malignancies. Using high-resolution single nucleotide polymorphism (SNP) arrays, we identified recurrent deletions of the SETD2 locus in 3% (8/261) of chronic lymphocytic leukaemia (CLL) patients. Further validation in two independent cohorts showed that SETD2 deletions were associated with loss of TP53, genomic complexity and chromothripsis. With next-generation sequencing we detected mutations of SETD2 in an additional 3.8% of patients (23/602). In most cases, SETD2 deletions or mutations were often observed as a clonal event and always as a mono-allelic lesion, leading to reduced mRNA expression in SETD2-disrupted cases. Patients with SETD2 abnormalities and wild-type TP53 and ATM from five clinical trials employing chemotherapy or chemo-immunotherapy had reduced progression-free and overall survival compared with cases wild type for all three genes. Consistent with its postulated role as a tumour suppressor, our data highlight SETD2 aberration as a recurrent, early loss-of-function event in CLL pathobiology linked to aggressive disease.

Loss of BAP1 Expression Is Very Rare in Pancreatic Ductal Adenocarcinoma.

BackgroundPancreatic cancer is both common and highly lethal and therefore new biomarkers or potential targets for treatment are needed. Loss of BRCA associated protein-1 (BAP1) expression has been found in up to a quarter of intrahepatic cholangiocarcinomas. Given the close anatomical relationship between intrahepatic cholangiocarcinoma and pancreatic ductal adenocarcinoma, we therefore sought to investigate the frequency of loss of BAP1 expression in pancreatic ductal adenocarcinoma.MethodsThe records of the department of Anatomical Pathology Royal North Shore Hospital, Sydney, Australia, were searched for cases of pancreatic ductal adenocarcinoma diagnosed between 1992 and 2014 with material available in archived formalin fixed paraffin embedded tissue blocks. Immunohistochemistry for BAP1 was performed on tissue microarray sections and if staining was equivocal or negative it was confirmed on whole sections. Negative staining for BAP1 was defined as loss of expression in all neoplastic nuclei, with preserved expression in non-neoplastic cells which acted as an internal positive control.ResultsLoss of BAP1 expression was found in only 1 of 306 (0.33%) pancreatic ductal adenocarcinomas. This case was confirmed to demonstrate diffuse loss of expression throughout all neoplastic cells in multiple blocks, consistent with BAP1 loss being an early clonal event. All other cases demonstrated positive expression of BAP1.ConclusionWe conclude that, in contrast to intrahepatic cholangiocarcinoma, loss of expression of BAP1 occurs very rarely in pancreatic ductal adenocarcinoma. Therefore BAP1 inactivation is unlikely to be a frequent driver abnormality in pancreatic adenocarcinoma.

Concurrent nuclear ERG and MYC protein overexpression defines a subset of locally advanced prostate cancer: Potential opportunities for synergistic targeted therapeutics.

Recurrent ERG gene fusions, the most common genetic alterations in prostate cancer, drive overexpression of the nuclear transcription factor ERG, and are early clonal events in prostate cancer progression. The nuclear transcription factor MYC is also frequently overexpressed in prostate cancer and may play a role in tumor initiation and/or progression. The relationship between nuclear ERG and MYC protein overexpression in prostate cancer, as well as the clinicopathologic characteristics and prognosis of ERG-positive/MYC high tumors, is not well understood. Immunohistochemistry (IHC) for ERG and MYC was performed on formalin-fixed, paraffin-embedded tissue from prostate cancer tissue microarrays (TMAs), and nuclear staining was scored semi-quantitatively (IHC product score range = 0-300). Correlation between nuclear ERG and MYC protein expression and association with clinicopathologic parameters and biochemical recurrence after radical prostatectomy was assessed. 29.1% of all tumor nodules showed concurrent nuclear ERG and MYC protein overexpression (i.e., ERG-positive/MYC high), including 35.0% of secondary nodules. Overall, there was weak positive correlation between ERG and MYC expression across all tumor nodules (rpb = 0.149, P = 0.045), although this correlation was strongest in secondary nodules (rpb = 0.520, P = 0.019). In radical prostatectomy specimens, ERG-positive/MYC high tumors were positively associated with the presence of extraprostatic extension (EPE), relative to all other ERG/MYC expression subgroups, however, there was no significant association between concurrent nuclear ERG and MYC protein overexpression and time to biochemical recurrence. Concurrent nuclear ERG and MYC protein overexpression is common in prostate cancer and defines a subset of locally advanced tumors. Recent data indicates that BET bromodomain proteins regulate ERG gene fusion and MYC gene expression in prostate cancer, suggesting possible synergistic targeted therapeutics in ERG-positive/MYC high tumors. Prostate 76:845-853, 2016. © 2016 Wiley Periodicals, Inc.

