Aron Charles Eklund

Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

BACKGROUND Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. METHODS To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. RESULTS Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. CONCLUSIONS Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).

A signature of chromosomal instability inferred from gene expression profiles predicts clinical outcome in multiple human cancers

We developed a computational method to characterize aneuploidy in tumor samples based on coordinated aberrations in expression of genes localized to each chromosomal region. We summarized the total level of chromosomal aberration in a given tumor in a univariate measure termed total functional aneuploidy. We identified a signature of chromosomal instability from specific genes whose expression was consistently correlated with total functional aneuploidy in several cancer types. Net overexpression of this signature was predictive of poor clinical outcome in 12 cancer data sets representing six cancer types. Also, the signature of chromosomal instability was higher in metastasis samples than in primary tumors and was able to stratify grade 1 and grade 2 breast tumors according to clinical outcome. These results provide a means to assess the potential role of chromosomal instability in determining malignant potential over a broad range of tumors.

Efficacy of Neoadjuvant Cisplatin in Triple-Negative Breast Cancer

PURPOSE Cisplatin is a chemotherapeutic agent not used routinely for breast cancer treatment. As a DNA cross-linking agent, cisplatin may be effective treatment for hereditary BRCA1-mutated breast cancers. Because sporadic triple-negative breast cancer (TNBC) and BRCA1-associated breast cancer share features suggesting common pathogenesis, we conducted a neoadjuvant trial of cisplatin in TNBC and explored specific biomarkers to identify predictors of response. PATIENTS AND METHODS Twenty-eight women with stage II or III breast cancers lacking estrogen and progesterone receptors and HER2/Neu (TNBC) were enrolled and treated with four cycles of cisplatin at 75 mg/m(2) every 21 days. After definitive surgery, patients received standard adjuvant chemotherapy and radiation therapy per their treating physicians. Clinical and pathologic treatment response were assessed, and pretreatment tumor samples were evaluated for selected biomarkers. Results Six (22%) of 28 patients achieved pathologic complete responses, including both patients with BRCA1 germline mutations;18 (64%) patients had a clinical complete or partial response. Fourteen (50%) patients showed good pathologic responses (Miller-Payne score of 3, 4, or 5), 10 had minor responses (Miller-Payne score of 1 or 2), and four (14%) progressed. All TNBCs clustered with reference basal-like tumors by hierarchical clustering. Factors associated with good cisplatin response include young age (P = .001), low BRCA1 mRNA expression (P = .03), BRCA1 promoter methylation (P = .04), p53 nonsense or frameshift mutations (P = .01), and a gene expression signature of E2F3 activation (P = .03). CONCLUSION Single-agent cisplatin induced response in a subset of patients with TNBC. Decreased BRCA1 expression may identify subsets of TNBCs that are cisplatin sensitive. Other biomarkers show promise in predicting cisplatin response.

PGC-1α, A Potential Therapeutic Target for Early Intervention in Parkinson’s Disease

