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Exosomes

Posted in Big Data, BioIT: BioInformatics, Cell Biology, Chemical Genetics, Clinical Genomics, Curation, Exosomes, Gene Regulation and Evolution, Genome Biology, tagged DNA, Exome sequencing, genome, mutations, Proteins on November 17, 2015| Leave a Comment »

Exosomes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Human Exomes Galore

A new database includes complete sequences of protein-coding DNA from 60,706 individuals.

By Karen Zusi | November 16, 2015

http://www.the-scientist.com//?articles.view/articleNo/44483/title/Human-Exomes-Galore/

The ability to sequence a person’s entire genome has led many researchers to hunt for the genetic causes of certain diseases. But without a larger set of genomes to compare mutations against, putting these variations into context is difficult. An international group of researchers has banked the full exomes of 60,706 individuals in a database called the Exome Aggregation Consortium (ExAC). The team’s analaysis, posted last month (October 30) on the preprint server bioRxiv, was presented at the Genome Science 2015 conference in Birmingham, U.K. (September 7).

Led by Daniel MacArthur from the Broad Institute of MIT and Harvard, the research team collected exomes from labs around the world for its dataset. “The resulting catalogue of human genetic diversity has unprecedented resolution,” the authors wrote in their preprint. Many of the variants observed in the dataset occurred only once.

“This is one of the most useful resources ever created for medical testing for genetic disorders,” Heidi Rehm, a clinical lab director at Harvard Medical School, told Science News.

Among other things, the team found 3,230 genes that are highly conserved across exomes, indicating likely involvement in critical cellular functions. Of these, 2,557 are not associated with diseases. The authors hypothesized that these genes, if mutated, either lead to embryonic death—before a problem can be diagnosed—or cause rare diseases that have not yet been genetically characterized.

“We should soon be able to say, with high precision: If you have a mutation at this site, it will kill you. And we’ll be able to say that without ever seeing a person with that mutation,” MacArthur said during his Genome Science talk, according to The Atlantic.

This is not the complete set of essential genes in the human body, David Goldstein, a geneticist at Columbia University in New York City, pointed out to Nature. Only by studying more exomes will researchers be able to refine that number, he noted.

Analysis of protein-coding genetic variation in 60,706 humans

, , , , , et al.

http://biorxiv.org/content/early/2015/10/30/030338 doi: http://dx.doi.org/10.1101/030338

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) sequence data for 60,706 individuals of diverse ethnicities. The resulting catalogue of human genetic diversity has unprecedented resolution, with an average of one variant every eight bases of coding sequence and the presence of widespread mutational recurrence. The deep catalogue of variation provided by the Exome Aggregation Consortium (ExAC) can be used to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; we identify 3,230 genes with near-complete depletion of truncating variants, 79% of which have no currently established human disease phenotype. Finally, we show that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human knockout variants in protein-coding genes.

Analysis of protein-coding genetic variation in 60,706 humans https://t.co/z0PtB4c8aY

Over the last five years, the widespread availability of high-throughput DNA sequencing technologies has permitted the sequencing of the whole genomes or exomes (the 18 protein-coding regions of genomes) of over half a million humans. In theory, these data represent a powerful source of information about the global patterns of human genetic variation, but in practice, are difficult to access for practical, logistical, and ethical reasons; in addition, the inconsistent processing complicates variant-calling pipelines used by different groups. Current publicly available datasets of human DNA sequence variation contain only a small fraction of all sequenced samples: the Exome Variant Server, created as part of the NHLBI Exome Sequencing Project (ESP)1, contains frequency information spanning 6,503 exomes; and the 1000 Genomes (1000G) Project, which includes individual-level genotype data from whole-genome and exome sequence data for 2,504 individuals2.

Databases of genetic variation are important for our understanding of human population history and biology1–5, but also provide critical resources for the clinical interpretation of variants observed in patients suffering from rare Mendelian diseases6,7. The filtering of candidate variants by frequency in unselected individuals is a key step in any pipeline for the discovery of causal variants in Mendelian disease patients, and the efficacy of such filtering depends on both the size and the ancestral diversity of the available reference data.

Here, we describe the joint variant calling and analysis of high-quality variant calls across 60,706 human exomes, assembled by the Exome Aggregation Consortium (ExAC; exac.broadinstitute.org). This call set exceeds previously available exome-wide variant databases by nearly an order of magnitude, providing unprecedented resolution for the analysis of very low-frequency genetic variants. We demonstrate the application of this data set to the analysis of patterns of genetic variation including the discovery of widespread mutational recurrence, the inference of gene-level constraint against 10 truncating variation, the clinical interpretation of variation in Mendelian disease genes, and the discovery of human “knockout” variants in protein-coding genes.

…..

Deleterious variants are expected to have lower allele frequencies than neutral ones, due to negative selection. This theoretical property has been demonstrated previously in human population sequencing data18,19 and here (Figure 1d, Figure 1e). This allows inference of the degree of natural selection against specific functional classes of variation: however, mutational recurrence as described above indicates that allele frequencies observed in ExAC-scale samples are also skewed by mutation rate, with 10 more mutable sites less likely to be singletons (Figure 2c and Extended Data Figure 4d). Mutation rate is in turn non-uniformly distributed across functional classes – for instance, stop lost mutations can never occur at CpG dinucleotides (Extended Data Figure 4e). We corrected for mutation rates (Supplementary Information) by creating a mutability-adjusted proportion singleton (MAPS) metric. This metric reflects (as expected) strong selection against predicted PTVs, as well as missense variants predicted by conservation-based methods to be deleterious (Figure 2e).

The deep ascertainment of rare variation in ExAC also allows us to infer the extent of 19 selection against variant categories on a per-gene basis by examining the proportion of 20 variation that is missing compared to expectations under random mutation. Conceptually similar approaches have been applied to smaller exome datasets13,20 but have been underpowered, particularly for the analysis of depletion of PTVs. We compared the observed number of rare (MAF <0.1%) variants per gene to an expected number derived from a selection neutral, sequence-context based mutational model13. The model performs extremely well in predicting the number of synonymous variants, which should be under minimal purifying selection, per gene (r = 0.98; Extended Data Figure 5).

……

Critically, we note that LoF-intolerant genes include virtually all known severe haploinsufficient human disease genes (Figure 3b), but that 79% of LoF-intolerant genes have not yet been assigned a human disease phenotype despite the clear evidence for extreme selective constraint (Supplementary Information 4.11). These likely represent either undiscovered severe dominant disease genes, or genes in which loss of a single copy results in embryonic lethality.

The most highly constrained missense (top 25% missense Z scores) and PTV (pLI ≥0.9) genes show higher expression levels and broader tissue expression than the least constrained genes24 (Figure 3c). These most highly constrained genes are also depleted for eQTLs (p < 10-9 for missense and PTV; Figure 3d), yet are enriched within genome-wide significant trait-associated loci (χ2 p < 10-14, Figure 3e). Intuitively, genes intolerant of PTV variation are dosage sensitive: natural selection does not tolerate a 50% deficit in expression due to the loss of single allele. It is therefore unsurprising that these genes are also depleted of common genetic variants that have a large enough effect on expression to be detected as eQTLs with current limited sample sizes. However, smaller changes in the expression of these genes, through weaker eQTLs or functional variants, are more likely to contribute to medically relevant phenotypes. Therefore, highly constrained genes are dosage-sensitive, expressed more broadly across tissues (as expected for core cellular processes), and are enriched for medically relevant variation.

Finally, we investigated how these constraint metrics would stratify mutational classes according to their frequency spectrum, corrected for mutability as in the previous section (Figure 3f). The effect was most dramatic when considering stop-gained variants in the LoF-intolerant set of genes. For missense variants, the missense Z score offers information additional to Polyphen2 and CADD classifications, indicating that gene-level measures of constraint offer additional information to variant-level metrics in assessing potential pathogenicity.

We assessed the value of ExAC as a reference dataset for clinical sequencing approaches, which typically prioritize or filter potentially deleterious variants based on functional consequence and allele frequency6. To simulate a Mendelian variant analysis, we filtered variants in 100 ExAC exomes per continental population against ESP (the previous default reference data set for clinical analysis) or the remainder of ExAC, removing variants present at ≥0.1% allele frequency, a filter recommended for dominant 16 disease variant discovery6. Filtering on ExAC reduced the number of candidate protein-altering variants by 7-fold compared to ESP, and was most powerful when the highest 18 allele frequency in any one population (“popmax”) was used rather than average (“global”) allele frequency (Figure 4a). ESP is not well-powered to filter at 0.1% AF without removing many genuinely rare variants, as AF estimates based on low allele counts are both upward-biased and imprecise (Figure 4b). We thus expect that ExAC will provide a very substantial boost in the power and accuracy of variant filtering in Mendelian disease projects.

…….

The above curation efforts confirm the importance of allele frequency filtering in analysis of candidate disease variants. However, literature and database errors are prevalent even at lower allele frequencies: the average ExAC exome contains 0.89 reportedly Mendelian variants in well-characterized dominant disease genes at <1% popmax AF and 0.20 at <0.1% popmax AF. This inflation likely results from a combination of false reports of pathogenicity and incomplete penetrance, as we show for PRNP in the accompanying work [Minikel et al, submitted]. The abundance of rare functional variation in many disease genes in ExAC is a reminder that such variants should not be assumed to be causal or highly penetrant without careful segregation or case-control analysis28,7.

We investigated the distribution of PTVs, variants predicted to disrupt protein-coding genes through the introduction of a stop codon or frameshift or the disruption of an essential splice site; such variants are expected to be enriched for complete loss-of-function of the impacted genes. Naturally-occurring PTVs in humans provide a model for the functional impact of gene inactivation, and have been used to identify many genes in 6 which LoF causes severe disease31, as well as rare cases where LoF is protective against disease32.