Pan-cancer analysis of intratumor heterogeneity as a prognostic determinant of survival.

As tumors accumulate genetic alterations, an evolutionary process occurs in which genetically distinct subclonal populations of cells co-exist, resulting in intratumor genetic heterogeneity (ITH). The clinical implications of ITH remain poorly defined. Data are limited with respect to whether ITH is an independent determinant of patient survival outcomes, across different cancer types. Here, we report the results of a pan-cancer analysis of over 3300 tumors, showing a varied landscape of ITH across 9 cancer types. While some gene mutations are subclonal, the majority of driver gene mutations are clonal events, present in nearly all cancer cells. Strikingly, high levels of ITH are associated with poorer survival across diverse types of cancer. The adverse impact of high ITH is independent of other clinical, pathologic and molecular factors. High ITH tends to be associated with lower levels of tumor-infiltrating immune cells, but this association is not able to explain the observed survival differences. Together, these data show that ITH is a prognostic marker in multiple cancers. These results illuminate the natural history of cancer evolution, indicating that tumor heterogeneity represents a significant obstacle to cancer control.

Evolutionary dynamics of Enterococcus faecium reveals complex genomic relationships between isolates with independent emergence of vancomycin resistance.

Enterococcus faecium, a major cause of hospital-acquired infections, remains problematic because of its propensity to acquire resistance to vancomycin, which currently is considered first-line therapy. Here, we assess the evolution and resistance acquisition dynamics of E. faecium in a clinical context using a series of 132 bloodstream infection isolates from a single hospital. All isolates, of which 49 (37 %) were vancomycin-resistant, underwent whole-genome sequencing. E. faecium was found to be subject to high rates of recombination with little evidence of sequence importation from outside the local E. faecium population. Apart from disrupting phylogenetic reconstruction, recombination was frequent enough to invalidate MLST typing in the identification of clonal expansion and transmission events, suggesting that, where available, whole-genome sequencing should be used in tracing the epidemiology of E. faecium nosocomial infections and establishing routes of transmission. Several forms of the Tn1549-like element–vanB gene cluster, which was exclusively responsible for vancomycin resistance, appeared and spread within the hospital during the study period. Several transposon gains and losses and instances of in situ evolution were inferred and, although usually chromosomal, the resistance element was also observed on a plasmid background. There was qualitative evidence for clonal expansions of both vancomycin-resistant and vancomycin-susceptible E. faecium with evidence of hospital-specific subclonal expansion. Our data are consistent with continuing evolution of this established hospital pathogen and confirm hospital vancomycin-susceptible and vancomycin-resistant E. faecium patient transmission events, underlining the need for careful consideration before modifying current E. faecium infection control strategies.

Myelodysplastic Syndrome (MDS)-Determining Clonal Events at Presentation of Aplastic Anemia (AA)