Abnormal expression of genes for energy regulation in Parkinson’s disease patients identifies a master regulator as a possible therapeutic target for early intervention. Getting to the Root of Parkinson’s Disease Parkinson’s disease (PD) is a debilitating neurodegenerative disorder that results in the loss of dopamine neurons in the substantia nigra of the brain. Degeneration of these movement-related neurons predictably causes rigidity, slowness of movement, and resting tremor, but patients also show cognitive changes. Although gene mutations have been identified in several families with PD, the cause of the more common sporadic form is not known. Certain environmental factors, such as exposure to the pesticide rotenone, combined with a genetic susceptibility, are thought to confer risk for developing PD. A key pathological feature seen in postmortem brain tissue from PD patients is Lewy bodies, neuronal inclusions containing clumps of the α-synuclein protein (which is mutated in familial PD), as well as damaged mitochondria. Taking a systems biology approach to pinpoint the root cause of PD, Zheng et al. now implicate altered activity of the master transcription factor PGC-1α and the genes it regulates in the early stages of PD pathogenesis. To detect new sets of genes that may be associated with PD, the investigators did a meta-analysis of 17 independent genome-wide gene expression microarray studies that had been performed on a total of 322 postmortem brain tissue samples and 88 blood samples. The samples came from presymptomatic and symptomatic PD patients, as well as from control individuals who did not show any neurological deficits at autopsy. Nine genome-wide expression studies were conducted either on dopaminergic neurons obtained by laser capture from substantia nigra (three studies) or on substantia nigra homogenates (six studies). The authors then used a powerful tool called Gene Set Enrichment Analysis to sift through 522 gene sets (a gene set is a group of genes involved in one biological pathway or process). At the end of this tour-de-force analysis, they identified 10 gene sets that were all associated with PD. The gene sets with the strongest association contained nuclear genes encoding subunits of the electron transport chain proteins found in mitochondria. These genes all showed decreased expression in substantia nigra dopaminergic neurons (obtained by laser capture) even in the earliest stages of PD. Furthermore, a second gene set associated with PD and also underexpressed in the earliest stages of PD encodes enzymes involved in glucose metabolism. These results are compelling because many studies have already implicated dysfunctional mitochondria and altered energy metabolism as well as defective glucose metabolism in PD. The authors realized that these gene sets had in common the master transcriptional regulator, PGC-1α, and surmised that disruption of PGC-1α expression might be a root cause of PD. They tested this hypothesis in cultured dopaminergic neurons from embryonic rat midbrain forced to express a mutant form of α-synuclein. Overexpression of PGC-1α in these neurons resulted in activation of electron transport genes and protection against neuronal damage induced by mutant α-synuclein. In other cultured neurons treated with rotenone, overexpression of PGC-1α also was protective, blocking pesticide-induced neuronal cell death. These exciting findings identify altered expression of PGC-1α and the genes it regulates as key players during early PD pathogenesis. This potential new target could be exploited therapeutically to interfere with the pathological process during the earliest stages before permanent damage and neuronal loss occurs. Parkinson’s disease affects 5 million people worldwide, but the molecular mechanisms underlying its pathogenesis are still unclear. Here, we report a genome-wide meta-analysis of gene sets (groups of genes that encode the same biological pathway or process) in 410 samples from patients with symptomatic Parkinson’s and subclinical disease and healthy controls. We analyzed 6.8 million raw data points from nine genome-wide expression studies, and 185 laser-captured human dopaminergic neuron and substantia nigra transcriptomes, followed by two-stage replication on three platforms. We found 10 gene sets with previously unknown associations with Parkinson’s disease. These gene sets pinpoint defects in mitochondrial electron transport, glucose utilization, and glucose sensing and reveal that they occur early in disease pathogenesis. Genes controlling cellular bioenergetics that are expressed in response to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) are underexpressed in Parkinson’s disease patients. Activation of PGC-1α results in increased expression of nuclear-encoded subunits of the mitochondrial respiratory chain and blocks the dopaminergic neuron loss induced by mutant α-synuclein or the pesticide rotenone in cellular disease models. Our systems biology analysis of Parkinson’s disease identifies PGC-1α as a potential therapeutic target for early intervention.

Molecular markers of early Parkinson's disease based on gene expression in blood

Parkinsons disease (PD) progresses relentlessly and affects five million people worldwide. Laboratory tests for PD are critically needed for developing treatments designed to slow or prevent progression of the disease. We performed a transcriptome-wide scan in 105 individuals to interrogate the molecular processes perturbed in cellular blood of patients with early-stage PD. The molecular multigene marker here identified is associated with risk of PD in 66 samples of the training set comprising healthy and disease controls [third tertile cross-validated odds ratio of 5.7 (P for trend 0.005)]. It is further validated in 39 independent test samples [third tertile odds ratio of 5.1 (P for trend 0.04)]. Insights into disease-linked processes detectable in peripheral blood are offered by 22 unique genes differentially expressed in patients with PD versus healthy individuals. These include the cochaperone ST13, which stabilizes heat-shock protein 70, a modifier of α-synuclein misfolding and toxicity. ST13 messenger RNA copies are lower in patients with PD (mean ± SE 0.59 ± 0.05) than in controls (0.96 ± 0.09) (P = 0.002) in two independent populations. Thus, gene expression signals measured in blood can facilitate the development of biomarkers for PD.

A large-scale analysis of tissue-specific pathology and gene expression of human disease genes and complexes

Heritable diseases are caused by germ-line mutations that, despite tissuewide presence, often lead to tissue-specific pathology. Here, we make a systematic analysis of the link between tissue-specific gene expression and pathological manifestations in many human diseases and cancers. Diseases were systematically mapped to tissues they affect from disease-relevant literature in PubMed to create a disease–tissue covariation matrix of high-confidence associations of >1,000 diseases to 73 tissues. By retrieving >2,000 known disease genes, and generating 1,500 disease-associated protein complexes, we analyzed the differential expression of a gene or complex involved in a particular disease in the tissues affected by the disease, compared with nonaffected tissues. When this analysis is scaled to all diseases in our dataset, there is a significant tendency for disease genes and complexes to be overexpressed in the normal tissues where defects cause pathology. In contrast, cancer genes and complexes were not overexpressed in the tissues from which the tumors emanate. We specifically identified a complex involved in XY sex reversal that is testis-specific and down-regulated in ovaries. We also identified complexes in Parkinson disease, cardiomyopathies, and muscular dystrophy syndromes that are similarly tissue specific. Our method represents a conceptual scaffold for organism-spanning analyses and reveals an extensive list of tissue-specific draft molecular pathways, both known and unexpected, that might be disrupted in disease.

