Alterations In Human Cancers Research Articles

Targeted therapy in cancer.

To describe the emergence of targeted therapies that have led to significant breakthroughs in cancer therapy and completed or ongoing clinical trials of novel agents for the treatment of patients with advanced cancer. The literature was systematically reviewed, based on clinical experience and the use of technologies that improved our understanding of carcinogenesis. Genomics and model systems have enabled the validation of novel therapeutic strategies. Tumor molecular profiling has enabled the reclassification of cancer and elucidated some mechanisms of disease progression or resistance to treatment, the heterogeneity between primary and metastatic tumors, and the dynamic changes of tumor molecular profiling over time. Despite the notable technologic advances, there is a gap between the plethora of preclinical data and the lack of effective therapies, which is attributed to suboptimal drug development for "driver" alterations of human cancer, the high cost of clinical trials and available drugs, and limited access of patients to clinical trials. Bioinformatic analyses of complex data to characterize tumor biology, function, and the dynamic tumor changes in time and space may improve cancer diagnosis. The application of discoveries in cancer biology in clinic holds the promise to improve the clinical outcomes in a large scale of patients with cancer. Increased harmonization between discoveries, policies, and practices will expedite the development of anticancer drugs and will accelerate the implementation of precision medicine. Combinations of targeted, immunomodulating, antiangiogenic, or chemotherapeutic agents are in clinical development. Innovative adaptive study design is used to expedite effective drug development.

Abstract 669: BGB-283 effectively enhances MEK inhibitor induced tumor suppression in RAS mutant cancers

Abstract Mutation in K-RAS, which drives constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway, is one of the most frequent genetic alterations in human cancers. Pharmacological targeting RAS mutants directly is extremely challenging and targeted inhibition of K-RAS downstream effector MEK has shown limited clinical activity in suppressing the progression of K-RAS mutant tumors. The reduced sensitivity of K-RAS-mutated cancer cells to MEK inhibitors (MEKi) is associated with feedback phosphorylation of MEK and rebound of phosphorylated ERK levels after prolonged treatment. A synthetic lethal screen provided the rational of targeting both RAF1 and MEK in K-RAS mutated cancers. In this study, we investigated whether combining BGB-283, a potent inhibitor to RAF dimers, and a series of MEKi could achieve better therapeutic effect of the latter. We confirmed that selumetinib (AZD6244) induced strong MEK phosphorylation in K-RAS mutated cancer cells. Either knockdown of B-RAF/C-RAF or addition of BGB-283 in the presence of selumetinib potently inhibited RAF-dependent MEK phosphorylation and led to sustained inhibition of ERK activity. Furthermore, BGB-283 and selumetinib showed synergistic effect in inhibiting proliferation of several non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines harboring RAS mutations. In vivo, combined dosing of BGB-283 and AZD6244 also exhibited enhanced antitumor activity in K-RAS mutant NSCLC and CRC xenograft models compared to monotherapies. Pharmacodynamic (PD) analysis revealed improved inhibition of ERK phosphorylation in dually treated tumors. Together, these findings suggested that combining RAF dimer inhibitor BGB-283 and MEKi could be a promising strategy in treating RAS mutated cancers. Citation Format: Xi Yuan, Zhiyu Tang, Rong Du, Shing-Hu Cheung, Jing Wei, Yuan Zhao, Yunguang Du, Rui Hao, Xiaoxia Hu, Wenfeng Gong, Yong Liu, Yajuan Gao, Min Wei, Changyou Zhou, Lai Wang, Lusong Luo. BGB-283 effectively enhances MEK inhibitor induced tumor suppression in RAS mutant cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 669. doi:10.1158/1538-7445.AM2015-669

