Abstract RNA-seq is a powerful tool to detect tissue-specific gene expression, splicing, and genetic variations associated with disease states. However, current RNA-seq approaches have limitations due to poor signal from low-abundant transcripts. Furthermore, tissue-derived RNA samples are often highly degraded, thereby limiting gene detection and suffering from potential sequencing artifacts. Here we show that target capture of sequencing libraries tagged with unique molecular identifiers (UMIs) using the xGen™ Prism DNA library prep kit, optimized for low-input and degraded samples, can overcome these obstacles. Directional and UMI-tagged RNA-seq libraries were constructed with RNA extracted from a FFPE RNA Fusion Reference Standard and captured with different designs of the xGen Pan-Cancer Panel spiked with probes for fusion genes. The first design strategy used IDT's stocked Pan Cancer V1.5 panel targeted to gDNA coordinates. The second design involved extracting all associated RefSeq NM transcripts associated with the gene list. Probes were designed to each transcript and duplicate probes were removed based on exact sequence match. The third strategy leveraged a multi-strain design, which created probes from fasta inputs, but removed probes with 90% or greater homology. Normalized expression was highly correlated (> 85%) between captured and uncaptured samples regardless of rRNA depletion prior to library prep. Captured samples had a greater depth of coverage with over 90% on target bases. In addition, our panel design strategies identified low frequency fusions with deep sequencing regardless of rRNA depletion prior to library prep. The multi-strain design was more effective in reducing redundant capture probes compared the other design strategies. Enhanced coverage and PCR de-duplication with UMIs allowed us to reproducibly measure expression over a wide range of RNA inputs (5-500 ng). We show that target capture of RNA-seq libraries reliably maintains expression information present in uncaptured libraries while increasing coverage for poorly expressed genes and low frequency fusions. In addition, the target captured libraries without rRNA depletion prior to library prep have comparable on-target rate and target coverage with rRNA-depleted, target captured libraries. The addition of UMIs to differentiate between PCR duplicates and unique starting molecules also makes it possible to reliably analyze even highly amplified libraries. (For research use only). Citation Format: Tzu-Chun Chen, Katelyn Larkin, Shale Dames, Hsiao-Yun Huang, Kevin Lai, Jessica Sheu, Timothy Barnes, Katia Star, Manqing Hong, Bosun Min, Ryan Demeter, Ashley Dvorak, Ushati Das Chakravarty, Patrick Lau, Steven Henck. High conversion library preparation with optimal hybridization capture panel design strategy in RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 327.

The impact of intratumoral heterogeneity (ITH) and the resultant neoantigen landscape on T cell immunity are poorly understood. ITH is a widely recognized feature of solid tumors and poses distinct challenges related to the development of effective therapeutic strategies, including cancer neoantigen vaccines. Here, we performed deep targeted DNA sequencing of multiple metastases from melanoma patients and observed ubiquitous sharing of clonal and subclonal single nucleotide variants (SNVs) encoding putative HLA class I-restricted neoantigen epitopes. However, spontaneous antitumor CD8+ T cell immunity in peripheral blood and tumors was restricted to a few clonal neoantigens featuring an oligo-/monoclonal T cell-receptor (TCR) repertoire. Moreover, in various tumors of the 4 patients examined, no neoantigen-specific TCR clonotypes were identified despite clonal neoantigen expression. Mature dendritic cell (mDC) vaccination with tumor-encoded amino acid-substituted (AAS) peptides revealed diverse neoantigen-specific CD8+ T responses, each composed of multiple TCR clonotypes. Isolation of T cell clones by limiting dilution from tumor-infiltrating lymphocytes (TILs) permitted functional validation regarding neoantigen specificity. Gene transfer of TCRαβ heterodimers specific for clonal neoantigens confirmed correct TCR clonotype assignments based on high-throughput TCRBV CDR3 sequencing. Our findings implicate immunological ignorance of clonal neoantigens as the basis for ineffective T cell immunity to melanoma and support the concept that therapeutic vaccination, as an adjunct to checkpoint inhibitor treatment, is required to increase the breadth and diversity of neoantigen-specific CD8+ T cells.