Historically, the evolution rate of aplastic anemia (AA) to MDS approaches 15% in 10 yrs; thus, AA can be considered a major predisposition factor for secondary MDS (sMDS). The likely etiology includes expansion of a preexisting clone or truly late clonogenic events. In both instances, progression can be a result of a clonal escape, but confirmation of the presence of mutant cells at presentation would indicate that initial autoimmune processes may represent a tumor surveillance reaction.We studied 326 patients with AA and 47 patients with paroxysmal nocturnal hemoglobinuria (PNH) and identified 36 cases (progression rate: 11% in median follow up of 6 years) that evolved to MDS or AML (median time to progression: 3.2 yrs.; transplanted patients were not censored). Cytogenetic analysis upon progression showed abnormal karyotype in 83% of cases; 7% had complex karyotype and -7/del(7q) was present in 62% of cases. The presence of a PNH clone was detected in 17% of cases that transformed to sMDS vs. 35% in non-progressors (P =.1). For comparison, we have also analyzed primary de novo cases of MDS (pMDS) with (N=94) and without (N=557) -7/del(7q). In contrast to sMDS, -7/del(7q) was present in 14.4% of cases in pMDS.Because sMDS following AA or PNH included a high proportion of patients with -7/del(7q), we compared sMDS with -7/del(7q) to pMDS with -7/del(7q) for coexisting mutational events. Mutations in RUNX1, CBL, SETBP1 and ASXL1 appeared to be more frequent in sMDS vs. pMDS (28.6% vs. 2.1%, 21.4% vs. 2.1%, 21.4% vs. 5.3%, 21.4% vs. 10.6%, P =.003, P =.02, P =.07, P =.37, respectively). In contrast, TP53 and DMT3A were more common in pMDS (7.1% for sMDS vs. 17%, 0% for sMDS vs. 8.5%, P =.69, P =.59). Similarly, there were several other distinctive differences between all sMDS and pMDS irrespective of the cytogenetics: mutations in SF3B1, SRSF2, NPM1, DNMT3A were common in primary AML but entirely absent from cases after AA; mutations in RUNX1 and SETBP1 appeared to be more frequent in sMDS vs. pMDS (26.3% vs. 8.3%, 21.1% vs. 3.2%, 15.8% vs. 3.9%, P =.03, P =.005, respectively).Whole exome NGS was performed after progression, with confirmed somatic mutations subsequently tracked back by targeted deep NGS applied to serial samples starting at initial presentation. Confirmed mutational events and chromosomal aberrations were found in 19/36 patients with sMDS; 17/19 cases of sMDS had at least 1 confirmed somatic mutation. Remarkably, in retrospective analysis in 6/7 cases studied serially, at least one of the identified mutations was detectable at presentation when deep targeted sequencing (depth 5,000~20,000 reads) was performed. In 5 of these cases the alterations appeared to be ancestral events for sMDS evolution. When anadditional 77 AA or PNH cases were studied by deep sequencing, somatic mutations were present in 48% of AA patients at presentation. Detection of clonal events at presentation was associated with an increased risk of subsequent MDS evolution (14/37 mutant cases vs. 3/40 nonclonal cases evolved, P =.002). Mutations found at both initial presentation and upon evolution were suggestive of a slow expansion of previously cryptic clones (ASXL1, CUX1, TET2, CBL, RUNX1, and SETBP1). Patients with these genes (n=18) had worseoverall survival compared to patients without these mutations (P =.03). To assess the potential impact of immunosuppressive therapies (IST), we also investigated a subset (out of 77) of 53 patients (39 responders and 14 refractory cases) following IST. Clonal somatic events were identified in 27 of them, but there was no association between the response to IST and somatic mutations at presentation.Our results demonstrate that while subclonal mutations indicative of oligoclonal hematopoiesis are frequent in AA, the presence of permissive ancestral somatic events at the outset of AA predisposes patients to sMDS, a feature that had diagnostic and prognostic implications. DisclosuresSekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

Impact of Eltrombopag on Expansion of Clones with Somatic Mutations in Refractory Aplastic Anemia