Chromosomal instability determines taxane response

Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these “CIN-survival” genes is associated with poor outcome in estrogen receptor–positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents.

GATA transcription factors directly regulate the Parkinson's disease-linked gene α-synuclein

Increased α-synuclein gene (SNCA) dosage due to locus multiplication causes autosomal dominant Parkinsons disease (PD). Variation in SNCA expression may be critical in common, genetically complex PD but the underlying regulatory mechanism is unknown. We show that SNCA and the heme metabolism genes ALAS2, FECH, and BLVRB form a block of tightly correlated gene expression in 113 samples of human blood, where SNCA naturally abounds (validated P = 1.6 × 10−11, 1.8 × 10−10, and 6.6 × 10−5). Genetic complementation analysis revealed that these four genes are co-induced by the transcription factor GATA-1. GATA-1 specifically occupies a conserved region within SNCA intron-1 and directly induces a 6.9-fold increase in α-synuclein. Endogenous GATA-2 is highly expressed in substantia nigra vulnerable to PD, occupies intron-1, and modulates SNCA expression in dopaminergic cells. This critical link between GATA factors and SNCA may enable therapies designed to lower α-synuclein production.

Paradoxical Relationship between Chromosomal Instability and Survival Outcome in Cancer

Chromosomal instability (CIN) is associated with poor prognosis in human cancer. However, in certain animal tumor models elevated CIN negatively impacts upon organism fitness, and is poorly tolerated by cancer cells. To better understand this seemingly contradictory relationship between CIN and cancer cell biological fitness and its relationship with clinical outcome, we applied the CIN70 expression signature, which correlates with DNA-based measures of structural chromosomal complexity and numerical CIN in vivo, to gene expression profiles of 2,125 breast tumors from 13 published cohorts. Tumors with extreme CIN, defined as the highest quartile CIN70 score, were predominantly of the estrogen receptor negative (ER(-)), basal-like phenotype and displayed the highest chromosomal structural complexity and chromosomal numerical instability. We found that the extreme CIN/ER(-) tumors were associated with improved prognosis relative to tumors with intermediate CIN70 scores in the third quartile. We also observed this paradoxical relationship between CIN and prognosis in ovarian, gastric, and non-small cell lung cancer, with poorest outcome in tumors with intermediate, rather than extreme, CIN70 scores. These results suggest a nonmonotonic relationship between gene signature expression and HR for survival outcome, which may explain the difficulties encountered in the identification of prognostic expression signatures in ER(-) breast cancer. Furthermore, the data are consistent with the intolerance of excessive CIN in carcinomas and provide a plausible strategy to define distinct prognostic patient cohorts with ER(-) breast cancer. Inclusion of a surrogate measurement of CIN may improve cancer risk stratification and future therapeutic approaches.

Telomeric Allelic Imbalance Indicates Defective DNA Repair and Sensitivity to DNA-Damaging Agents

UNLABELLED DNA repair competency is one determinant of sensitivity to certain chemotherapy drugs, such as cisplatin. Cancer cells with intact DNA repair can avoid the accumulation of genome damage during growth and also can repair platinum-induced DNA damage. We sought genomic signatures indicative of defective DNA repair in cell lines and tumors and correlated these signatures to platinum sensitivity. The number of subchromosomal regions with allelic imbalance extending to the telomere (N(tAI)) predicted cisplatin sensitivity in vitro and pathologic response to preoperative cisplatin treatment in patients with triple-negative breast cancer (TNBC). In serous ovarian cancer treated with platinum-based chemotherapy, higher levels of N(tAI) forecast a better initial response. We found an inverse relationship between BRCA1 expression and N(tAI) in sporadic TNBC and serous ovarian cancers without BRCA1 or BRCA2 mutation. Thus, accumulation of telomeric allelic imbalance is a marker of platinum sensitivity and suggests impaired DNA repair. SIGNIFICANCE Mutations in BRCA genes cause defects in DNA repair that predict sensitivity to DNA damaging agents, including platinum; however, some patients without BRCA mutations also benefit from these agents. NtAI, a genomic measure of unfaithfully repaired DNA, may identify cancer patients likely to benefit from treatments targeting defective DNA repair.