BackgroundMetastasis is the main cause of cancer patient deaths and remains a poorly characterized process. It is still unclear when in tumor progression the ability to metastasize arises and whether this ability is inherent to the primary tumor or is acquired well after primary tumor formation. Next-generation sequencing and analytical methods to define clonal heterogeneity provide a means for identifying genetic events and the temporal relationships between these events in the primary and metastatic tumors within an individual.Methods and FindingsWe performed DNA whole genome and mRNA sequencing on two primary tumors, each with either four or five distinct tissue site-specific metastases, from two individuals with triple- negative/basal-like breast cancers. As evidenced by their case histories, each patient had an aggressive disease course with abbreviated survival. In each patient, the overall gene expression signatures, DNA copy number patterns, and somatic mutation patterns were highly similar across each primary tumor and its associated metastases. Almost every mutation found in the primary was found in a metastasis (for the two patients, 52/54 and 75/75). Many of these mutations were found in every tumor (11/54 and 65/75, respectively). In addition, each metastasis had fewer metastatic-specific events and shared at least 50% of its somatic mutation repertoire with the primary tumor, and all samples from each patient grouped together by gene expression clustering analysis. TP53 was the only mutated gene in common between both patients and was present in every tumor in this study. Strikingly, each metastasis resulted from multiclonal seeding instead of from a single cell of origin, and few of the new mutations, present only in the metastases, were expressed in mRNAs. Because of the clinical differences between these two patients and the small sample size of our study, the generalizability of these findings will need to be further examined in larger cohorts of patients.ConclusionsOur findings suggest that multiclonal seeding may be common amongst basal-like breast cancers. In these two patients, mutations and DNA copy number changes in the primary tumors appear to have had a biologic impact on metastatic potential, whereas mutations arising in the metastases were much more likely to be passengers.

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader. © 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

There is interest in using leukemia-gene panels and next-generation sequencing to assess acute myelogenous leukemia (AML) response to induction chemotherapy. Studies have shown that patients with AML in morphologic remission may continue to have clonal hematopoiesis with populations closely related to the founding AML clone and that this confers an increased risk of relapse. However, it remains unknown how induction chemotherapy influences the clonal evolution of a patient’s nonleukemic hematopoietic population. Here, we report that 5 of 15 patients with genetic clearance of theirfoundingAMLcloneafterinductionchemotherapyhadaconcomitantexpansionofa hematopoieticpopulationunrelatedtotheinitialAML.Thesepopulationsfrequentlyharboredsomaticmutationsingenesrecurrently mutated in AML or myelodysplastic syndromes and were detectable at very low frequencies at the time of AML diagnosis. These resultssuggestthatnonleukemichematopoietic stemandprogenitor cells,harboringspecificaging-acquiredmutations, mayhavea competitive fitness advantage after induction chemotherapy, expand, and persist long after the completion of chemotherapy. Although the clinical importance of these “rising” clones remains to be determined, it will be important to distinguish them from leukemia-related populations when assessing for molecular responses to induction chemotherapy. ( Blood . 2016;127(7):893-897)

We report the findings from a patient who presented with a concurrent mediastinal germ cell tumor (GCT) and acute myeloid leukemia (AML). Bone marrow pathology was consistent with a diagnosis of acute megakaryoblastic leukemia (AML M7), and biopsy of an anterior mediastinal mass was consistent with a nonseminomatous GCT. Prior studies have described associations between hematological malignancies, including AML M7 and nonseminomatous GCTs, and it was recently suggested that a common founding clone initiated both cancers. We performed enhanced exome sequencing on the GCT and the AML M7 from our patient to define the clonal relationship between the two cancers. We found that both samples contained somatic mutations in PTEN (C136R missense) and TP53 (R213 frameshift). The mutations in PTEN and TP53 were present at ∼100% variant allele frequency (VAF) in both tumors. In addition, we detected and validated five other shared somatic mutations. The copy-number analysis of the AML exome data revealed an amplification of Chromosome 12p. We also identified a heterozygous germline variant in FANCA (S858R), which is known to be associated with Fanconi anemia but is of uncertain significance here. In summary, our data not only support a common founding clone for these cancers but also suggest that a specific set of distinct genomic alterations (in PTEN and TP53) underlies the rare association between GCT and AML. This association is likely linked to the treatment resistance and extremely poor outcome of these patients. We cannot resolve the clonal evolution of these tumors given limitations of our data.