Among the 7,404,909 HQ variants in ExAC, we found 179,774 high-confidence PTVs (as 10 defined in Supplementary Information Section 6), 121,309 of which are singletons. This 11 corresponds to an average of 85 heterozygous and 35 homozygous PTVs per individual (Figure 5a). The diverse nature of the cohort enables the discovery of substantial numbers of novel PTVs: out of 58,435 PTVs with an allele count greater than one, 33,625 occur in only one population. However, while PTVs as a category are extremely rare, the majority of the PTVs found in any one person are common, and each individual 16 has only ~2 singleton PTVs, of which 0.14 are found in PTV-constrained genes (pLI 17 >0.9). The site frequency spectrum of these variants across the populations represented in ExAC recapitulates known aspects of demographic models, including an increase in intermediate-frequency (1%-5%) PTVs in Finland33 and relatively common (>0.1%) PTVs in Africans (Figure 5b).

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Discussion Here we describe the generation and analysis of the most comprehensive catalogue of 29 human protein-coding genetic variation to date, incorporating high-quality exome sequencing data from 60,706 individuals of diverse geographic ancestry. The resulting call set provides unprecedented resolution for the analysis of very low-frequency protein-coding variants in human populations, as well as a powerful resource for the clinical interpretation of genetic variants observed in disease patients. The complete frequency CC-BY-ND 4.0 International license for this preprint is the author/funder. It is made available under a bioRxiv preprint first posted online October 30, 2015;
http://dx.doi.org/10.1101/030338 ; The copyright holder and annotation data from this call-set has been made freely available through a public website [exac.broadinstitute.org]

The ExAC resource provides the largest database to date for the estimation of allele frequency for protein-coding genetic variants, providing a powerful filter for analysis of candidate pathogenic variants in severe Mendelian diseases. Frequency data from ESP1 have been widely used for this purpose, but those data are limited by population diversity and by resolution at allele frequencies ≤0.1%. ExAC therefore provides 21 substantially improved power for Mendelian analyses, although it is still limited in power at lower allele frequencies, emphasizing the need for more sophisticated pathogenic variant filtering strategies alongside on-going data aggregation efforts. ExAC also highlights an unexpected tolerance of many disease genes to functional variation, and reveals that the literature and public databases contain an inflated number of reportedly pathogenic variants across the frequency spectrum, indicating a need for stringent criteria for assertions of pathogenicity.

Finally, we show that different populations confer different advantages in the discovery of gene-disrupting PTVs, providing guidance for projects seeking to identify human “knockouts” to understand gene function. Individuals of African ancestry have more PTVs (140 on average), with this enrichment most pronounced at allele frequencies above 1% (Figure 5b). Finnish individuals, as a result of a population bottleneck, are depleted at the lowest (<0.1%) allele frequencies but have a peak in frequency at 1-5% (Figure 5b). However, these differences are diminished when considering only LoF-constrained (pLI > 0.9) genes (Extended Data Figure 10). Sampling multiple populations would likely be a fruitful strategy for a researcher investigating common PTV variation. However, discovery of homozygous PTVs is markedly enhanced in the South Asia samples, which come primarily from a Pakistani cohort with 38.3% of individuals self- reporting as having closely related parents, emphasizing the extreme value of consanguineous cohorts for “human knockout” discovery (Figure 5d) [Saleheen et al., to 8 be co-submitted].

…..

While the ExAC dataset dramatically exceeds the scale of previously available frequency reference datasets, much remains to be gained by further increases in sample size. Indeed, the fact that even the rarest transversions have mutational rates13 on the order of 1 x 10-9 implies that almost all possible non-lethal SNVs likely exist in some person on Earth. ExAC already includes >70% of all possible protein-coding CpG transitions at well-covered sites; order of magnitude increases in sample size will eventually lead to saturation of other classes of variation.

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Geneticist George Church: A Future Without Limits

Posted in BioIT: BioInformatics, BioIT: BioInformatics, NGS, Clinical & Translational, Pharmaceutical R&D Informatics, Clinical Genomics, Cancer Informatics, BioTechnology - Venture Creation, Genomic Testing: Methodology for Diagnosis, Interviews with Key Opinion Leaders (KOLs), Investment in Technological Breakthrough, Massachusetts Academy of Sciences (MAS) , Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Personalized Medicine Coalition, Precision Cancer Medicine, RNA Biology, Cancer and Therapeutics, Scoop.it, Transcriptomics, Translational Science, tagged biotech, Exome sequencing, gene, George Church, MIT, Personalized medicine, precision medicine, variants, Venture capital, WES on October 24, 2014| Leave a Comment »

UPDATED 12/05/2020

In the future, George Church believes, almost everything will be better because of genetics. If you have a medical problem, your doctor will be able to customize a treatment based on your specific DNA pattern. When you fill up your car, you won’t be draining the world’s dwindling supply of crude oil, because the fuel will come from microbes that have been genetically altered to produce biofuel. When you visit the zoo, you’ll be able to take your children to the woolly mammoth or passenger pigeon exhibits, because these animals will no longer be extinct. You’ll be able to do these things, that is, if the future turns out the way Church envisions it—and he’s doing everything he can to see that it does.

UPDATED 12/05/2020

George Church backs a startup solution to the massive gene therapy manufacturing bottleneck

Source: https://endpts.com/george-church-backs-a-startup-solution-to-the-massive-gene-therapy-manufacturing-bottleneck/
Jason Mast: Associate Editor
George Church and his graduate students have spent the last decade seeding startups on the razor’s edge between biology and science fiction: gene therapy to prevent aging, CRISPRed pigs that can be used to harvest organs for transplant, and home kits to test your poop for healthy or unhealthy bacteria. (OK, maybe they’re not all on that razor’s edge.)

But now a new spinout from the Department of Genetics’ second floor is tackling a far humbler problem — one that major company after major company has stumbled over as they tried to get cures for rare diseases and other gene therapies into the clinic and past regulators: How the hell do you build these?

CEO Lex Vovner of 64x Bio

“There’s a lot happening for new therapies but not enough attention around this problem,” Lex Rovner, who was a post-doc at Church’s lab from 2015 to 2018, told Endpoints News. “And if we don’t figure out how to fix this, many of these therapies won’t even reach patients.”

This week, with Church and a couple other prominent scientists as co-founders, Rovner launched 64x Bio to tackle one key part of the manufacturing bottleneck. They won’t be looking to retrofit plants or build gene therapy factories, as Big Pharma and big biotech are now spending billions to do. Instead, with $4.5 million in seed cash, they will try to engineer the individual cells that churn out a critical component of the therapies.

George Church
The goal is to build cells that are fine-tuned to do nothing but spit out the viral vectors that researchers and drug developers use to shuttle gene therapies into the body. Different vectors have different demands; 64x Bio will look to make efficient cellular factories for each.

“While a few general ways to increase vector production may exist, each unique vector serotype and payload poses a specific challenge,” Church said in an emailed statement. “Our platform enables us to fine tune custom solutions for these distinct combinations that are particularly hard to overcome.”

Before joining Church’s lab, Rovner did her graduate work at Yale, where she studied how to engineer bacteria to produce new kinds of protein for drugs or other purposes. And after leaving Church’s lab in 2018, she initially set out to build a manufacturing startup with a broad focus.

Yet as she spoke with hundreds of biotech executives on LinkedIn and in coffee shops around Cambridge, the same issue kept popping up: They liked their gene therapy technology in the lab but they didn’t know how to scale it up.

“Everyone kept saying the same thing,” Rovner said. “We basically realized there’s this huge problem.”

The issue would soon make headlines in industry publications: bluebird delaying the launch of Zynteglo, Novartis delaying the launch of Zolgensma in the EU, Axovant delaying the start of their Parkinson’s trial.

Part of the problem, Rovner said, is that gene therapies are delivered on viral vectors. You can build these vectors in mammalian cell lines by feeding them a small circular strand of DNA called a plasmid. The problem is that mammalian cells have, over billions of years, evolved tools and defenses precisely to avoid making viruses. (Lest the mammal they live in die of infection).

There are genetic mutations that can turn off some of the internal defenses and unleash a cell’s ability to produce virus, but they’re rare and hard to find. Other platforms, Rovner said, try to find these mutations by using CRISPR to knock out genes in different cells and then screening each of them individually, a process that can require hundreds of thousands of different 100-well plates, with each well containing a different group of mutant cells.

“It’s just not practical, and so these platforms never find the cells,” Rovner said.

64x Bio will try to find them by building a library of millions of mutant mammalian cells and then using a molecular “barcoding” technique to screen those cells in a single pool. The technique, Rovner said, lets them trace how much vector any given cell produces, allowing researchers to quickly identify super-producing cells and their mutations.

The technology was developed partially in-house but draws from IP at Harvard and the Wyss Institute. Harvard’s Pam Silver and Wyss’s Jeffrey Way are co-founders.

The company is now based in SoMa in San Francisco. With the seed cash from Fifty Years, Refactor and First Round Capital, Rovner is recruiting and looking to raise a Series A soon. They’re in talks with pharma and biotech partners, while they try to validate the first preclinical and clinical applications.

Gene therapy is one focus, but Rovner said the platform works for anything that involves viral vector, including vaccines and oncolytic viruses. You just have to find the right mutation.

“It’s the rare cell you’re looking for,” she said.

AUTHOR
Jason Mast
Associate Editor
jason@endpointsnews.com
@JasonMMast
Jason Mas

In 2005 he launched the Personal Genome Project, with the goal of sequencing and sharing the DNA of 100,000 volunteers. With an open-source database of that size, he believes, researchers everywhere will be able to meaningfully pursue the critical task of correlating genetic patterns with physical traits, illnesses, and exposure to environmental factors to find new cures for diseases and to gain basic insights into what makes each of us the way we are. Church, tagged as subject hu43860C, was first in line for testing. Since then, more than 13,000 people in the U.S., Canada, and the U.K. have volunteered to join him, helping to establish what he playfully calls the Facebook of DNA.

Church has made a career of defying the impossible. Propelled by the dizzying speed of technological advancement since then, the Personal Genome Project is just one of Church’s many attempts to overcome obstacles standing between him and the future.