Contributions of DNA methylation aberrancies in shaping the cancer epigenome

Genetic alterations, including DNA sequence and copy number alterations, have been characterized in human cancers for over several decades. However, it is becoming increasingly evident that in addition to these events, epigenetic changes are widespread in virtually every cancer type. The scientific literature is rich with reports of changes in DNA methylation, chromatin modifications and nucleosome occupancy, all of which contribute to the aberrant gene expression profiles described in human cancers. Whilst genetic and epigenetic alterations have been historically characterized on a candidate gene and cancer-type basis, the unprecedented acceleration in microarray and next-generation sequencing technologies used to profile large collections of primary specimens has provided a panoramic, genome-wide view of cancer genomes and epigenomes. For the first time, the relationships between individual alterations can be understood, with the ultimate goal of improving cancer detection, monitoring, surveillance and most importantly, treatment. A central aspect of these involves characterizing DNA methylation alterations in human cancers. DNA methylation aberrancies are present in every tumor type, serve as stable biomarkers of disease, are associated with patient clinical features, and are reversible through the application of DNA methylation inhibitors. DNA methylation inhibitors are currently used in treating patients with myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML), and new inhibitors are currently in clinical trials for potential cancer treatment. This review highlights DNA methylation changes and their significance in human cancers from mechanistic, biomarker and treatment perspectives.

Proteomics for Early Diagnostics

Advances in molecular biology over the past decade have helped to enhance understanding of the complex interplay between genetic, transcriptional, and translational alterations in human cancers. These molecular changes are the basis for an evolving field of high-throughput cancer discovery techniques using microscopic amounts of patient-based materials. In recent years, there has been an explosion in the development of new tools to analyze the proteome of cells, blood, and other body fluids. The information that is expected from such technologies may soon exert a dramatic change in the pace of cancer research and impact dramatically on the care of cancer patients.

Mutant p53 - Heat Shock Response Oncogenic Cooperation: A New Mechanism of Cancer Cell Survival.

The main tumor suppressor function of p53 as a “guardian of the genome” is to respond to cellular stress by transcriptional activation of apoptosis, growth arrest, or senescence in damaged cells. Not surprisingly, mutations in the p53 gene are the most frequent genetic alteration in human cancers. Importantly, mutant p53 (mutp53) proteins not only lose their wild-type tumor suppressor activity but also can actively promote tumor development. Two main mechanisms accounting for mutp53 proto-oncogenic activity are inhibition of the wild-type p53 in a dominant-negative fashion and gain of additional oncogenic activities known as gain-of-function (GOF). Here, we discuss a novel mechanism of mutp53 GOF, which relies on its oncogenic cooperation with the heat shock machinery. This coordinated adaptive mechanism renders cancer cells more resistant to proteotoxic stress and provides both, a strong survival advantage to cancer cells and a promising means for therapeutic intervention.

The TP53 intron 6 G13964C polymorphism and risk of thyroid and breast cancer development in the Iranian Azeri population.

TP53 mutations are the most common genetic alterations in human cancers. There are also several polymorphisms in both exons and introns of TP53 that may influence its anti-tumor functions and increase the risk of cancer development. Associations of the TP53 intron 6 G13964C polymorphism with increased risk of development of several cancers have been investigated in numerous studies, but the results were controversial and conflicting. In this study, we aimed to investigate the probable association of this polymorphism with risk of both thyroid and breast cancers among the Iranian-Azeri population. We performed two separate case control studies on associations of the intron 6 polymorphism with two different kinds of cancer. In one case-control study, a total of 75 patients with thyroid carcinoma and 180 controls were analyzed and the other study included 170 patients with breast cancer and 135 healthy women. The intron 6 genotype was determined by RFLP-PCR and the SPSS 16 program was applied for data analysis. For thyroid cancer, the frequencies of GG genotype were 96.0% in patients and 93.3% in controls. The GC genotype had a frequency of 4.0% in patients and 6.7% in controls. In the study on breast cancer, the frequency of GG and GC genotypes in patients were 95.3% and 4.7%, respectively. In breast related control group, the frequency of GG genotype was 93.3% and the frequency of GC genotype was 6.7%. None of the cases and controls had the CC genotype. There was no significant association between the TP53 intron 6 G13964C polymorphism and risk of development of both thyroid and breast cancer in Iranian-Azeri patients.

NOTCH mutations: multiple faces in human malignancies.

NOTCH proteins have been implicated in multiple cellular functions, such as stem cell maintenance and cell fate determination. Initially identified as proto-oncogenes because they promote the development of certain types of leukemia, inactivating mutations of NOTCH were later reported. Together with the potential distinct functions of NOTCH family members, their ligands and associated niches, the precise roles of NOTCH in human cancers, particularly solid tumors, remain unsettled. In oral squamous cell carcinoma (OSCC), mutations of NOTCH1 are found in 10% to 15% tumors from Caucasian patients, mostly inactivating mutations. Recent studies of OSCC from Chinese patients, however, showed mutation rates of NOTCH1 about 50% with a considerable portion of potential activating mutations. These findings add another twist into the already complex picture of NOTCH alterations in human cancers, calling for further investigation to uncover what role exactly these molecules play in cancer initiation and progression to develop strategies targeting NOTCH signaling for cancer prevention and treatment.