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1-RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader.

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome, an acquired clonal abnormality resulting from translocation of chromosomes 9 and 22, and the generation of the BCR-ABL fusion oncogene.The development of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of CML, as TKI therapy leads to inhibition of BCR-ABL activity, suppression of the BCR-ABL-containing clone and restoration of normal hematopoiesis in the vast majority of cases.Although TKI therapy is remarkably effective in clearing the BCR-ABL-containing clone in CML, it has been associated with the development of non-BCR-ABL rearranged (Ph -) clonal cytogenetic abnormalities (CCA). 1-4CCA have been observed in 3-10% of cases after complete cytogenetic resolution of the BCR-ABL fusion oncogene, occurring 6-18 months after initiation of TKI therapy. 2,3hese abnormalities-suggestive of clonal hematopoiesis-are often transient and not associated with CML progression.However, as routine cytogenetic monitoring during TKI therapy is rarely performed, the occurrence of CCA may be underestimated. 2,4evelopment of Ph -MDS/AML (myelodysplastic syndrome/ acute myeloid leukemia) following TKI therapy has been reported to occur infrequently and, in contrast to Ph -CCA, is associated with poor outcomes. 1,2,5,6Analysis of two CML patient cohorts treated with TKI therapy reported 2/985 and 3/1701 patients subsequently developed MDS/AML. 1,5Both studies were published with relatively short follow-up, raising the possibility that they may also underestimate the prevalence of Ph -AML.The relationship between CML and other clonal abnormalities that arise after TKI therapy is unclear, but they have been theorized to result from the unmasking of a premalignant clone that existed before acquisition of BCR-ABL. 2,3,5,7This scenario raises the possibility of a 'multi-hit' mechanism of CML development in which an early mutation(s) occurs and predisposes the premalignant clone to acquisition (or tolerance) of BCR-ABL.A high frequency of somatic mutations was reported in genes associated with myeloid malignancies in Ph -cells of CML patients treated with TKI. 7 Reports published prior to the TKI era suggested that multiple events are required for CML pathogenesis (postulated by Phil Fialkow and as reviewed by Deininger et al.). 4 In this scenario, one could hypothesize that the early mutation(s) that cooperates with BCR-ABL could also cooperate with mutations that lead to AML.Thus, a premalignant clone could increase susceptibility to both leukemias.Alternatively, it is possible that some CML patients harbor an abnormal bone marrow stroma that predisposes them to both the acquisition of BCR-ABL and other genetic aberrations (that could lead to MDS or AML), which could arise in distinct founding clones.Here we report two patients with CML treated with TKIs who achieved complete molecular remission but subsequently developed Ph -AML (Case Synopses).Both patients achieved durable complete molecular remissions before being referred to our center with AML.Consistent with the multi-hit model of leukemogenesis, we hypothesized that both malignancies were

Tumors are typically sequenced to depths of 75-100× (exome) or 30-50× (whole genome). We demonstrate that current sequencing paradigms are inadequate for tumors that are impure, aneuploid or clonally heterogeneous. To reassess optimal sequencing strategies, we performed ultra-deep (up to ~312×) whole genome sequencing (WGS) and exome capture (up to ~433×) of a primary acute myeloid leukemia, its subsequent relapse, and a matched normal skin sample. We tested multiple alignment and variant calling algorithms and validated ~200,000 putative SNVs by sequencing them to depths of ~1,000×. Additional targeted sequencing provided over 10,000× coverage and ddPCR assays provided up to ~250,000× sampling of selected sites. We evaluated the effects of different library generation approaches, depth of sequencing, and analysis strategies on the ability to effectively characterize a complex tumor. This dataset, representing the most comprehensively sequenced tumor described to date, will serve as an invaluable community resource (dbGaP accession id phs000159).