“It’s not for everyone,” he says. “But I see a trend here. Openness has changed since many of us were young. People didn’t use to talk about sexuality or cancer in polite society. This is the Facebook generation.” If individuals were told which diseases or medical conditions they were genetically predisposed to, they could adjust their behavior accordingly, he reasoned. Although universal testing still isn’t practical today, the cost of sequencing an individual genome has dropped dramatically in recent years, from about $7 million in 2007 to as little as $1,000 today.

“It’s all too easy to dismiss the future,” he says. “People confuse what’s impossible today with what’s impossible tomorrow.”, especially through the emerging discipline of “synthetic” biology. The basic idea behind synthetic biology, he explained, was that natural organisms could be reprogrammed to do things they wouldn’t normally do, things that might be useful to people. In pursuit of this, researchers had learned not only how to read the genetic code of organisms but also how to write new code and insert it into organisms. Besides making plastic, microbes altered in this way had produced carpet fibers, treated wastewater, generated electricity, manufactured jet fuel, created hemoglobin, and fabricated new drugs. But this was only the tip of the iceberg, Church wrote. The same technique could also be used on people.

“Every cell in our body, whether it’s a bacterial cell or a human cell, has a genome,” he says. “You can extract that genome—it’s kind of like a linear tape—and you can read it by a variety of methods. Similarly, like a string of letters that you can read, you can also change it. You can write, you can edit it, and then you can put it back in the cell.”

This April, the Broad Institute, where Church holds a faculty appointment, was awarded a patent for a new method of genome editing called CRISPR (clustered regularly interspersed short palindromic repeats), which Church says is one of the most effective tools ever developed for synthetic biology. By studying the way that certain bacteria defend themselves against viruses, researchers figured out how to precisely cut DNA at any location on the genome and insert new material there to alter its function. Last month, researchers at MIT announced they had used CRISPR to cure mice of a rare liver disease that also afflicts humans. At the same time, researchers at Virginia Tech said they were experimenting on plants with CRISPR to control salt tolerance, improve crop yield, and create resistance to pathogens.

The possibilities for CRISPR technology seem almost limitless, Church says. If researchers have stored a genetic sequence in a computer, they can order a robot to produce a piece of DNA from the data. That piece can then be put into a cell to change the genome. Church believes that CRISPR is so promising that last year he co-founded a genome-editing company, Editas, to develop drugs for currently incurable diseases.

Source: news.nationalgeographic.com

See on Scoop.it – Cardiovascular and vascular imaging

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Finding the Genetic Links in Common Disease: Caveats of Whole Genome Sequencing Studies

Posted in Alzheimer's Disease, Biological Networks, Gene Regulation and Evolution, Cardiovascular Pharmacogenomics, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Etiology, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Testing: Methodology for Diagnosis, Health Economics and Outcomes Research, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmacogenomics, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, tagged Alzheimers Disease, American Society of Human Genetics, Aviva Lev-Ari, Biology, Conditions and Diseases, Congenital disorder of glycosylation, DNA, DNA Sequencing, Exome sequencing, Full genome sequencing, gene, genetic variants, genetics, Genome-wide association study, health, Heart disease, metabolic syndrome, Parkinsons disease, Personalized medicine, Single Nucleotide polymorphisms, Single-nucleotide polymorphism, type 2 diabetes on May 15, 2013| 5 Comments »

Finding the Genetic Links in Common Disease: Caveats of Whole Genome Sequencing Studies

Writer and Reporter: Stephen J. Williams, Ph.D.

In the November 23, 2012 issue of Science, Jocelyn Kaiser reports (Genetic Influences On Disease Remain Hidden in News and Analysis)[1] on the difficulties that many genomic studies are encountering correlating genetic variants to high risk of type 2 diabetes and heart disease. At the recent American Society of Human Genetics annual 2012 meeting, results of several DNA sequencing studies reported difficulties in finding genetic variants and links to high risk type 2 diabetes and heart disease. These studies were a part of an international effort to determine the multiple genetic events contributing to complex, common diseases like diabetes. Unlike Mendelian inherited diseases (like ataxia telangiectasia) which are characterized by defects mainly in one gene, finding genetic links to more complex diseases may pose a problem as outlined in the article:

Variants may be so rare that massive number of patient’s genome would need to be analyzed
For most diseases, individual SNPs (single nucleotide polymorphisms) raise risk modestly
Hard to find isolated families (hemophilia) or isolated populations (Ashkenazi Jew)
Disease-influencing genes have not been weeded out by natural selection after human population explosion (~5000 years ago) resulted in numerous gene variants
What percentage variants account for disease heritability (studies have shown this is as low as 26% for diabetes with the remaining risk determined by environment)

Although many genome-wide-associations studies have found SNPs that have causality to increasing risk diseases such as cancer, diabetes, and heart disease, most individual SNPs for common diseases raise risk by about only 20-40% and would be useless for predicting an individual’s chance they will develop disease and be a candidate for a personalized therapy approach. Therefore, for common diseases, investigators are relying on direct exome sequencing and whole-genome sequencing to detect these medium-rare risk variants, rather than relying on genome-wide association studies (which are usually fine for detecting the higher frequency variants associated with common diseases).

Three of the many projects (one for heart risk and two for diabetes risk) are highlighted in the article:

1. National Heart, Lung and Blood Institute Exome Sequencing Project (ESP)[2]: heart, lung, blood

Sequenced 6,700 exomes of European or African descent
Majority of variants linked to disease too rare (as low as one variant)
Groups of variants in the same gene confirmed link between APOC3 and higher risk for early-onset heart attack
No other significant gene variants linked with heart disease

2. T2D-GENES Consortium: diabetes

Sequenced 5,300 exomes of type 2 diabetes patients and controls from five ancestry groups
SNP in PAX4 gene associated with disease in East Asians
No low-frequency variant with large effect though

3. GoT2D: diabetes

After sequencing 2700 patient’s exomes and whole genome no new rare variants above 1.5% frequency with a strong effect on diabetes risk

A nice article by Dr. Sowmiya Moorthie entitled Involvement of rare variants in common disease can be found at the PGH Foundation site http://www.phgfoundation.org/news/5164/ further discusses this conundrum, and is summarized below:

“Although GWAs have identified many SNPs associated with common disease, they have as yet had little success in identifying the causative genetic variants. Those that have been identified have only a weak effect on disease risk, and therefore only explain a small proportion of the heritable, genetic component of susceptibility to that disease. This has led to the common disease-common variant hypothesis, which predicts that common disease-causing genetic variants exist in all human populations, but each individual variant will necessarily only have a small effect on disease susceptibility (i.e. a low associated relative risk).

An alternative hypothesis is the common disease, many rare variants hypothesis, which postulates that disease is caused by multiple strong-effect variants, each of which is only found in a few individuals. Dickson et al. in a paper in PLoS Biology postulate that these rare variants can be indirectly associated with common variants; they call these synthetic associations and demonstrate how further investigation could help explain findings from GWA studies [Dickson et al. (2010) PLoS Biol. 8(1):e1000294][3]. In simulation experiments, 30% of synthetic associations were caused by the presence of rare causative variants and furthermore, the strength of the association with common variants also increased if the number of rare causative variants increased. “

Figure from Dr. Moorthie’s article showing the problem of “finding one in many”.

(please click to enlarge)

Indeed, other examples of such issues concerning gene variant association studies occur with other common diseases such as neurologic diseases and obesity, where it has been difficult to clearly and definitively associate any variant with prediction of risk.

For example, Nuytemans et. al.[4] used exome sequencing to find variants in the vascular protein sorting 3J (VPS35) and eukaryotic transcription initiation factor 4 gamma1 (EIF4G1) genes, tow genes causally linked to Parkinson’s Disease (PD). Although they identified novel VPS35 variants none of these variants could be correlated to higher risk of PD. One EIF4G1 variant seemed to be a strong Parkinson’s Disease risk factor however there was “no evidence for an overall contribution of genetic variability in VPS35 or EIF4G1 to PD development”.

These negative results may have relevance as companies such as 23andme (www.23andme.com) claim to be able to test for Parkinson’s predisposition. To see a description of the LLRK2 mutational analysis which they use to determine risk for the disease please see the following link: https://www.23andme.com/health/Parkinsons-Disease/. This company and other like it have been subjects of posts on this site (Personalized Medicine: Clinical Aspiration of Microarrays)

However there seems to be more luck with strategies focused on analyzing intronic sequence rather than exome sequence. Jocelyn Kaiser’s Science article notes this in a brief interview with Harry Dietz of Johns Hopkins University where he suspects that “much of the missing heritability lies in gene-gene interactions”. Oliver Harismendy and Kelly Frazer and colleagues’ recent publication in Genome Biology http://genomebiology.com/content/11/11/R118 support this notion[5]. The authors used targeted resequencing of two endocannabinoid metabolic enzyme genes (fatty-acid-amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in 147 normal weight and 142 extremely obese patients.

These patients were enrolled in the CRESCENDO trial and patients analyzed were of European descent. However, instead of just exome sequencing, the group resequenced exome AND intronic sequence, especially focusing on promoter regions. They identified 1,448 single nucleotide variants but using a statistical filter (called RareCover which is referred to as a collapsing method) they found 4 variants in the promoters and intronic areas of the FAAH and MGLL genes which correlated to body mass index. It should be noted that anandamide, a substrate for FAAH, is elevated in obese patients. The authors did note some issues though mentioning that “some other loci, more weakly or inconsistently associated in the original GWASs, were not replicated in our samples, which is not too surprising given the sample size of our cohort is inadequate to replicate modest associations”.

PLEASE WATCH VIDEO on the National Heart, Lung and Blood Institute Exome Sequencing Project

https://www.youtube.com/watch?v=-Qr5ahk1HEI

REFERENCES

http://www.phgfoundation.org/news/5164/ PHG Foundation

1. Kaiser J: Human genetics. Genetic influences on disease remain hidden. Science 2012, 338(6110):1016-1017.

2. Tennessen JA, Bigham AW, O’Connor TD, Fu W, Kenny EE, Gravel S, McGee S, Do R, Liu X, Jun G et al: Evolution and functional impact of rare coding variation from deep sequencing of human exomes. Science 2012, 337(6090):64-69.