Control of cardiac repolarization by phosphoinositide 3-kinase signaling to ion channels.

Upregulation of phosphoinositide 3-kinase (PI3K) signaling is a common alteration in human cancer, and numerous drugs that target this pathway have been developed for cancer treatment. However, recent studies have implicated inhibition of the PI3K signaling pathway as the cause of a drug-induced long-QT syndrome in which alterations in several ion currents contribute to arrhythmogenic drug activity. Surprisingly, some drugs that were thought to induce long-QT syndrome by direct block of the rapid delayed rectifier (IKr) also seem to inhibit PI3K signaling, an effect that may contribute to their arrhythmogenicity. The importance of PI3K in regulating cardiac repolarization is underscored by evidence that QT interval prolongation in diabetes mellitus also may result from changes in multiple currents because of decreased insulin activation of PI3K in the heart. How PI3K signaling regulates ion channels to control the cardiac action potential is poorly understood. Hence, this review summarizes what is known about the effect of PI3K and its downstream effectors, including Akt, on sodium, potassium, and calcium currents in cardiac myocytes. We also refer to some studies in noncardiac cells that provide insight into potential mechanisms of ion channel regulation by this signaling pathway in the heart. Drug development and safety could be improved with a better understanding of the mechanisms by which PI3K regulates cardiac ion channels and the extent to which PI3K inhibition contributes to arrhythmogenic susceptibility.

Functional repair of p53 mutation in colorectal cancer cells using trans-splicing.

Mutation in the p53 gene is arguably the most frequent type of gene-specific alterations in human cancers. Current p53-based gene therapy contains the administration of wt-p53 or the suppression of mutant p53 expression in p53-defective cancer cells. . We hypothesized that trans-splicing could be exploited as a tool for the correction of mutant p53 transcripts in p53-mutated human colorectal cancer (CRC) cells. In this study, the plasmids encoding p53 pre-trans-splicing molecules (PTM) were transfected into human CRC cells carrying p53 mutation. The plasmids carrying p53-PTM repaired mutant p53 transcripts in p53-mutated CRC cells, which resulted in a reduction in mutant p53 transcripts and an induction of wt-p53 simultaneously. Intratumoral administration of adenovirus vectors carrying p53 trans-splicing cassettes suppressed the growth of tumor xenografts. Repair of mutant p53 transcripts by trans-splicing induced cell-cycle arrest and apoptosis in p53-defective colorectal cancer cells in vitro and in vivo. In conclusion, the present study demonstrated for the first time that trans-splicing was exploited as a strategy for the repair of mutant p53 transcripts, which revealed that trans-splicing would be developed as a new therapeutic approach for human colorectal cancers carrying p53 mutation.

CS-02 * SHP2 PROMOTES TUMOR PROGRESSION IN GBM VIA RAS ACTIVATION

Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and have also been identified in several familial/developmental syndromes. Ras is also an important factor in many cancers where it is not mutated but functionally activated through inappropriate function of other signal transduction elements. One such example is the overexpression of growth factor receptors in glioblastoma (GBM). Elucidating the mechanistic details of the GTPase reaction has been an important scientific goal and the subject of intensive research for many years. However to this day, targeting Ras directly remains elusive. Our laboratory recently demonstrated that Src binds to and phosphorylates Ras on a conserved tyrosine residue markedly reducing the binding of effector Raf to Ras and concomitantly increasing the rate of GTP hydrolysis leading to Ras inactivation. Furthermore we demonstrate that tyrosine phosphorylated Ras can be dephosphorylated by the phosphatase SHP2 resulting in Ras activation. We also demonstrate that increased Ras activity in human primary GBM cell lines and astrocytes derived from GBM-prone mice, as well as in human GBM patient samples results from elevated SHP2 activity. Finally we show that the highly specific SHP2 inhibitor II-B08 promotes tyrosine phosphorylation of Ras leading to attenuation of downstream proliferative MAPK/ERK signaling and reduction in tumor burden in mouse models of GBM.