The DNA methyltransferases DNMT3A and DNMT3B are primarily responsible for de novo methylation of specific cytosine residues in CpG dinucleotides during mammalian development. While loss-of-function mutations in DNMT3A are highly recurrent in acute myeloid leukemia (AML), DNMT3A mutations are almost never found in AML patients with translocations that create oncogenic fusion genes such as PML-RARA, RUNX1-RUNX1T1, and MLL-AF9. Here, we explored how DNMT3A is involved in the function of these fusion genes. We used retroviral vectors to express PML-RARA, RUNX1-RUNX1T1, or MLL-AF9 in bone marrow cells derived from WT or DNMT3A-deficient mice. Additionally, we examined the phenotypes of hematopoietic cells from Ctsg-PML-RARA mice, which express PML-RARA in early hematopoietic progenitors and myeloid precursors, with or without DNMT3A. We determined that the methyltransferase activity of DNMT3A, but not DNMT3B, is required for aberrant PML-RARA-driven self-renewal ex vivo and that DNMT3A is dispensable for RUNX1-RUNX1T1- and MLL-AF9-driven self-renewal. Furthermore, both the PML-RARA-driven competitive transplantation advantage and development of acute promyelocytic leukemia (APL) required DNMT3A. Together, these findings suggest that PML-RARA requires DNMT3A to initiate APL in mice.

Abstract Most tumors are heterogeneous, and the presence of distinct subclonal populations has been shown to be common in every type of cancer assayed to date. Even very low-frequency populations may harbor mutations that offer a selective advantage during therapy, a fact that carries profound clinical implications. Despite their importance, the landscape of low-frequency clonal SNVs remains largely unexplored, due to standard sequencing assays that only achieve mean coverages of 75-100x in exomes, or 30-50x in whole genomes. While these approaches are adequate for characterizing samples with high cellularity, normal cytogenetics, and homogeneous tumor cell populations, most tumors are impure, aneuploid, and heterogeneous to various degrees. To reassess optimal tumor sequencing and analysis strategies, we performed ultra-deep sequencing of an acute myeloid leukemia (AML) with complex sub-clonal architecture, and known canonical driver mutations in DNMT3A, NPM1, FLT3, and IDH1. This patient's tumor, matched normal, and post-chemotherapy relapse sample were whole-genome sequenced to ~350x coverage, whole exome sequenced to ~300X coverage, and a panel of 260 recurrently mutated AML genes were sequenced to ~10,000X coverage. We then tested 10 alignment algorithms and 7 state-of-the-art somatic single-nucleotide variant callers on these data. To validate the calls, 200,000 putative SNVs were assayed on a custom targeted capture array to mean depths of ~1000x. This exceptionally comprehensive data set allowed the investigation of the use of multiple sequencing libraries of varying fragment size distributions, the effect of alignment algorithm choice on variant calling, optimal strategies for the combination of multiple variant callers and the effect of increasing sequence depth on subclonal definition. This deep sequencing and validation resulted in a many-fold increase in variant calls, and led to the discovery of additional known or suspected driver mutations in FLT3, IDH2, TP53, RUNX1, FOXP1 and KRT1. We also observed an 80-fold increase in the number of single-nucleotide variants detected at a VAF of less than 10%, including many at a frequency of 1% or lower. We applied the sciClone algorithm to trace the clonal evolution of this tumor through treatment and relapse, showing that many variants previously thought to be relapse-specific were in fact present in the tumor at very low levels. One of these subclones, containing a mutation in IDH2, became heavily enriched after the therapy-induced bottleneck and grew into the dominant clone in the relapse tumor. We demonstrate that the widely used practices of obtaining ~100X exome sequencing or 30-50X whole genome sequencing are inadequate to characterize tumors with even moderate heterogeneity, impurity, contamination, aneuploidy, or combinations of these. Similarly, current analysis strategies relying on a single alignment algorithm and variant caller, using data from a single sequence library, suffer from poor sensitivity and specificity. This dataset, generated from the most deeply sequenced and cross-validated tumor described to date, will serve as an invaluable community resource for benchmarking algorithms by providing an extremely high confidence set of low frequency mutations. Citation Format: Christopher A. Miller, Malachi Griffith, Avinash Ramu, Zachery L. Skidmore, Obi L. Griffith, Vincent Magrini, Ryan Demeter, Ha Dang, Jason Walker, David E. Larson, Robert S. Fulton, Christopher Maher, Elaine R. Mardis, Timothy J. Ley, Richard K. Wilson. Ultra-deep whole-genome sequencing reveals clinically relevant low-frequency subclones in an acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-13.