3. Dickson SP, Wang K, Krantz I, Hakonarson H, Goldstein DB: Rare variants create synthetic genome-wide associations. PLoS biology 2010, 8(1):e1000294.

4. Nuytemans K, Bademci G, Inchausti V, Dressen A, Kinnamon DD, Mehta A, Wang L, Zuchner S, Beecham GW, Martin ER et al: Whole exome sequencing of rare variants in EIF4G1 and VPS35 in Parkinson disease. Neurology 2013, 80(11):982-989.

5. Harismendy O, Bansal V, Bhatia G, Nakano M, Scott M, Wang X, Dib C, Turlotte E, Sipe JC, Murray SS et al: Population sequencing of two endocannabinoid metabolic genes identifies rare and common regulatory variants associated with extreme obesity and metabolite level. Genome biology 2010, 11(11):R118.

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Whole exome somatic mutations analysis of malignant melanoma contributes to the development of personalized cancer therapy for this disease

Posted in Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, tagged Cancer - General, Cancer research, Conditions and Diseases, Exome sequencing, gene sequencing, Ipilimumab, Melanoma, Personalized medicine, skin cancer, treatment, Vemurafenib on April 23, 2013| 3 Comments »

Whole exome somatic mutations analysis of malignant melanoma contributes to the development of personalized cancer therapy for this disease

Author: Ziv Raviv, PhD

Article-7.2.2. Whole exome somatic mutations analysis of malignant melanoma contributes to the development of personalized cancer therapy for this disease

Introduction

Cutaneous melanoma is a type of skin cancer that originates in melanocytes, the cells that are producing melanin. While being the least common type of skin cancer, melanoma is the most aggressive one with invasive characteristics and accounts for the majority of death incidences among skin cancers. Melanoma has an annual rate of 160,000 new cases and 48,000 deaths worldwide. Melanoma affects mainly Caucasians exposed to sun high UV irradiation. Among the genetic factors that characterize the disease, BRAF mutation (V600E) is found in most cases of melanoma (80%). Awareness toward risk factors of melanoma should lead to prevention and early detection*. There are several developmental stages (I-IV) of the disease, starting from local non-invasive melanoma, through invasive and high risk melanoma, up to metastatic melanoma. As with other cancers, the earlier stage melanoma is being detected, the better odds for full recovery are. Treatment is usually involving surgery to remove the local tumor and its margins, and when necessary also to remove the proximal lymph node(s) that drain the tumor. In high stages melanoma, adjuvant therapy is given in the form of chemotherapy (Dacarbazine and Temozolomide) and immunotherapy (IL-2 and IFN). Until recently no useful treatment was available for metastatic melanoma. However, research efforts had led to the development of two new drugs against metastatic melanoma: Vemurafenib (Zelboraf), a B-Raf inhibitor; and Ipilimumab (Yervoy), a monoclonal antibody that blocks the inhibitory signal of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Both drugs are now available for clinical use presenting good results.

Personalized therapy for melanoma

In an attempt to develop personalized therapies for malignant melanoma, a unique strategy has been taken by the group of Prof. Yardena Samuels at the NIH (now situated at the WIS) to identify recurring genetic alterations of metastatic cutaneous melanoma. The researchers approach employed the collections of hundreds of tumors samples taken from metastasized melanoma patients together with matched normal blood tissues samples. The samples are undergoing exome sequencing for the analysis of somatic mutations (namely mutations that evolved during the progress of the disease to the stage of metastatic melanoma, unlike genomic mutations that may have contribute to the formation of the disease). The discrimination of such tumor related somatic mutations is done by comparison to the exome sequencing of the patient’s matched blood cells DNA. In addition, the malignant cells derived from the removed cancer tissue of each patient are extracted to form a cell line and are grown in culture. These cells are easily cultivate in culture with no special media supplements, nor further genetic manipulations such as hTERT are needed, and are extremely aggressive as determined by various cell culture and in vivo tests. The ability to grow these primary tumor-derived cell lines in culture has a great value as a tool for studying and characterizing the biochemical, functional, and clinical aspects of the mutated genes identified.

In one study [1] Samuels and her colleagues performed this sequencing process for mutation analysis for the protein tyrosine kinase (PTK) gene family, as PTKs are frequently mutated in cancer. Using high-throughput gene sequencing to analyze the entire PTK gene family, the researchers have identified 30 somatic mutations affecting the kinase domains of 19 PTKs and subsequently evaluated the entire coding regions of the genes encoding these 19 PTKs for somatic mutations in 79 melanoma samples. The most frequent mutations were found in ERBB4, a member of the EGFR/ErbB family of receptor tyrosine kinase (RTK), were 19% of melanoma patients had such mutations. Seven missense mutations in the ERBB4 gene were found to induce increased kinase activity and transformation capability. Melanoma derived cell lines that were expressing these mutant ERBB4 forms had reduced cell growth after silencing ERBB4 by RNAi or after treatment with the ERBB inhibitor Lapatinib. Lapatinib is already in use in the clinic for the treatment of HER2 (ErbB2) positive breast cancers patients. Following this study, a clinical trial is now conducted with this drug to evaluate its effect in cutaneous metastatic melanoma patients harboring mutations in ERBB4.

In another study of this group [2], the scientists employed the exome sequencing method to analyze the somatic mutations of 734 G protein coupled receptors (GPCRs) in melanoma. GPCRs are regulating various signaling pathways including those that affect cell growth and play also important role in human diseases. This screen revealed that GRM3 gene that encode the metabotropic glutamate receptor 3 (mGluR3), was frequently mutated and that one of its mutations clustered within one position. Mutant GRM3 was found to selectively regulate the phosphorylation of MEK1 leading to increased anchorage-independent cell growth and cellular migration. Tumor derived melanoma cells expressing mutant GRM3 exhibited reduced cell growth and migration upon knockdown of GRM3 by RNAi or by treatment with the selective MEK inhibitor, Selumetinib (AZD-6244), a drug that is being testing in clinical trials. Altogether, the results of this study point to the increased violent characteristics of melanomas bearing mutational GRM3.

In a third study, melanoma samples were examined for somatic mutations in 19 human genes that encode ADAMTS proteins [3]. Some of the ADAMTS genes have been suggested before to have implication in tumorigenesis. ADAMTS18, which was previously found to be a candidate cancer gene, was found in this study to be highly mutated in melanoma. ADAMTS18 mutations were biologically examined and were found to induce an increased proliferation of melanoma cells, as well as increased cell migration and metastasis. Moreover, melanoma cells expressing these mutated ADAMTS18 had reduced cell migration after RNAi-mediated knockdown of ADAMTS18. Thus, these results suggest that genetic alteration of ADAMTS18 plays a major role in melanoma tumorigenesis. Since ADAMTS genes encode extracellular proteins, their accessibility to systematically delivered drugs makes them excellent therapeutic targets.

Conclusive remarks

The above illustrated research approach intends to discover frequent melanoma-specific mutations by employing high-throughput whole exome and genome sequencing means. For the most highly mutated genes identified, the biochemical, functional, and clinical aspects are being characterized to examine their relevancy to the disease outcomes. This approach therefore introduces new opportunities for clinical intervention for the treatment of cutaneous melanoma. In addition to the discovery of novel highly mutated genes, this approach may also help determine which pathways are altered in melanoma and how these genes and pathways interact. Finding melanoma-associated highly mutated genes could lead to personalized therapeutics specifically targeting these altered genes in individual melanomas. Along with the opportunity to develop new agents to treat melanoma, the approach takes advantage of existing anti-cancer drugs, utilizing them to treat these mutated genes melanoma individuals. In addition to their potential for therapeutics, the discovery of highly mutated genes in melanoma patients may lead to the discovery of new markers that may assist the diagnosis of the disease. The implications of these screenings findings on other types of cancer bearing common pathways similar to melanoma should be examined as well. Finally, this elegant approach should be adopted in research efforts of other cancer types.

* Special review will be published further in the cancer prevention section of Pharmaceutical Intelligence

References

1. Prickett TD, Agrawal NS, Wei X, Yates KE, Lin JC, Wunderlich JR, Cronin JC, Cruz P, Rosenberg SA, Samuels Y (2009) Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4. Nat Genet 41 (10):1127-1132

2. Prickett TD, Wei X, Cardenas-Navia I, Teer JK, Lin JC, Walia V, Gartner J, Jiang J, Cherukuri PF, Molinolo A, Davies MA, Gershenwald JE, Stemke-Hale K, Rosenberg SA, Margulies EH, Samuels Y (2011) Exon capture analysis of G protein-coupled receptors identifies activating mutations in GRM3 in melanoma. Nat Genet 43 (11):1119-1126

3. Wei X, Prickett TD, Viloria CG, Molinolo A, Lin JC, Cardenas-Navia I, Cruz P, Rosenberg SA, Davies MA, Gershenwald JE, Lopez-Otin C, Samuels Y (2010) Mutational and functional analysis reveals ADAMTS18 metalloproteinase as a novel driver in melanoma. Mol Cancer Res 8 (11):1513-1525

Related articles on melanoma on this open access online scientific journal:

1. In focus: Melanoma Genetics. Curator: Ritu Saxena, Ph.D.

2. In focus: Melanoma therapeutics. Author and Curator: Ritu Saxena, Ph.D.

3. A New Therapy for Melanoma. Reporter- Larry H Bernstein, M.D.

4. Thymosin alpha1 and melanoma. Author, Editor: Tilda Barliya, Ph.D.

5. Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes. Reporter and Curator: Dr. Sudipta Saha, Ph.D.

6. How Genome Sequencing is Revolutionizing Clinical Diagnostics. Reporter: Aviva Lev-Ari, PhD, RN.

7. Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing. Curator and Reporter: Stephen J. Williams, Ph.D.