The cyclin D1-CDK4 oncogenic interactome enables identification of potential novel oncogenes and clinical prognosis

Overexpression of cyclin D1 and its catalytic partner, CDK4, is frequently seen in human cancers. We constructed cyclin D1 and CDK4 protein interaction network in a human breast cancer cell line MCF7, and identified novel CDK4 protein partners. Among CDK4 interactors we observed several proteins functioning in protein folding and in complex assembly. One of the novel partners of CDK4 is FKBP5, which we found to be required to maintain CDK4 levels in cancer cells. An integrative analysis of the extended cyclin D1 cancer interactome and somatic copy number alterations in human cancers identified BAIAPL21 as a potential novel human oncogene. We observed that in several human tumor types BAIAPL21 is expressed at higher levels as compared to normal tissue. Forced overexpression of BAIAPL21 augmented anchorage independent growth, increased colony formation by cancer cells and strongly enhanced the ability of cells to form tumors in vivo. Lastly, we derived an Aggregate Expression Score (AES), which quantifies the expression of all cyclin D1 interactors in a given tumor. We observed that AES has a prognostic value among patients with ER-positive breast cancers. These studies illustrate the utility of analyzing the interactomes of proteins involved in cancer to uncover potential oncogenes, or to allow better cancer prognosis.

Large hypomethylated blocks as a universal defining epigenetic alteration in human solid tumors.

BackgroundOne of the most provocative recent observations in cancer epigenetics is the discovery of large hypomethylated blocks, including single copy genes, in colorectal cancer, that correspond in location to heterochromatic LOCKs (large organized chromatin lysine-modifications) and LADs (lamin-associated domains).MethodsHere we performed a comprehensive genome-scale analysis of 10 breast, 28 colon, nine lung, 38 thyroid, 18 pancreas cancers, and five pancreas neuroendocrine tumors as well as matched normal tissue from most of these cases, as well as 51 premalignant lesions. We used a new statistical approach that allows the identification of large hypomethylated blocks on the Illumina HumanMethylation450 BeadChip platform.ResultsWe find that hypomethylated blocks are a universal feature of common solid human cancer, and that they occur at the earliest stage of premalignant tumors and progress through clinical stages of thyroid and colon cancer development. We also find that the disrupted CpG islands widely reported previously, including hypermethylated island bodies and hypomethylated shores, are enriched in hypomethylated blocks, with flattening of the methylation signal within and flanking the islands. Finally, we found that genes showing higher between individual gene expression variability are enriched within these hypomethylated blocks.ConclusionThus hypomethylated blocks appear to be a universal defining epigenetic alteration in human cancer, at least for common solid tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0061-y) contains supplementary material, which is available to authorized users.

Chromosome Breakage Induced by the Genotoxic Agents Mitomycin C and Cytosine arabinoside is Concentration and p53 Dependent

The p53 tumor suppressor protein plays an essential role in cellular integrity and inactivation of the TP53 gene by mutation is the most frequent alteration in human cancer. As loss of p53 function is associated with increased genetic instability, it is important in genotoxicity testing to explore the role of p53 competency. In vitro model systems for genotoxicity testing are sometimes prone to misleading positive results; some of this loss of predictivity may be caused by p53 inactivation in some cell models. To explore whether impaired p53 function plays a role in mutation sensitivity, TK6 cells (p53 competent) and NH32 cells (p53 deficient) were treated with two known genotoxicants, mitomycin C (MMC) and cytosine arabinoside (araC). Chromosomal damage was assessed in the low dose region by an automated micronucleus system and p53 activity was investigated by gene and protein expression analysis. Cell cycle progression studies were also assessed. Low levels of micronucleus and p53 induction were observed in TK6 cells treated with MMC. On the other hand, higher levels of micronucleus and p53 induction were shown in TK6 cells treated with araC and a G1/S arrest was observed after araC treatment. p53 deficient NH32 cells showed an increased sensitivity of micronucleus(MN) induction after araC treatment compared with TK6 cells and less of an active G1/S phase checkpoint. Thus, impaired p53 function sensitizes cells to genotoxicants and plays a central role in the DNA damage response. This data has clear importance for safety assessment of genotoxicity and shows how crucial p53 competence is.