Abstract Acute myeloid leukemia is heterogeneous with respect to clinical outcome and molecular pathogenesis. Approximately 20% of AML cases are refractory to induction chemotherapy, and about 50% of patients ultimately relapse within a time interval that ranges from months to years. At the molecular level, diverse chromosomal abnormalities and genetic mutations have been observed across patients1. Although several clinical factors (age, white blood cell count), cytogenetic aberrations (t[15;17] translocation, loss of chromosome 5) 2-4, and genetic mutations (DNMT3A, FLT3) have been associated with differences in survival 5,6, these factors are of limited prognostic utility. Moreover, few studies have integrated sequence data with clinical and cytogentic factors to build predictive models of patient outcome. Here, we sought to identify genomic predictors of refractory disease or early relapse. We used whole genome and exome sequencing to analyze the genomes of 71 adult de novo AML patients treated with anthracycline and cytarabine-based induction chemotherapy. Of these, 34 had refractory disease or relapsed within 6 months, 12 relapsed in 6-12 months, and 25 had a long first remission (>12 months). We also developed an enhanced exome sequencing (EES) approach to identify and follow leukemia-associated variants over time. In 12 additional patients that achieved morphologic remission after induction chemotherapy, we used EES to identify and track variants at time of diagnosis, time of morphologic remission (roughly 30 days later), and a final time point corresponding to eventual relapse (n=8) or extended remission (n=4). No novel coding or non-coding variants present at the time of diagnosis were found to be predictive of refractory disease or early relapse. Using EES, however, we were able to detect leukemia-associated variants in the initial remission bone marrow in all eight patients who eventually relapsed. One persistent leukemia-associated variant was also detected in one patient still in remission, but all other variants in that patient were eliminated. We also detected 64 somatic variants that became enriched following chemotherapy, but were not detected in the original leukemic cells. These may represent relapse-specific variants or oligoclonal hematopoiesis after bone marrow recovery. Overall, our data suggest that the persistence of leukemia-associated variants after bone marrow recovery from cytotoxic therapy is strongly correlated with relapse, and may be used to complement more traditional, morphologic measures of leukemic cell clearance. 1. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England Journal of Medicine 2013;368:2059-74. 2. Byrd JC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325-36. 3. Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116:354-65. 4. Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. The New England Journal of Medicine 2008;358:1909-18. 5. Kihara R, Nagata Y, Kiyoi H, et al. Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients. Leukemia 2014;28:1586-95. 6. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. The New England Journal of Medicine 2010;363:2424-33. This abstract is also presented as a poster at the Translation of the Cancer Genome conference. Citation Format: Jeffery M. Klco, Christopher A. Miller, Malachi Griffith, Allegra Petti, David H. Spencer, Shamika Ketkar-Kulkarni, Lukas D. Wartman, Matthew Christopher, Tamara L. Lamprecht, Jacqueline E. Payton, Jack Baty, Sharon E. Heath, Obi L. Griffith, Dong Shen, Jasreet Hundal, Gue Su Chang, Robert S. Fulton, Michelle O'laughlin, Catrina Fronick, Vincent Magrini, Ryan Demeter, David E. Larson, Shashikant Kulkarni, Bradley A. Ozenberger, John S. Welch, Matthew J. Walker, Timothy A. Graubert, Peter Westervelt, Jerald P. Radich, Daniel C. Link, Elaine R. Mardis, John F. DiPersio, Richard K. Wilson. Genomic approaches for risk assessment in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR03.