Despite documented success of immunosuppressive therapy (IST) in the treatment of aplastic anemia (AA), a significant minority of patients remain refractory, most responses are incomplete, and allogeneic stem cell transplantation is not available for older patients or those with significant comorbidities. Until introduction of the cMpl agonist eltrombopag, anabolic steroids were the most commonly used salvage drugs. At least theoretically, engaging growth factor receptors with eltrombopag has the potential to promote the evolution or expansion of mutant clones and thereby increase the rate of progression to secondary MDS, a feared complication of AA occurring in 10-20% of patients. Recently we and others reported detection of clonogenic somatic mutations typical of MDS in patients with AA and PNH. Subsequent study demonstrated that mutations characteristic of sMDS can be found in some patients at presentation of AA and may constitute risk for subsequent progression to MDS. As the risk of MDS evolution was a prominent concern when filgrastim was more widely used in management of AA, now similar questions have been raised regarding use of eltrombopag, be it as salvage therapy or to complement IST.Recently, one of our primary refractory patients receiving eltrombopag progressed to AML. This clinical observation led to investigation of the impact of eltrombopag on evolution and clonal expansion using deep sequencing of a cohort of patients with AA. DNA from bone marrow cells was sequenced before and after initiation of eltrombopag to evaluate clonal expansion or evolution using a targeted multi-amplicon deep sequencing panel of the top 60 most commonly mutated genes in MDS.Among 208 AA patients treated at Cleveland Clinic, we identified 13 patients (median age 68 yrs.) who were treated with eltrombopag for IST-refractory AA; median duration of treatment was 85 wks. The overall response rate, defined as sustained improvement in blood counts and transfusion independence after 12 weeks of therapy, was 46% (6/13), while 38% (5/13) of patients showed stable disease with intermittent transfusions (one of whom underwent HSCT). Among the two non-responders, one patient developed a PNH clone and another progressed to AML (see below). Expansion of PNH granulocytes after eltrombopag treatment was observed in 2 patients. Two patients had chromosomal abnormalities at initial diagnosis, one with t (10; 18) in 2 metaphases, and one with an extranumeral Y chromosome.Use of next generation sequencing (NGS) allows for the quantitative detection of clonal events. We hypothesized that serial analysis by NGS before and after eltrombopag therapy may provide clues as to potential effects of this drug on clonal evolution. Sequencing analysis before eltrombopag treatment revealed the presence of a sole clonal mutational event in 3/13 cases, including CEBPA, EZH2, and BCOR. In the patient with a CEBPA mutation, the mutation persisted during treatment with minimal clonal expansion evidenced by a change in VAF from 53% to 65%. In the second patient, NGS results revealed the initial presence of an EZH2 mutation. A post eltrombopag sample clearly identified acquisition of additional clonal events in genes highly associated with advanced disease and clonal evolution (RUNX1 and U2AF1), as well as slight expansion of a persistent EZH2 clone from 2 to 8%. The third patient harbored a BCOR mutation which expanded markedly, increasing from 8% to 21%, and was accompanied by a hematological response. Sequencing results after eltrombopag treatment revealed the acquisition of new somatic mutations in 5/13 (38%) cases: 2 new CEBPA mutations, 1 new BCOR mutation, and, as discussed, one case with an initial EZH2 mutation in which RUNX1 and U2AF1 mutations were later discovered. In the 5th patient, evolution to AML was observed and accompanied by a large DNMT3A and U2AF1 clone that was absent on initial evaluation.In conclusion, we did observe occasional expansion of clones with potentially leukemogenic mutations during treatment with eltrombopag. At our institution a case control study of patients with refractory aplastic anemia without treatment with eltrombopag is ongoing; ideally a prospective trial would be needed to confirm results. Our results suggest that the initial detection of certain somatic mutations (CBL, SETBP1 and RUNX1) associated with post-AA MDS may contraindicate use of eltrombopag in AA. DisclosuresSekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