P53 suppresses carcinoma progression by inhibiting mTOR pathway activation

Genetic alterations in human cancers and murine models indicate that Rb and p53 have critical tumor suppressive functions in retinoblastoma, a tumor of neural origin, and neuroendocrine tumors including small cell lung cancer and medullary thyroid cancer (MTC). Rb inactivation is the initiating lesion in retinoblastoma and current models propose that induction of apoptosis is a key p53 tumor suppressive function. Genetic studies in mice, however, indicate that other undefined p53 tumor suppressive functions are operative in vivo. How p53 loss cooperates with Rb inactivation to promote carcinogenesis is also not fully understood. In the current study, genetically engineered mice were generated to determine the role of Rb and p53 in MTC pathogenesis and test the hypothesis that p53 suppresses carcinogenesis by inhibiting mTOR signaling. Conditional Rb ablation resulted in thyroid tumors mimicking human MTC, and additional p53 loss led to rapid tumor progression. p53 suppressed tumorigenesis by inhibiting cell cycle progression, but did not induce apoptosis. On the contrary, p53 loss led to increased apoptosis that had to be overcome for tumor progression. mTOR activity was markedly increased in p53 deficient tumors and rapamycin treatment suppressed tumor cell growth identifying mTOR inhibition as a critical p53 tumor suppressive function. Rapamycin treatment did not result in AKT/MAPK activation providing evidence that this feedback mechanism operative in other cancers is not a general response to mTORC1 inhibition. Together, these studies provide mechanistic links between genetic alterations and aberrant signaling pathways critical in carcinogenesis, and identify essential Rb and p53 tumor suppressive functions in vivo.

Characterizing DNA methylation alterations from The Cancer Genome Atlas

The Cancer Genome Atlas (TCGA) Research Network is an ambitious multi-institutional consortium effort aimed at characterizing sequence, copy number, gene (mRNA) expression, microRNA expression, and DNA methylation alterations in 30 cancer types. TCGA data have become an extraordinary resource for basic, translational, and clinical researchers and have the potential to shape cancer diagnostic and treatment strategies. DNA methylation changes are integral to all aspects of cancer genomics and have been shown to have important associations with gene expression, sequence, and copy number changes. This Review highlights the knowledge gained from DNA methylation alterations in human cancers from TCGA.

Emerging role of the KRAS-PDK1 axis in pancreatic cancer.

Pancreatic cancer is a highly aggressive tumour that is very resistant to treatments and it is rarely diagnosed early because of absence of specific symptoms. Therefore, the prognosis for this disease is very poor and it has the grim supremacy in terms of unfavourable survival rates. There have been great advances in survival rates for many types of cancers over the past few decades but hardly any change for pancreatic cancer. Mutations of the Ras oncogene are the most frequent oncogenic alterations in human cancers. The frequency of KRAS mutations in pancreatic cancer is around 90%. Given the well-established role of KRAS in cancer it is not surprising that it is one of the most attractive targets for cancer therapy. Nevertheless, during the last thirty years all attempts to target directly KRAS protein have failed. Therefore, it is crucial to identify downstream KRAS effectors in order to develop specific drugs able to counteract activation of this pathway. Among the different signalling pathways activated by oncogenic KRAS, the phosphoinositide 3-Kinase (PI3K) pathway is emerging as one of the most critical KRAS effector. In turn, PI3K activates several parallel pathways making the identification of the precise effectors activated by KRAS/PI3K more difficult. Recent data identify 3-phosphoinositide-dependent protein kinase 1 as a key tumour-initiating event downstream KRAS interaction with PI3K in pancreatic cancer.

Genome-wide profiling of copy number alterations in cancer: focus on melanoma

Thanks to a never-before detailed view of the human genome, the last decade has brought to light the notion of DNA copy number variation (CNV) as the pivotal force contributing to population genomic diversity and evolution. It is as well clear now that cancer typically results in loosened control over genomic integrity and that the acquisition of somatic copy number alterations (SCNAs), whether confined to specific genes or affecting entire chromosome arms, is likely to be a fundamental prerequisite to the adaptive pressure that drives oncogenesis. This review gives a brief overview of key developments in genome-wide SCNA profiling, with specific emphasis on array-based techniques and deep-sequencing, which indeed enabled us to identify the large majority of genomic regions undergoing frequent alteration in human cancers and defining recognizable clinical phenotype. Alongside with the prospective to take advantage for future personalized precision medicine, high-throughput SCNA analysis have already proven diagnostic and prognostic potential, particularly for those clinically unpredictable and therapy-refractory tumors, such us cutaneous melanoma. Biomedical Reviews 2013; 24: 11-24.