Our group (Welch, Cell 2012) previously showed that hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations with age. This produces a genetically heterogeneous HSPC population with each HSPC possessing its own unique set of mutations. Later work from our group (Xie, Nature Medicine 2014) and others (Genovese, Jaiswal, NEJM 2014) demonstrated that some these mutations may provide HSPCs with a fitness advantage, allowing them to clonally expand over time in healthy individuals. We recently published data (Wong, Nature 2015) suggesting that cytotoxic therapy can select for HSPC clones with TP53 mutations, resulting in their clonal expansion and contributing to the subsequent development of therapy-related AML/MDS. From these data, we hypothesized that the intensive cytoreductive chemotherapy used to treat AML poses a significant selection pressure on a patient's non-malignant HSPC population, favoring HSPCs with specific somatic mutations and potentially resulting in oligoclonal hematopoiesis even after elimination of the founding AML clone.

IMPORTANCE Tests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML. OBJECTIVES To determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML. DESIGN, SETTING, AND PARTICIPANTS Whole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases. EXPOSURES Whole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data. MAIN OUTCOMES AND MEASURES Mutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival. RESULTS Analysis of comprehensive genomic data from the 71 patients did not improve outcome assessment over current standard-of-care metrics. In an analysis of 50 patients with both presentation and documented remission samples, 24 (48%) had persistent leukemia-associated mutations in at least 5% of bone marrow cells at remission. The 24 with persistent mutations had significantly reduced event-free and overall survival vs the 26 who cleared all mutations. Patients with intermediate cytogenetic risk profiles had similar findings. [table: see text]. CONCLUSIONS AND RELEVANCE The detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.

The relationships between clonal architecture and functional heterogeneity in acute myeloid leukemia (AML) samples are not yet clear. We used targeted sequencing to track AML subclones identified by whole-genome sequencing using a variety of experimental approaches. We found that virtually all AML subclones trafficked from the marrow to the peripheral blood, but some were enriched in specific cell populations. Subclones showed variable engraftment potential in immunodeficient mice. Xenografts were predominantly comprised of a single genetically defined subclone, but there was no predictable relationship between the engrafting subclone and the evolutionary hierarchy of the leukemia. These data demonstrate the importance of integrating genetic and functional data in studies of primary cancer samples, both in xenograft models and in patients.

The use of massively parallel sequencing for studying RNA expression has greatly enhanced our understanding of the transcriptome through the myriad ways these data can be characterized. In particular, clinical samples provide important insights about RNA expression in health and disease, yet these studies can be complicated by RNA degradation that results from the use of formalin as a clinical preservative and by the limited amounts of RNA often available from these precious samples. In this study we describe the combined use of RNA sequencing with an exome capture selection step to enhance the yield of on-exon sequencing read data when compared with RNA sequencing alone. In particular, the exome capture step preserves the dynamic range of expression, permitting differential comparisons and validation of expressed mutations from Limited and FFPE preserved samples, while reducing the data generation requirement. We conclude that cDNA hybrid capture has the potential to significantly improve transcriptome analysis from low-yield FFPE material.

Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions—the population frequency of individual clones, their genetic composition, and their evolutionary relationships—which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.

Acute promyelocytic leukemia (APL) is an AML subtype that is characterized by aberrant expansion of immature myeloid progenitors and precursors that are arrested at the promyelocyte stage. Almost all APL cases are characterized by the t(15;17)(q22;q11.2) translocation that creates the PML-RARA fusion oncogene. Human APL cells are known to have a canonical expression signature and a specific methylation phenotype that is unique to this form of AML. Our laboratory previously created a mouse model of APL by expressing a human PML-RARA cDNA from the mouse Cathepsin G (Ctsg) locus (Ctsg-PML-RARA), which activates human PML- RARA expression in early myeloid progenitor cells, with peak expression in promyelocytes. After a long latent period (6-12 months), ~60% of these mice develop a clonal, APL-like myeloid malignancy. The long latent period is probably due to the requirement for cooperating mutations that synergize with PML-RARA to accelerate the disease.

Material Supplemental http://genome.cshlp.org/content/suppl/2013/01/02/gr.142604.112.DC1.html References http://genome.cshlp.org/content/23/3/431.full.html#ref-list-1 This article cites 66 articles, 18 of which can be accessed free at: License Commons Creative . http://creativecommons.org/licenses/by-nc/3.0/ described at a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as ). After six months, it is available under http://genome.cshlp.org/site/misc/terms.xhtml first six months after the full-issue publication date (see This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the