Determinants of Phenotypic Commitment and Clonal Progression--Conclusions from the Study of Clonal Architecture in CMML

CMML is heterogeneous clinically (a varying degree of dysplastic or proliferative clinical features) and in terms of its molecular pathogenesis. Analysis of the spectrum of genomic lesions in CMML may contribute to understanding of the pathogenesis and help identify certain mutations as diagnostic biomarkers. Apart of the mere presence of a somatic lesion, phenotypic features may be shaped by initial hits. Conversely sub-clonal events may determine the phenotype or progression. Finally, initial hits may predetermine e.g., mutator phenotype, or differentiation block and therefore selecting for specific sub-clonal hits.We have selected a large CMML cohort to establish generalizable pathogenetic and clinical associations to account for molecular heterogeneity and serial samples have to be analyzed to assess clonal dynamics and hierarchy. The study group consisted of 242 patients, including 150 CMML cases (96 CMML-1, 27 CMML-2, 27 post CMML sAML) and JMML (N =92); 15 patients were studied serially. We also used comparison cohorts of M4/M5 AML (N =64) and advanced (N= 231) and low risk MDS (N= 199) serving as risk adjusted match for CMML subtypes. The CMML entity was further sub-classified based on clinical parameters and pathomorphologic features, 57% dysplastic (MD-CMML) and 43% proliferative form (MP-CMML). Analysis was performed using WES (paired germ line/tumor samples) and multiamplicon NGS targeting top 60 most commonly affected genes. For clonal architecture analysis, cross-sectional variant allelic frequency (VAF) concept based analysis was performed including assessment of affected genes by ranking of the corresponding clonal burden rather than the absolute cellular frequency. The results of this analysis were confirmed in serial samples to identify expanding, declining and stable subclones.Comparison of mutational spectra between the disease entity show profound differences in morphologically similar entities as particularly evident in comparison of CMML to JMML (TET2, ASXL1) or to lesser degree low risk MDS and CMML1 while progression in advanced cases was often associated with similar spectrum of additional subclonal events. However, the differences were more striking when clonal hierarchy was assessed to identify dominant/codominant and subclonal mutational events. We found that top 4 dominant/codominant clonal events, included TET2 (56%), SRSF2 (42%), ASXL1 (46%), DNMT3A (45% of patients), while in MDS corresponding frequency of these dominant events was TET2 (15%) SRSF2 (8%), ASXL1 (11%) and DNMT3A (8%), with most common ancestral events ranked SF3B1, TET2, ASXL1 etc. The clinical importance of these dominant events in CMML is highlighted by their impact on survival in KM analysis (p=.018).Our analysis also demonstrated that for certain founding events not pathognomonic for CMML either codominant or subsequent subclonal events determine the phenotypic features (1st generation) or progression (2nd generation). For instance initial TET2 in CMML was followed often by SRSF2 or in conjunction, RAS pathway mutations while MDS was driven by TET2, SF3B1, TP53, and many other events. Progression in our cohort was driven in both CMML and MDS by ASXL1, RUNX1, NPM1. When other common mutations were categorized by their role in individual patients 27% EZH2, 20% of CBL and 22% of SETBP1 were dominant. Serial analysis further qualified the cross-sectional analysis and allowed for categorization of subclonal events. For instance, CMML-1 cases initially presented with dominant TET2 followed by subtype specific subclonal SRSF2 and progression event IDH2 progressed to sAML with new NPM1 acquisition and expansion of IDH2 c lone. In our serial sample analysis we observed that increasing ASXL1 and RUNX1 clones correspond to clinical progression, ancestral events may remain stable (TET2, SRSF2, SETBP1), while non-permissive subclones can smolder or even decline.In sum, deep NGS allows for identification of specific ancestral events, which may determine the subsequent secondary mutational events in CMML. Classification of CMML based on ancestral events and subclonal events rather than on the global mutational spectrum correlates with clinical features and prognosis and may contribute to further clinical resolution of CMML based on the presence of specific founder mutations ultimately help establish therapeutic interventions. DisclosuresSekeres:Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