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Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

Posted in Biological Networks, Gene Regulation and Evolution, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Prevention: Research & Programs, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Health Economics and Outcomes Research, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, tagged Aviva Lev-Ari, Biology, Cancer - General, Cancer Research UK, Cancer stem cell, carcinoid, DNA, Eukaryotic, everolimus, Evolution, Exome sequencing, gene expression, genetics, genome sequence, Mammalian target of rapamycin, medicine, mTOR, neuroendocrine tumor, New England Journal of Medicine, ovarian cancer, Personalized medicine, renal cancer, research, Targeted therapy on April 10, 2013| 14 Comments »

Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing[1]

Curator and Reporter: Stephen J. Williams, Ph.D.

UPDATED 3/11/2023

The updates to this article are an expansion of the original article on 4/10/2013 on tumor heterogeneity in renal cancer, extended to other solid tumor types.

Evaluating Tumor Evolution via Genomic Profiling of Individual Tumor Spheroids in a Malignant Ascites

Scientific Reports volume 8, Article number: 12724 (2018) Cite this article

Abstract

Epithelial ovarian cancer (EOC) is a silent but mostly lethal gynecologic malignancy. Most patients present with malignant ascites and peritoneal seeding at diagnosis. In the present study, we used a laser-aided isolation technique to investigate the clonal relationship between the primary tumor and tumor spheroids found in the malignant ascites of an EOC patient. Somatic alteration profiles of ovarian cancer-related genes were determined for eight spatially separated samples from primary ovarian tumor tissues and ten tumor spheroids from the malignant ascites using next-generation sequencing. We observed high levels of intra-tumor heterogeneity (ITH) in copy number alterations (CNAs) and single-nucleotide variants (SNVs) in the primary tumor and the tumor spheroids. As a result, we discovered that tumor cells in the primary tissues and the ascites were genetically different lineages. We categorized the CNAs and SNVs into clonal and subclonal alterations according to their distribution among the samples. Also, we identified focal amplifications and deletions in the analyzed samples. For SNVs, a total of 171 somatic mutations were observed, among which 66 were clonal mutations present in both the primary tumor and the ascites, and 61 and 44 of the SNVs were subclonal mutations present in only the primary tumor or the ascites, respectively. Based on the somatic alteration profiles, we constructed phylogenetic trees and inferred the evolutionary history of tumor cells in the patient. The phylogenetic trees constructed using the CNAs and SNVs showed that two branches of the tumor cells diverged early from an ancestral tumor clone during an early metastasis step in the peritoneal cavity. Our data support the monophyletic spread of tumor spheroids in malignant ascites.

Conclusion

In this study, we performed genome-wide sequence analysis of the primary tumor and the associated tumor spheroids in the malignant ascites of an EOC patient. We analyzed genetic heterogeneity in the primary tumor and tumor spheroids through multi-region sequencing and the laser-aided cell isolation technique¹¹. From the sequencing data, we discovered clonal or subclonal somatic CNAs and SNVs, based on which we constructed phylogenetic trees and inferred the evolutionary history of tumor cells in the patient. As a result, we found that the tumor cells in the malignant ascites were an independent lineage from the primary tumor. The phylogenetic analysis showed that the lineage branched before the evolution of the cancer cells at the primary tissues, which suggests that analyzing malignant ascites might be used to detect ovarian cancer or metastasis in the early stage. In summary, the genetic plasticity and similarity between a primary tumor and associated tumor spheroids are still not clear, and yet, the nature of the similarity may have profound implications for both tumor progression and therapeutic outcomes in ovarian cancer. Therefore, future prospective studies profiling the genomic information of primary ovarian tumors, distant metastatic tumors, and tumor spheroids to determine the direction of tumor evolution and metastasis of ovarian cancer are warranted.

Tumor evolution and intratumor heterogeneity of an epithelial ovarian cancer investigated using next-generation sequencing

BMC Cancer volume 15, Article number: 85 (2015) Cite this article

Abstract

Background

The extent to which metastatic tumors further evolve by accumulating additional mutations is unclear and has yet to be addressed extensively using next-generation sequencing of high-grade serous ovarian cancer.

Methods

Eleven spatially separated tumor samples from the primary tumor and associated metastatic sites and two normal samples were obtained from a Stage IIIC ovarian cancer patient during cytoreductive surgery prior to chemotherapy. Whole exome sequencing and copy number analysis were performed. Omental exomes were sequenced with a high depth of coverage to thoroughly explore the variants in metastatic lesions. Somatic mutations were further validated by ultra-deep targeted sequencing to sort out false positives and false negatives. Based on the somatic mutations and copy number variation profiles, a phylogenetic tree was generated to explore the evolutionary relationship among tumor samples.

Results

Only 6% of the somatic mutations were present in every sample of a given case with TP53 as the only known mutant gene consistently present in all samples. Two non-spatial clusters of primary tumors (cluster P1 and P2), and a cluster of metastatic regions (cluster M) were identified. The patterns of mutations indicate that cluster P1 and P2 diverged in the early phase of tumorigenesis, and that metastatic cluster M originated from the common ancestral clone of cluster P1 with few somatic mutations and copy number variations.

Conclusions

Although a high level of intratumor heterogeneity was evident in high-grade serous ovarian cancer, our results suggest that transcoelomic metastasis arises with little accumulation of somatic mutations and copy number alterations in this patient.

Discussion

We have shown the range of genomic diversity at nucleotide, copy number and gene expression scales present in six patients with HGSC. These samples were resected prior to treatment, revealing intratumoural diversity in the tumours’ natural evolutionary state. Our results were consistent with recent reports 39 that considerable genomic diversity is measurable in HGSCs at the level of copy number. However, in some cases (cases 3 and 5) we observed large-scale copy number differences in spatially separated samples that were not evident at the level of mutational profiling, indicative of different mutational mechanisms operating independently in different parts of a tumour. Together, copy number and mutational profiling identified well-characterized mutations in genes such as PIK3CA, CTNNB1 and PDGFRB, as well as homozygous deletions in NF1, that were not present in all samples of the same case. With the exception of TP53, our data suggest that well-known, actionable driver mutations may only be partially represented if only a single sample is considered per patient. Our results establish that embedded within the context of extreme intra- and intertumour heterogeneity, TP53 mutation remains the most stable genomic feature in HGSC. However, as efforts towards precision medicine informed by mutational landscapes of tumours reach maturity, it will be imprudent to ignore the degree to which regional genomic variation is present in primary samples prior to any treatment-related selective pressures. Moreover, studies of evolution in cancer through analysis of serial biopsies (eg through pre- and post-treatment samples, or comparisons of primary and metastatic samples), will need to account for evidence of divergent profiles simply due to regional sampling bias.

Mutational profiling revealed for the first time that mutations beyond TP53 are present in FT lesions. In case 4, we used the full spectrum of mutations to establish that evolutionary trajectories of two histologically and karyotypically distinct tumours within a single patient arose from a common aetiology, with 26.2% of all mutations (including TP53) conserved in the tubal lesion. Our results indicate how histologically distinct cell populations in the same patient can be linked by common aetiology in the FT. In contrast to HGSC, endometrioid tumours are thought to develop from atypical ovarian endometriosis, with the FT merely acting as a conduit for endometrial epithelium to spread to the ovary (reviewed in 40). The possibility of the FT playing a causative role in rare cases of endometrioid carcinoma, or mixed endometrioid–serous carcinoma, has not yet been explored. Mutations in case 5 revealed the FT lesion to be a metastatic implant more closely related to a lymph node metastasis than to the ovarian samples. Thus, future investigations into aetiological underpinnings of HGSC will likely benefit from mutational comparison to extra-tubal sites to definitively establish the evolutionary origin of FT lesions.

We noted an extreme case of unexpected genomic conservation. In case 6, samples were obtained at primary surgery and after 42 months, with 21 cycles of multi-agent chemotherapy in between. The two samples exhibited near-identical genomic landscapes. Coupled with the long survivorship of this patient, this observation represents an intriguing anecdote. Larger studies will be needed to establish if stable clonal population structure is a feature of long survivorship.

Finally, as proof of concept, examination of plasma ctDNA suggested that representative mutations in the ancestral clone and mutations present in subclones could be detected in ctDNA, although sensitivity to detect mutations varied from patient to patient. This implies that clinical sensitivity analyses will need to be undertaken to establish the generalizability of ctDNA analysis to clonally diverse tumours.

In summary, our study unveils the extensive genomic diversity in primary, untreated, high-grade serous cancers of the ovary prior to treatment-related selection pressures, and illuminates highly individualized evolutionary trajectories that will require detailed consideration if curative therapeutic strategies are to be achieved.