Increased Local Disorder of DNA Methylation Forms the Basis of High Intra-Leukemic Epigenetic Heterogeneity and Enhances CLL Evolution

We recently demonstrated that genetic heterogeneity contributes to evolution and clinical outcome in chronic lymphocytic leukemia (CLL) (Landau et al., Cell 2013). As both genetic and epigenetic alterations contribute to the cellular phenotype and its fitness for selection, we hypothesized that heterogeneity in DNA methylation impacts leukemic evolution as well.We analyzed existing CLL methylation data (Kulis et al., Nat Gen 2012), and found higher heterogeneity in 450K arrays of 139 CLLs compared to 23 normal B cell samples, with a higher proportion of sites having intermediate levels of methylation (13.7% vs 10.7%, P<0.0001). Next, we re-analyzed whole genome bisulfite sequencing data from 2 CLL and 3 normal B cell samples to distinguish between 2 possible explanations for heterogeneity: 1) a mixture of cell subpopulations with ordered but differing methylation states, and 2) increased proportion of cells with ‘noisy' or disordered methylation states. We found that the bulk of heterogeneity resulted from the latter, as evidenced by a high proportion in CLL of individual sequencing reads with a discordant methylation state in neighboring CpGs.To validate this observation, we applied reduced representation bisulfite sequencing (RRBS) to generate genome-scale information of methylation at critical sites (e.g., CG islands) of 108 genetically-characterized CLLs. As predicted, we found higher proportions of discordant reads in CLL than either samples from 30 normal B cells or from 35 diverse healthy human tissues (average of 24.7% vs. 17.6% or 16.3%, respectively, P<0.00001). Similarly high proportions of discordant reads were also found in 31 cancer cell lines. Samples with somatic mutations in methylation modulators (e.g., DNMT3A) had increased proportion of discordant reads (P<0.001). Importantly, the presence of methylation ‘noise' was associated with reduced correlation between promoter methylation and gene silencing (P<0.0001), and with substantial allele specific expression, both measured by RNA-seq of 48 CLLs. These findings suggest that much of DNA methylation heterogeneity in CLL stems from locally disordered methylation, and shed light on how epigenetic alterations in human cancer arise in part from epigenetic drift.Increased epigenetic ‘noise' is expected to result in a more plastic evolutionary landscape that facilitates the emergence of fitness-enhancing genetic and epigenetic alterations. Indeed, the increase in discordant reads in CLL compared to normal samples was most prominent in genomic regions important to transcription (promoters, CG islands [P <1*e-14] vs. introns, CGI shelves [P <1*e-2]). In addition, significantly higher levels of discordant reads were found in promoters of genes silenced in CLL, in differentially methylated regions and in genes important to pluripotency potential (e.g., stem cell modules, Q<1*e-14). Finally, samples with increased disordered methylation had higher numbers of subclonal, but not clonal mutations (P<0.001 vs. P=0.26, respectively).To observe the relationship between genetic and epigenetic evolution, we used RRBS to longitudinally study 14 CLLs with characterized patterns of genetic evolution (median time between samples 3.5 yrs; 6 unevolved, 8 evolved). While unevolved CLLs revealed largely unchanged epigenetic landscapes, evolved CLLs demonstrated decreased methylation over time in promoters enriched for stem cell and MYC targets gene sets (N=347, Q<0.0001), and increased methylation in TP53 targets (N=336, Q<0.00001). Moreover, in evolved CLLs, specific promoters with disordered methylation across all samples (e.g., TERT, WT1), revealed changes over time in methylation proportions corresponding to increases in subclone size inferred from the genetic analysis.Increased heterogeneity was found to be associated with shorter retreatment-free survival in a cohort of 46 patients treated after sampling (P=0.03, HR of 3.13), in a regression model including established risk indicators (IGHV unmutated status, del(17p), del(11q) and treatment prior to sample).Our data suggest that heterogeneity in CLL DNA methylation results from stochastic drift that subsequently undergoes positive selection. Thus, we propose that locally disordered DNA methylation may have a similar role to genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories. Disclosures:No relevant conflicts of interest to declare.

Mining the epigenetic landscape in ALL

The significance of epigenomic aberrations in cancer development has been underscored by the discovery of mutations in key chromatin modifiers, most notably in hematological malignancies. A new study of pediatric acute lymphoblastic leukemia (ALL) demonstrates the usefulness of mapping global epigenetic signatures and applying these data in a framework to identify and characterize underlying somatic genetic alterations in human cancers.