Clonal Events in Normal Aging Hematopoiesis

Chronological aging of the hematopoietic compartment is associated with decreased bone marrow cellularity, reduced lymphopoiesis, increased anemia, a myeloid proliferation bias and an increased incidence of myeloid cancers. Beerman et al. proposed that this age-related myeloid lineage favoritism may be explained by clonal expansion of intrinsically myeloid-biased hematopoietic stem cells with robust self-renewal potential(1). This age-associated clonal expansion was initially suspected by X-chromosome inactivation (XCI) studies performed in the normal aging population, which documented a skewed XCI pattern in a significant proportion of women over 60 year-old(2). More recently, genome wide approaches led several groups to document au augmented prevalence of acquired clonal copy number changes (3,4,5) or clonal somatic mutations with increasing age (6,7,8,9). The most frequently mutated genes are the same as those documented in myeloid cancers, such as TET2, DNMT3A, ASXL1, PPM1D, GNAS, TP53, JAK2 and SF3B1 among others. The prevalence of these age-associated mutations may reach > 10% of older individuals, and is associated with an 11-12 fold increased relative risk of developing hematological malignancies. However, the actual problematic is to define the prognostic significance of these clonal mutations in the aging population. Steensma et al. proposed to consider these mutations as «Clonal Hematopoiesis of Indeterminate Potential (CHIP)»(10). The goal of our research group is to define the oncogenic penetrance of CHIP by applying a precision medicine approach in a large prospective cohort (n=4000) of aging individuals comprised of related and unrelated subjects. The variables under investigation include, clonality by XCI in women, deep sequencing (NGS) of myeloid cancer associated genes, epigenetic markers (5hmC, 5mC), telomere length, blood counts, heritability and outcome. PRELIMINARY RESULTS. XCI analyses Acquired skewing of XCI predominantly affects the myeloid lineage with a prevalence of 41.4% for PMN and is age dependent (r=0.15, P<10-4), in contrast to T cells 22.5%. These results support the idea of an age-associated clonal myeloid expansion. NGS of myeloid gene panel. We documented a prevalence of 17.9% of mutated individuals. Mutations were mainly documented in TET2 and DNMT3A which accounted for 90% of all identified mutations. Other significantly mutated genes included JAK2, ASXL1, CBL, TP53 and KRAS. Double mutations were identified in 2.5% of individuals (14% of the mutated individuals) and half of them had concomitant mutation in TET2 and DNMT3A. Age and XCI skewing was similar between subjects with mutation in TET2 or DNMT3A, but slightly higher in double mutants. Epigenetic markers. Subjects with mutation in TET2 had a significant reduction in 5hmC level that correlated with Variable Allele Frequency (VAF) of the mutation. No specific global epigenetic phenotype was documented in the DNMT3A mutation subgroup. We also documented an age-associated reduction in 5hmC that was independent of acquired mutation in the TET2 gene. Taken together these results indicate that age-associated clonal mutations involves predominantly two genes (TET2 and DNMT3A), suggesting that alteration of epigenetic maintenance is a central to the initiation of clonal dominance. Completion of investigation of the aging cohort and prospective follow-up will help characterize the link between aging hematopoiesis and the development of myeloid cancers. 1. Beerman I, Maloney WJ, Weissmann IL, et al. Stem cells and the aging hematopoietic system. Curr Opin Immunol. 2010;22(4):500-506. 2. Busque L, Mio R, Mattioli J, et al. Non-random X-inactivation patterns in normal females: lyonization ratios vary with age. Blood. 1996;88(1):59-65. 3. Forsberg LA, Rasi C, Razzaghian HR, et al. Age-related somatic structural changes in the nuclear genome of human blood cells. AJHG, 2012;90:217-228. 3. Laurie CC, Laurie CA, Rice K, et al. Detectable clonal mosaicism from birth to old age and its relationship to cancer. Nat Genet. 2012;44(6):642-650. 4. Jacobs KB, Yeager M, Zhou W, et al. Detectable clonal mosaicism and its relationship to aging and cancer. Nat Genet. 2012;44(6):651-658. 5. Busque L, Patel JP, Figueroa ME, et al. Recurrent somatic TET2 mutation in normal elderly individuals with clonal hematopoiesis. Nat Genet. 2012;444(11):1179-1181. 6. Xie M, Lu C, Wang J, et al. Age-related mutations associated with clonal hematopoietic expansion and malignancies. Nat Med. 2014;20(12):1472-1478. 7. Genovese G, Kähler AK, Handsaker RE, et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med. 2014;371(26):2477-2487. 8. Jaiswal S, Fontanillas P, Flannick J, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med. 2014;371(26):2488-2498. 9.Steensma DP, Bejar R, Jaiswal S, et al. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood. 2015;126(1):9-16 Disclosures Busque: Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