Updated 3/04/2023 articles on specific tumor type intratumoral heterogeneity

Complex Patterns of Genomic Heterogeneity Identified in 42 Tumor Samples and ctDNA of a Pulmonary Atypical Carcinoid Patient

Source: Tamsin J. Robb, Peter Tsai, Sandra Fitzgerald, Paula Shields, Pascalene S. Houseman, Rachna Patel, Vicky Fan, Ben Curran, Rexson Tse, Jacklyn Ting, Nicole Kramer, Braden J. Woodhouse, Esther Coats, Polona Le Quesne Stabej, Jane Reeve, Kate Parker, Ben Lawrence, Cherie Blenkiron, Cristin G. Print; Complex Patterns of Genomic Heterogeneity Identified in 42 Tumor Samples and ctDNA of a Pulmonary Atypical Carcinoid Patient. Cancer Research Communications 3 January 2023; 3 (1): 31–42. https://doi.org/10.1158/2767-9764.CRC-22-0101

Tumor evolution underlies many challenges facing precision oncology, and improving our understanding has the potential to improve clinical care. This study represents a rare opportunity to study tumor heterogeneity and evolution in a patient with an understudied cancer type. A patient with pulmonary atypical carcinoid, a neuroendocrine tumor, metastatic to 90 sites, requested and consented to donate tissues for research. 42 tumor samples collected at rapid autopsy from 14 anatomically distinct sites were analyzed through DNA whole-exome sequencing and RNA sequencing, and five analyzed through linked-read sequencing. Targeted DNA sequencing was completed on two clinical tissue biopsies and one blood plasma sample. Chromosomal alterations and gene variants accumulated over time, and specific chromosomal alterations preceded the single predicted gene driver variant (ARID1A). At the time of autopsy, all sites shared the gain of one copy of Chr 5, loss of one copy of Chr 6 and 21, chromothripsis of one copy of Chr 11, and 39 small variants. Two tumor clones (carrying additional variants) were detected at metastatic sites, and occasionally in different regions of the same organ (e.g., within the pancreas). Circulating tumor DNA (ctDNA) sequencing detected shared tumor variants in the blood plasma and captured marked genomic heterogeneity, including all metastatic clones but few private tumor variants. This study describes genomic tumor evolution and dissemination of a pulmonary atypical carcinoid donated by a single generous patient. It highlights the critical role of chromosomal alterations in tumor initiation and explores the potential of ctDNA analysis to represent genomically heterogeneous disease. Significance: DNA sequencing data from tumor samples and blood plasma from a single patient highlighted the critical early role of chromosomal alterations in atypical carcinoid tumor development. Common tumor variants were readily detected in the blood plasma, unlike emerging tumor variants, which has implications for using ctDNA to capture cancer evolution.

In this article Robb et. al analyzes the tumor evolution of variants and mutations over time in a single patient. Tumor samples were obtained from liquid biopsy as ctDNA, which is unlike the previous study in renal cancer, where multiple fine needle aspirates needed to be conducted to determine intratumoral heterogeneity with respect to spatial distribution within a solid tumor. In this current study the authors were able to study the evolution of tumor heterogeneity in a single patient.

Introduction

Tumor evolution underlies many of the most pressing challenges facing clinical precision oncology today, and a better understanding of this process has the potential to improve clinical care. We present a unique example of tumor evolution in an uncommon tumor type not previously featured in such studies, a pulmonary atypical carcinoid [an intermediate grade neuroendocrine tumor (NET)].

NETs arise from hormone-producing cells of the neuroendocrine system located throughout the body, and are highly heterogeneous, both genetically and pathologically. While once considered rare tumors (1) and thought to be indolent in nature (2), we now recognize that NETs have an age-adjusted incidence of 6.2 cases per 100,000 in the country where this study was undertaken, New Zealand (3), similar to the incidence of ovarian and cervical cancers (4). Pulmonary atypical carcinoids are mitotically active well-differentiated NETs, with one in five presenting with distant metastatic disease at diagnosis (5). They feature relatively few DNA variants, with recurrent variants occurring in chromatin remodeling genes such as MEN1 and ARID1A [reported in 25% and 10% of studied atypical carcinoids, respectively (6)]. In general, NETs feature few small DNA variants and often instead have large-scale chromosomal changes; for example, around 25% of pancreatic NETs lose a suite of 10 chromosomes, and a further 40% feature the loss of chromosome (Chr) 11 (7). Few genomic studies have been completed on pulmonary atypical carcinoids; however, comparative genomic hybridization studies identified recurrent deletions in Chr 11q (harboring MEN1) in around 60% of cases (8–10).

Tumor development is an evolutionary process with similarities to Darwinian natural selection, where tumor cells may be under multiple simultaneous “selective pressures” (11), including immune attack (12) and drug treatment (13). There are multiple debated models for the generation, propagation, and selection of variants throughout tumor development, including linear, branching, punctuated, and neutral evolution (11, 14), each of which may occur in different tumors or at different timepoints within the same patient. Tumor evolution has been associated with key clinical challenges, including cancer metastasis (15), immune evasion (12, 16), and drug resistance (13, 17, 18).

Sequencing the cell-free DNA (cfDNA) in a patient’s blood plasma to identify ctDNA variants derived from tumor cells has been postulated to better represent the total disease burden (including potential tumor heterogeneity) than single tumor biopsies or resections (19, 20). However, we do not yet fully understand the detectability of ctDNA shed from different anatomic sites around a patient’s body—and early results suggest that some tumor sites may be more easily detected than others (21).

In this study, we completed multimodal genomic analysis on samples collected at autopsy from 41 metastatic tumor sites from a single patient with an uncommon pulmonary atypical carcinoid, alongside a blood plasma sample and clinical biopsies, to catalog driver genomic alterations and hypothesize their order of accumulation. We compared the genomic variants identified in the tumors with those detected in the patient’s blood plasma to consider how well the ctDNA analysis represented the genomic tumor heterogeneity, and in turn, infer metastatic sites that clinical ctDNA assays may poorly detect. A potential limitation, though, as stated by authors

“studies that analyze many tumors from a single patient, while revealing detailed and valuable insights about that patient’s cancer, cannot necessarily be generalized to other patients. Furthermore, all samples except for two clinical biopsies were collected at a single timepoint (at autopsy), limiting the extent of computational tumor evolution inferences that could be made, and the small size of these biopsies limited the scope of genomic assays that could be applied

In their conclusions they allude to the branched evolution that was seen with the renal cancer study:

Models of Tumor Evolution and Metastatic Dissemination

We can hypothesize a putative genomic evolution process (Fig. 7) that is consistent with the genomic heterogeneity observed at autopsy and in the two clinical biopsies. The early lung tumor represented by the diagnostic biopsy featured gain of Chr 5, loss of Chr 21 as well as nine small variants, none of which are known or predicted cancer drivers. Genomic features of the tumors collected at autopsy are consistent with further mutation after the lung biopsy was undertaken, followed by an evolutionary event such as a selective sweep, that fixed into the genomes of subsequent tumors a loss of function variant in ARID1A as well as 29 small non-driver variants and additional chromosomal alterations including Chr 11 chromothripsis and Chr 6 loss. There are plausible selective advantages to the tumor of each, including Chr 6 LOH of the HLA genes responsible for presenting peptides to T cells, hypothetically reducing the immune system’s ability to recognize neoantigens presented by the tumor cells (47). The data are consistent with subsequent branching events, including the accumulation of a second somatic “hit” to EPS8L2 (where the first was somatic LOH on Chr 11), before two parallel lineages developed, metastatic group 1 and 2 (Fig. 7). In metastatic group 1, some variants of unknown significance deserve further study. For instance, truncation of tumor suppressor ETV6 [a common fusion gene partner in breast and thyroid cancer (48)], following the LOH of a region of Chr 12 may be functionally significant given this gene’s role in development (49). The missense SLIT1 variant was the only additional protein-affecting variant carried by tumors in metastatic group 2 and may play a role in angiogenesis and migration (50). Interestingly, the two metastatic groups in the pancreas appear to have arisen by two independent seeding events. However, in the literature there is not overwhelming evidence for consistent genetic drivers of metastatic progression (51, 52) and we have no evidence in our data to suggest the additional genomic variants accumulated by each of the two metastatic groups (e.g., in EPS8L2, ETV6, and SLIT1) were necessary to drive their metastasis. Given a general propensity of cancers to metastasize to lung (53), it is possible that the presence of multiple metastatic groups in the lung may be the result of tertiary metastatic seeding events returning tumor cells that have evolved elsewhere to the site of the lung primary tumor.

4/10/2013

Genomic instability is considered a hallmark and necessary for generating the mutations which drive tumorigenesis. Multiple studies had suggested that there may be multiple driver mutations and a plethora of passenger mutations driving a single tumor. This diversity of mutational spectrum is even noticed in cultured tumor cells (refer to earlier post Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell). Certainly, intratumor heterogeneity has been a concern to clinicians in determining the proper personalized therapy for a given cancer patient, and has been debated if multiple biopsies of a tumor is required to acquire a more complete picture of a tumor’s mutations. In the New England Journal of Medicine, lead author Dr. Marco Gerlinger in the laboratory of Dr. Charles Swanton of the Cancer Research UK London Research Institute, and colleagues reported the results of a study to determine if intratumoral differences exist in the mutational spectrum of primary and metastatic renal carcinomas, pre- and post-treatment with the mTOR (mammalian target of rapamycin) inhibitor, everolimus (Afinitor®)[1].

The authors compared exome sequencing of multiregion biopsies from four patients with metastatic renal-cell carcinoma who had been enrolled in the Personalized RNA Interference to Enhance the Delivery of Individualized Cytotoxic and Targeted Therapeutics clinical trial of everolimus (E-PREDICT) before and after cytoreductive surgery.

Biopsies taken:

Multiregion spatial biopsy of primary tumor (representing 9 regions of the tumor)
Chest-wall metastases
Perinephric metastases
Germline DNA as control

Multiple platforms were used to determine aberrations as follows:

Illumina Genome Analyzer IIx and Hiseq: for sequencing and mutational analysis
Illumina Omni 2.5: for SNP (single nucleotide polymorphism)-array-based allelic imbalance detection for chromosomal imbalance and ploidy analysis
Affymetrix Gene 1.0 Array: for mRNA analysis

A phylogenetic reconstruction of all somatic mutations occurring in primary disease and associated metastases was performed to determine the clonal evolution of the metastatic disease given the underlying heterogeneity of the tumor. Basically the authors wanted to know if the mutational spectra of one metastasis could be found in biopsies taken from the underlying primary tumor or if the mutational landscape of metastases had drastically changed.

Results

Multiregion exon-capture sequencing of DNA from pretreatment biopsy samples of the primary tumor, chest wall metastases, and perinephrous metastasis revealed 128 mutations classified as follows:

40 ubiquitous mutations
59 mutations shared by several but not all regions
29 mutations unique to specific regions
31 mutations shared by most primary tumor regions
28 mutations shared by most metastatic regions

The authors mapped these mutations out with respect to their location, in order to determine how the metastatic lesions evolved from the primary tumor, given the massive heterogeneity in the primary tumor. Construction of this “phylogenetic tree” (see Merlo et. al[2]) showed that the disease evolves in a branched not linear pattern, with one branch of clones evolving into a metastatic disease while another branch of clones and mutations evolve into the primary disease.