Cancer detection using whole-genome sequencing of cell free DNA

Biomarkers are molecules whose detection provide valuable information regarding human disease. They may herald the presence of a disease or provide information regarding the course of disease and response to treatment. Historically, cancer biomarkers have been based on aberrant protein expression in a subset of tumor types (e.g. CEA in colorectal cancer, PSA in prostate cancer, and CA19-9 in ovarian cancer). Although protein biomarkers have been useful in cancer over the past several decades, their correlation with disease could be improved by the development of more sensitive, specific, and widely applicable cancer biomarkers. We have recently developed a non-invasive approach using whole-genome sequencing to identify chromosomal alterations in the circulation of cancer patients [1]. Cell-free DNA analyses of the circulation have the potential to alter the management of cancer care, leading to earlier diagnoses, identification of disease recurrence, and monitoring responses to therapy. Human cancer arises from the sequential accumulation of genetic mutations. Dozens of genetic changes may be observed in each tumor's genome and are comprised of small sequence alterations as well as structural alterations, including copy number changes and rearrangements. Over the past decade genome sequencing has elucidated complex cancer genome landscapes, with unique collections of somatic genetic alterations in each patient. The phenomena of chromosomal instability is thought to be near-universal in human cancers and responsible for the development of somatic structural alterations. Recent analyses of structural alterations in breast cancers have shown multiple alterations in every cancer genome evaluated [2]. Additionally, analyses of 81 colorectal and breast cancer cell lines and xenografts using SNP arrays showed at least five whole-chromosome or chromosome arm alterations and an average of 14 focal copy number changes per case [3]. Although the same genes may not be altered in patients with a particular tumor type, the universal phenomena of somatic genetic alterations may be exploited to develop biomarkers that are specific to each patient's tumor. Structural alterations, including rearrangements and chromosome arm gains and losses, are especially attractive biomarkers as they are dramatic changes that readily distinguish tumor-derived DNA from normal DNA. When primary tumor material is available, patient-specific rearrangements may be identified with Personalized Analysis of Rearranged Ends (PARE) or related methods [4,5]. Using massively parallel sequencing, PARE identifies rearrangements in the tumor and allows development of personalized PCR biomarker analyses to detect patient-specific rearrangements in cell-free DNA present in the circulation. PARE may be used to monitor disease progression, determine response to therapy, and evaluate presence of residual disease after potentially curative surgery or other clinical interventions. Given the potential applications of these approaches, we recently examined whether we could extend this method to identify structural alterations directly in patient plasma without prior knowledge of disease status or tumor genotype [1]. Cell-free DNA isolated from patient plasma was used to generate libraries for massively parallel sequencing. The sequencing data obtained from whole genome sequencing of cell-free DNA permitted detection of both rearrangements as well as larger chromosomal abnormalities such as gains or losses of whole chromosomes or chromosome arms. In this proof of principle study, we sequenced cell-free DNA from the plasma of seven colorectal and three breast cancer patients and successfully detected structural alterations in the plasma of all cases analyzed. When these same analyses were applied to plasma samples from 10 healthy individuals as well as 28 normal lymphocyte samples, no structural alterations were identified. Several of the rearranged sequences detected in patient plasma were in regions associated with clinically actionable genes. As genomic rearrangements co-occur with copy number alterations, the detected rearrangements could be used to infer the presence of copy number changes in nearby genes. For example, rearrangements associated with the ERBB2 oncogene were identified in the plasma of a colorectal cancer patient and shown to be the result of ERBB2 amplification in the patient's tumor. These results indicate the potential application of this approach to identify clinically relevant tumor-specific genetic changes in the absence of available tumor tissue [1]. Although this initial study analyzed patients with late-stage disease, simulations of detection of circulating tumor DNA in earlier-stage disease indicate that the sensitivity of this approach is largely dependent on the amount of sequence data obtained. Currently, cell-free DNA analyses could be used to provide information about patient's tumor and guide clinical management. As the cost of sequencing continues to decrease, this approach and related methods [6,7] may provide a feasible avenue for profiling circulating DNA for early tumor detection. Figure 1 Schematic of cell-free genomic analyses to detect structural alterations in human cancer.