High Risk Multiple Myeloma Demonstrates Marked Spatial Genomic Heterogeneity Between Focal Lesions and Random Bone Marrow; Implications for Targeted Therapy and Treatment Resistance

High Risk Multiple Myeloma Demonstrates Marked Spatial Genomic Heterogeneity Between Focal Lesions and Random Bone Marrow; Implications for Targeted Therapy and Treatment Resistance

Genomic Disruption of the Histone Methyltransferase SETD2 in Chronic Lymphocytic Leukemia

Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and have been found disrupted at the genomic level in a spectrum of human tumors including hematological malignancies. In CLL, recurring genomic lesions targeting chromatin modifiers are emerging in the literature (Puente 2015, Nature) but their biological and clinical significance remain uncertain. We studied 802 CLL patients, divided into a discovery [n=261, sampled pre-treatment] and two validation cohorts [n=541, 431 pre-treatment and 110 ultra-high risk], using high-resolution SNP-arrays [n=572, Affymetrix SNP6.0 and HumanOmniS-8] and high-throughput re-sequencing [n=320, Haloplex and TruSeq] to identify genomic lesions targeting HMT genes. In our discovery cohort, we identified nine novel regions of copy-number changes; the prime finding was a recurrent deletion of chromosome 3p in 4% of patients between genomic positions 47.12-47.36Mb. This region included SETD2, KIF9 and KLHL18 (Fig 1A), with SETD2 being the most significantly under-expressed gene (p=0.001) in 3p-deleted [n=6] versus non-deleted cases [n=8]. Further validation in two independent cohorts showed that SETD2 deletions were enriched in ultra high-risk CLL and associated with loss of TP53 (p=0.003), genomic complexity (in TP53 wild-type cases, p=0.01) and chromothripsis.Next, we screened for somatic mutations in SETD2 using targeted re-sequencing and identified non-synonymous mutations in four (4.5%) discovery cases (p.D99G, p.W1306X, p.Q1545K, p.E1955Q), and 7/231 (3%) in the pre-treatment validation cases (p.A50T, p.P167L, p.E670K, p.M1742L, p.M1889T (x2), p.I2295M) (Fig 1B). Mutations were somatic in all samples tested [n=5]. To study the clonal nature of the SETD2 deletions, we assigned each genomic CNA with a relative copy-number by normalizing CNA intensity values from array features, and could infer that the 3p deletion was in the dominant clonal population in 14/21 cases with data available for analysis. Employing either the ABSOLUTE algorithm for our discovery cohort or manually correcting for tumor sample purity and local copy-number changes in our validation cohort, we observed that 10/11 SETD2 mutations exhibited a clonal cell fraction. These data strongly imply that SETD2 aberrations represent early clonal events in the pathobiology of CLL. Next, we extended our gene expression analysis to include additional wild-type, deleted, and mutated patients, showing reduced expression both in deleted and mutant cases (p=0.035), thus confirming haplo-insufficiency also for the SETD2-mutant cases. W e reviewed the DNA methylation status of the SETD2 gene body and promoter regions (15 & 9 CpG probes, respectively) from published data, and found no correlation between SETD2 methylation and RNA expression, suggesting that DNA methylation does not play a substantial role in regulating SETD2 expression in CLL.Finally, we analyzed the impact of SETD2 lesions on treatment-free survival (TFS) and overall survival (OS). For TFS and OS, we observed a significantly worse outcome (TFS: 44 vs. 105 months; p=0.004, OS: 85 vs. 199 months; p=0.002) in SETD2 deleted cases that were wild-type for TP53/ATM, compared to cases wild type for TP53/ATM/SETD2 (Fig 1D). Mutant SETD2 cases (wild-type for TP53/ATM) and those wild-type for TP53/ATM/SETD2 exhibited median TFS of 74 and 106 months respectively, differences that did not reach significance (p=0.1). Whilst these data suggest that SETD2 lesions may be clinically relevant, further investigations in larger materials are warranted to understand their full impact on survival.In conclusion, we report for the first time somatic deletions and mutations in SETD2, a gene found disrupted invarious human solid and hematological tumors, in ~7% of CLL patients requiring treatment. These are likely to be early clonal events and associate with TP53 dysfunction, genomic complexity and chromothripsis, with deletions enriched in ultra high-risk CLL. [Display omitted] DisclosuresTausch:Gilead: Other: Travel support. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015. Hillmen:Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Dyer:Gilead: Research Funding; Roche Pharmaceuticals: Speakers Bureau; ONO Pharmaceuticals: Research Funding. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Schuh:Acerta Pharma BV: Research Funding. Strefford:Roche: Research Funding.

Molecular Alterations in Refractory Acute Myeloid Leukemia and Persistent Preleukemic Lesions Are Disclosed Using Mesenchymal Stromal Cells As Germline Control