One of the major themes of the study is shown by results that an average of 70 somatic mutations were found in a single biopsy (a little more than just half of all tumor mutations) yet only 34% of the mutations in multiregion biopsies were detected in all tumor regions.

This indicated to the authors that “a single biopsy was not representative of the mutational landscape of the entire bulk tumor”. In addition, microarray studies concluded that gene-expression signatures from a single biopsy would not be able to predict outcome.

Everolimus therapy did not change the mutational landscape. Interestingly, allelic composition and ploidy analyses revealed an extensive intratumor heterogeneity, with ploidy heterogeneity in two of four tumors and 26 of 30 tumor samples containing divergent allelic-imbalances. This strengthens the notion that multiple clones with diverse genomic instability exist in various regions of the tumor.

The intratumor heterogeneity reveals a convergent tumor evolution with associated heterogeneity in target function

Genes commonly mutated in clear cell carcinoma[3, 4] (and therefore considered the prevalent driver mutations for renal cancer) include:

VHL: the Von Hippel–Lindau tumor suppressor
KDM5C: a lysine-specific demethylase 5C, commonly known as JARD1C
SETD2: a histone-lysine N-methyltransferase
MTOR

Only VHL mutations were found in all regions of a given tumor, however there were three distinct SETD2 mutations (frameshift, splice site, missense) which were located in different regions of the tumor.

SETD2 trimethylates histones at various lysine residues, such as lysine residue 36 (H3K36). The trimethylation of H3K36 is found on many actively transcribed genes. Immunohistochemistry showed trimethylated H3K36 was reduced in cancer cells but positive in most stromal cells and in SETD2 wild-type clear-cell carcinomas.

Interestingly most regions of the primary tumor, except one, contained a kinase-domain activating mutation in mTOR. Immunohistochemistry analysis of downstream target genes of mTOR revealed that mTOR activity was enhanced in regions containing this mutation. Therefore the intratumoral heterogeneity corresponded to therapeutic activity, leading to the impression that a single biopsy may result in inappropriate targeted therapy. Additional downstream biomarkers of activity confirmed both the intratumoral heterogeneity of mutational spectrum as well as an intratumoral heterogeneity of therapeutic-target function.

The authors conclude that “intratumor heterogeneity can lead to underestimation of the tumor genomics landscape from single tumor biopsies and may present major challenges to personalized-medicine and biomarker development”.

In an informal interview with Dr. Swanton, he had stressed the importance of performing these multi-region biopsies and the complications that intratumoral heterogeneity would present for personalized medicine, biomarker development, and chemotherapy resistance.

Q: Your data clearly demonstrates that multiple biopsies must be done to get a more complete picture of the tumor’s mutational landscape. In your study, what percentage of the tumor would be represented by the biopsies you had performed?

Dr. Swanton: Realistically this is a very difficult question to answer, the more biopsies we sequence, the more we find, in the near term it may be very difficult to ever formally address this in large metastatic tumours

Q: You have very nice data which suggest that genetic intratumor heterogeneity complicates the tumor biomarker field? do you feel then that quests for prognostic biomarkers may be impossible to attain?

Dr. Swanton: Not necessarily although heterogeneity is likely to complicate matters

Identifying clonally dominant lesions may provide better drug targets

Predicting resistance events may be difficult given the potential impact of tumour sampling bias and the concern that in some tumours a single biopsy may miss a relevant subclonal mutation that may result in resistance

Q: Were you able to establish the degree of genomic instability among the various biopsies?

Dr. Swanton: Yes, we did this by allelic imbalance analysis and found that the metastases were more genomically unstable than the primary region from which the metastasis derived

Q: I was actually amazed that there was a heterogeneity of mTOR mutations and SETD2 after everolimus therapy? Is it possible these clones obtained a growth advantage?

Dr. Swanton: We think so yes, otherwise we wouldn’t identify recurrent mutations in these “driver genes”

Dr. Swanton will present his results at the 2013 AACR meeting in Washington D.C. (http://www.aacr.org/home/scientists/meetings–workshops/aacr-annual-meeting-2013.aspx)

The overall points of the article are as follows:

Multiple biopsies of primary tumor and metastases are required to determine the full mutational landscape of a patients tumor
The intratumor heterogeneity will have an impact on the personalized therapy strategy for the clinician

Metastases arising from primary tumor clones will have a greater genomic instability and mutational spectrum than the tumor from which it originates

Tumors and their metastases do NOT evolve in a linear path but have a branched evolution and would complicate biomarker development and the prognostic and resistance outlook for the patient

A great video of Dr. Swanton discussing his research can be viewed here

VIEW VIDEO

Everolimus: an inhibitor of mTOR

The following information was taken from the New Medicine Oncology Database (http://www.nmok.net)

Developer	Designation	Description	Approved/Filed Indications
Novartis PharmaCurrent as of: August 30, 2012	Generic Name: Everolimus Brand Name: Afinitor Other Designation: RAD001, RAD001C	RAD001, an ester of the macrocytic immunosuppressive agent sirolimus (rapamycin), is an inhibitor of mammalian target of rapamycin (mTOR) kinase.Administration Route: intravenous (IV) • PO	• solid organ transplant • renal cell carcinoma (RCC), metastatic after failure of treatment with sunitinib, sorafenib, or sunitinib plus sorafenib • renal cell carcinoma, advanced, refractory to treatment with vascular endothelial growth factor (VEGF)-targeted therapy • treatment of progressive neuroendocrine tumors (NET) of pancreatic origin (PNET) in patients with inoperable, locally advanced or metastatic disease

Marker Designation Alias Gene Location	Marker Description	Indications
5’-AMP-activated Protein Kinase (AMPK)AMPK beta 1 (beta1 non-catalytic subunit) • HAMPKb (beta1 non-catalytic subunit) • MGC17785 (beta1 non-catalytic subunit) • AMPK2 (alpha1 catalytic subunit) • PRKAA (alpha1 catalytic subunit) • AMPK alpha 1 (alpha1 catalytic subunit) • AMPKa1 (	AMPK is a member of a metabolite-sensing protein kinase family found in all eukaryotes. It functions as a cellular fuel sensor and its activation strongly suppresses cell proliferation in non-malignant cells and cancer cells. AMPK regulates the cell cycle by upregulating the p53-p21 axis and modulating the TSC2-mTOR (mammalian target of rapamycin) pathway. The AMPK signaling network contains a number of tumor suppressor genes including LKB1, p53, TSC1 and TSC2, and modulates growth factor signaling involving proto-oncogenes including PI3K, Akt and ERK. AMPK activation is therefore therapeutic target for cancer (Motoshima H, etal, J Physiol, 1 Jul 2006; 574(Pt 1): 63–71).AMPK is a protein serine/threonine kinase consisting of a heterotrimeric complex of a catalytic alpha subunit and regulatory ß and gamma subunits. AMPK is activated by increased AMP/ATP ratio, under conditions such as glucose deprivation, hypoxia, ischemia and heat shock. It is also activated by several hormones and cytokines. AMPK inhibits ATP-consuming cellular events, protein synthesis, de novo fatty acid synthesis, and generation of mevalonate and the downstream products in the cholesterol synthesis pathway (Motoshima H, etal, J Physiol, 1 Jul 2006; 574(Pt 1): 63–71).	– ovarian cancer – brain cancer – liver cancer – leukemia – colon cancer
CREB regulated transcription coactivator 2 (CRTC2)TOR complex 2 (TORC2, mTORC2) • RP11-422P24.6 • transducer of regulated cAMP response element-binding protein (CREB)2 • transducer of CREB protein 2 • TOR1Location: 1q21.3	The mammalian target of rapamycin (mTOR) exists in two complexes, TORC1 and TORC2, which are differentially sensitive to rapamycin. cAMP response element-binding protein (CREB) regulated transcription coactivator 2 (CRTC2) or TORC2 is a multimeric kinase composed of mTOR, mLST8, mSin1, and rictor. The complex is insensitive to acute rapamycin exposure and functions in controlling cell growth and actin cytoskeletal assembly.TORC2 controls gene silencing, telomere length maintenance, and survival under DNA-damaging conditions. It is primaily located in the cytoplasm but also shuttles into the nucleus (Schonbrun M, etal, Mol Cell Biol, Aug 2009;29(16):4584-94).	– brain cancer
Hypoxia inducible factor 1 alpha (HIF1A)HIF1-alpha (HIF-1 alpha) • HIF-1A • PASD8 • MOP1 • bHLHe78Location: 14q21-q24	The alpha subunit of the hypoxia inducible factor 1 (HIF-1alpha) is a 826 amino acid antigen consisting of a basic helix-loop-helix (bHLH)-PAS domain at its N-terminus. HIF-1alpha is rapidly degraded by the proteasome under normal conditions, but is stabilized by hypoxia resulting in the transactivation of several proangiogenic genes. HIF-1alpha is responsible for inducing production of new blood vessels as needed when tumors outgrow existing blood supplies. HIF-1alpha serves as a transcriptional factor that regulates gene expression involved in response to hypoxia and promotes angiogenesis.HIF-1alpha is a proangiogenic transcription factor induced primarily by tumor hypoxia that is critically involved in tumor progression, metastasis and overall tumor survival. HIF-1alpha functions as a survival factor that is required for tumorigenesis in many types of malignancies, and is expressed in a majority of metastases and late-stage tumors. HIF-1alpha is overexpressed in brain, breast, colon, endometrial, head and neck, lung, ovarian, and pancreatic cancer, and is associated with increased microvessel density and/or VEGF expression	– prostate cancer – bladder cancer – nasopharyngeal cancer – head and neck cancer – kidney cancer – pancreatic cancer – endometrial cancer – breast cancer
Mammalian target of rapamycin (mTOR)FK506 binding protein 12-rapamycin associated protein 1 • RAFT1 • FK506 binding protein 12-rapamycin associated protein 2 • FRAP • FRAP1 • FRAP2 • RAPT1 • FKBP-rapamycin associated protein • FKBP12-rapamycin complex-associated protein 1 • rapamycin target protein • TOR • FLJ44809 • MTORC1 • MTORC2 • RPTOR • RAPTOR • KIAA1303 • mammalian target of rapamycin complex 1Location: 1p36.22	The mammalian target of rapamycin (mTOR) is a large serine/threonine protein (Mr 300,000) having heat repeats, and protein-protein interaction domains at its amino terminus, and a protein kinase domain at its carboxy terminus. mTOR is a member of the phosphoinositide 3-kinase (PI3K)-related kinase (PIKK) family and a central modulator of cell growth. It regulates cell growth, proliferation and survival by impacting on protein synthesis and transcription. mTOR is present in two multi-protein complexes, a rapamycin-sensitive complex, TOR complex 1 (TORC1), defined by the presence of Raptor and a rapamycin insensitive complex, TOR complex 2 (TORC2), with Rictor, Protor and Sin1. Rapamycin selectively inhibits mTORC1 by binding indirectly to the mTOR/Raptor complex via FKBP12, resulting in inhibition of p70S6kinase but not the mTORC2 substrate AKTSer473. Selective inhibition of p70S6K attenuates negative feedback loops to IRS1 and TORC2 resulting in an increase in pAKT which may limit the activity of rapamycin.In a hypoxic environment the increase in mass of solid tumors is dependent on the recruitment of mitogens and nutrients. As a function of nutrient levels, particularly essential amino acids, mTOR acts as a checkpoint for ribosome biogenesis and cell growth. Ribosome biogenesis has long been recognized in the clinics as a predictor of cancer progression; increase in size and number of nucleoli is known to be associated with the most aggressive tumors and a poor prognosis. In bacteria, ribosome biogenesis is independently regulated by amino acids and energy charge. The mTOR pathway is controlled by intracellular ATP levels, independent of amino acids, and mTOR itself is an ATP sensor (Kozma SC, etal, AACR02, Abs. 5628).	– breast cancer – pancreatic cancer – multiple myeloma – liver cancer – brain cancer – prostate cancer – kidney cancer – lymphoma
Signal transducer and activator of transcription 3 (STAT3)Stat-3 • acute-phase response factor (APRF) • FLJ20882 • HIESLocation: 17q21	Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family. STAT3, plays a critical role in hematopoiesis. STAT3 is located in the cytoplasm and translocated to the nucleus after tyrosine phosphorylation. In response to cytokines and growth and other activation factors, STAT family members are phosphorylated by the receptor associated kinases and then form homo- or heterodimers, which translocate to the cell nucleus where they act as transcription activators.	– multiple myeloma – hematologic malignancy – lymphoma
Sonic hedgehog homolog (SHH)Shh • HHG1 • HHG-1 • holoprosencephaly 3 (HPE3) • HLP3 • SMMCILocation: 7q36	Sonic hedgehog, a secreted hedgehog ligand, is a human homolog of the Drosophila segment polarity gene hedgehog, cloned by investigators at Harvard University (Marigo V, etal, Genomics, 1 Jul 1995;28 (1):44-51).The mammalian sonic hedgehog (Shh) pathway controls proliferation of granule cell precursors in the cerebellum and is essential for normal embryonic development. Shh signaling is disrupted in a variety of malignancies.	– pancreatic cancer – CNS cancer