Introduction:Over the last years, genome and exome sequencing approaches have increased our knowledge of molecular alterations in acute myeloid leukemia (AML). However, some important limitations still need to be addressed. First, insights into the spectrum of molecular alterations of patients with refractory AML are rare, partly due to the lack of remission samples as germline control. As these patients have a dismal prognosis, there remains an unmet need to improve therapeutic options and to identify druggable molecular lesions. Secondly, in AML patients achieving a complete remission (CR), preleukemic alterations may persist in CR and are underestimated in frequency and relevance. In this work, we investigated mesenchymal stromal cells (MSC) as germline control to decipher the spectrum of molecular alterations in refractory patients with induction failure and to disclose preleukemic hits in patients achieving CR.Patients and methods:Bone marrow (BM) aspirates at initial diagnosis (ID) were obtained from 18 AML patients (9 pts with subsequent induction failure and 9 pts that achieved CR after first induction). MSC were expanded to passage 4 and defined as CD73+/CD105+/CD271+/low/CD45-/CD33- plastic-adherent cells. For all patients, BM hematopoietic cells (BM-HC; n=18) as well as MSC (n=18) were analysed at the time of first diagnosis. All samples (n=45) were analysed by exome sequencing on a HiSeq2500 (100 bp paired end) with four samples per lane. For variant calling, MSC were used as germline control. We demanded a variant allele frequency (VAF) of >20%, coverage of >30 reads and translational consequences. In germline samples, the VAF had to be < 5%. For patients achieving CR, BM-HC at CR were also studied (n=9). We repeated the analysis with CR BM-HC as germline control and compared the two results. For all patients, clinical as well as molecular characteristics were available.Results:We obtained an average coverage of 96 reads per base for the protein coding regions. 96% of the target region was covered at least 10-fold.The use of MSC as germline control allowed us to detect somatic mutations at initial diagnosis of refractory AML. In 9 refractory AML samples, we found 90 single nucleotide variants (SNV) and indels, which resulted in a median of 11 alterations per sample (range: 3-17). The spectrum of mutations showed an unexpectedly high rate of mutations in the spliceosome gene SRSF2 (3/9). Other recurrent mutations affected TET2 (2/9) and WT1 (2/9). Genes frequently mutated in non-selected AML were only present in one refractory patient (DNMT3A, RUNX1, IDH2, ASXL1, TP53, NRAS) or not found mutated (IDH1, KRAS).To uncover preleukemic alterations in AML patients achieving CR (n=9), we compared MSC and BM-HC at CR as germline controls. Using MSC as germline, we called 97 SNVs and indels (median: 11 per sample; range: 4-18) in the leukemic cells at ID. Thirty-three additional SNVs were called in the leukemic BM by using MSC as germline, whereas these would have been missed using BM-HC at CR as germline (median: 3 SNVs per sample, range: 0-7). These represent preleukemic hits persistent in CR with a VAF between 5% (lower bound) and 75%. Recurrently mutated genes included genes recently associated with clonal haematopoiesis in the elderly population: DNMT3A (3/9; VAF: 18%, 24%, 75%) and TET2 (2/9; VAF: 13%, 23%). In addition, mutations in ASXL1 (VAF: 14%), SRSF2 (VAF: 15%), and RUNX1 (VAF: 5%) persisted in at least one patient in CR. This unbiased approach also allowed us to identify lesions, which have not yet been associated with AML, but account for clonal events in remission. Candidates included genes linked to cancer like PROX1 (VAF: 5%), or ERBB2 (VAF: 35%), but also genes involved in NF-kB activation such as CARD8 (VAF: 30%), or NLRC3 (VAF: 10%).Conclusion:The use of MSC allows to unravel molecular lesions in refractory AML by exome sequencing. Refractory AML patients showed a high rate of mutations in the spliceosome gene SRSF2 that needs further investigations as potential therapeutic target for patients with treatment failure. Moreover, the comparison of two different germline controls (MSC and BM-HC in CR) allowed detecting persistent preleukemic alterations. In addition to known hits like in DNMT3A, TET2, or ASXL1, we systematically identified a broader spectrum of premalignant events that indicate clonal hematopoietic expansion and thereby may provide insights into leukemic transformation. DisclosuresNo relevant conflicts of interest to declare.

Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts.

SummaryThe derivation of genetically modified induced pluripotent stem (iPS) cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes.

Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing.

BackgroundThe transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening.MethodsWe developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS.ResultsWe identified 1652 mutant sequences from 1006 independent mutations that underwent clonal expansion. This deep sequencing analysis of mutation spectrum demonstrated that BaP causes primarily guanine transversions (e.g. G:C → T:A), which is highly consistent with previous studies employing Sanger sequencing. Furthermore, we identified novel mutational hotspots in the lacZ transgene that were previously uncharacterized by Sanger sequencing. Deep sequencing also allowed for an unprecedented ability to correct for clonal expansion events, improving the sensitivity of the mutation reporter assay by 50 %.ConclusionThese results demonstrate that the high-throughput nature and reduced costs offered by NGS provide a sensitive and fast approach for elucidating and comparing mutagenic mechanisms of various agents among tissues and enabling improved evaluation of genotoxins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2004-4) contains supplementary material, which is available to authorized users.

Targeted sequencing reveals clonal genetic changes in the progression of early lung neoplasms and paired circulating DNA.

Lungs resected for adenocarcinomas often harbour minute discrete foci of cytologically atypical pneumocyte proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and fully invasive adenocarcinoma. Despite efforts to identify predictive markers of malignant transformation, alterations driving this progression are poorly understood. Here we perform targeted next-generation sequencing on multifocal AAHs and different zones of histologic progression within AISs and MIAs. Multiregion sequencing demonstrated different genetic drivers within the same tumour and reveal that clonal expansion is an early event of tumorigenesis. We find that KRAS, TP53 and EGFR mutations are indicators of malignant transition. Utilizing droplet digital PCR, we find alterations associated with early neoplasms in paired circulating DNA. This study provides insight into the heterogeneity of clonal events in the progression of early lung neoplasia and demonstrates that these events can be detected even before neoplasms have invaded and acquired malignant potential.

Genetics and Prognostication in Splenic Marginal Zone Lymphoma: Revelations from Deep Sequencing.

Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies. We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL. We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02-4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05-4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08-5.2; P = 0.03). We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.

Noncatalytic PTEN missense mutation predisposes to organ-selective cancer development in vivo.

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.