References:

1. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P et al: Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. The New England journal of medicine 2012, 366(10):883-892.

2. Merlo LM, Pepper JW, Reid BJ, Maley CC: Cancer as an evolutionary and ecological process. Nature reviews Cancer 2006, 6(12):924-935.

3. Varela I, Tarpey P, Raine K, Huang D, Ong CK, Stephens P, Davies H, Jones D, Lin ML, Teague J et al: Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 2011, 469(7331):539-542.

4. Dalgliesh GL, Furge K, Greenman C, Chen L, Bignell G, Butler A, Davies H, Edkins S, Hardy C, Latimer C et al: Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 2010, 463(7279):360-363.

Genomics in Medicine- Tomorrow’s Promise

Prostate Cancer: Androgen-driven “Pathomechanism” in Early-onset Forms of the Disease

CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics and Computational Genomics – Part IIB

Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell

Directions for Genomics in Personalized Medicine

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ http://pharmaceuticalintelligence.com

In Focus: Targeting of Cancer Stem Cells

Modulating Stem Cells with Unread Genome: microRNAs

What can we expect of tumor therapeutic response?

Read Full Post »

Genomics in Medicine – Tomorrow’s Promise

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Health Economics and Outcomes Research, Health Law & Patient Safety, International Global Work in Pharmaceutical, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Scientist: Career considerations, Technology Transfer: Biotech and Pharmaceutical, tagged American Civil Liberties Union, American Society of Human Genetics, DNA, DNA Sequencing, Exome sequencing, genetic testing, genomics, Mark Lemley, Myriad Genetic, Nucleic acid sequence on March 3, 2013| 3 Comments »

Genomics in Medicine – Tomorrow’s Promise

Reporter: Larry H Bernstein, MD, FCAP

Genomics in Medicine: Today’s Issues, Tomorrow’s Promise

KM Beima-Sofie, EH Dorfman, JM Kocarnik, MY Laurino

Feb 13, 2013 Medscape Genomic Medicine

http://www.medscape.com/viewarticle/778921_5

Genetic Sequencing Moves Beyond the Laboratory
Who Should Have Access to Genetic Information?
Noninvasive Prenatal Genetic Testing: Continuing Controversy
Looking Back to Look Ahead

What do you think about these issues before reading this piece?

The Broader Implications of Genetic Sciences

The 62nd annual meeting of the American Society of Human Genetics (ASHG), which was held in San Francisco, California, in November 2012, featured a diverse array of research in basic, clinical, and population science contributed by human geneticists across the globe.

Genetic Sequencing Moves Beyond the Laboratory

Several presentations at the meeting focused on the lessons learned from the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project. The goal of the project was to

develop and validate a cost-effective and high-throughput sequencing technology
capable of analyzing the DNA sequence in the exome, which
consists of all protein-coding regions in the human genome.

At previous ASHG meetings, presentations and discussions largely focused on

the development of sequencing technology and on applications of this technology for research.

Now that sequencing is an increasing reality, this year’s conference featured presentations on

what to do with the resulting information, in both research and clinical settings.

Issues discussed include the challenges of

interpreting sequence data,
determining which results should be returned to various parties, and
the potential impacts of different testing techniques.

Results from the NHLBI Exome Sequencing Project and other projects are fueling the discussion on
legal issues surrounding gene patenting, a hotly debated topic that is currently under consideration by the US Supreme Court. During a plenary session on gene discovery and patent law,

attorneys Hank Greely and Mark Lemley from Stanford University and Lori Andrews from ITT Chicago-Kent College of Law
were joined by molecular biologist Gert Matthijs from the Center for Human Genetics in Belgium in sharing their perspectives on this topic.

Of particular focus was the lawsuit brought by the American Civil Liberties Union against Myriad Genetics,

contesting the company’s patent of the BRCA1 and BRCA2 genes for hereditary breast and ovarian cancer.

At present, Myriad has exclusive rights to offer clinical genetic testing for these genes; one of the main arguments of the lawsuit is

that gene patents hinder the pursuit of confirmatory tests and limit the testing options available to women.

DNAPrint Genomics (Photo credit: Wikipedia)

English: Exome sequencing workflow: Part 2. Target exons are enriched, eluted and then amplified by ligation-mediated PCR. Amplified target DNA is then ready for high-throughput sequencing. (Photo credit: Wikipedia)

Cost per Megabase of DNA Sequence (Why biologists panic about compute) (Photo credit: dullhunk)

Genome sequencing of the Healthy (pharmaceuticalintelligence.com)
A human genome in minutes, and what it will mean to you (sciencegremlin.wordpress.com)
Four barriers that must fall before the personalized medicine revolution can start (medcitynews.com)
Directions for genomics in personalized medicine (pharmaceuticalintelligence.com)
What is the future for genomics in clinical medicine? (pharmaceuticalintelligence.com)
Genomics & Ethics: DNA Fragments are Products of Nature or Patentable Genes? (pharmaceuticalintelligence.com)
The Initiation and Growth of Molecular Biology and Genomics (pharmaceuticalintelligence.com)
CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics and Computational Genomics (pharmaceuticalintelligence.com)
2013 Genomics: The Era Beyond the Sequencing Human Genome: Francis Collins, Craig Venter, Eric Lander, et al. (pharmaceuticalintelligence.com)
Understanding the Role of Personalized Medicine (pharmaceuticalintelligence.com)
http://venturebeat.com/2013/01/27/the-personalized-medicine-revolution-is-almost-here/
ERCC1 Isoform Expression and DNA Repair in Non–Small-Cell Lung Cancer

Read Full Post »

Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes

Posted in Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Chemical Genetics, Genome Biology, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, tagged Cancer - General, chromatin remodelling, endometrial tumour, Exome sequencing, gene, recurrent somatic mutations, ubiquitin ligase complex on December 18, 2012| 5 Comments »

Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes

Reporter and Curator: Dr. Sudipta Saha, Ph.D

Endometrial cancer is the sixth most commonly diagnosed cancer in women worldwide, causing ~74,000 deaths annually. Serous endometrial cancers are a clinically aggressive subtype with a poorly defined genetic etiology2–4. Whole-exome sequencing was used to comprehensively search for somatic mutations within ~22,000 protein-encoding genes in 13 primary serous endometrial tumors. Subsequently 18 genes were resequenced, which were mutated in more than 1 tumor and/or were components of an enriched functional grouping, from 40 additional serous tumors.

The study provides new insights into the somatic mutations present in serous endometrial cancer exomes. However, it is important that the discovery screen is underpowered to detect all somatically mutated genes that drive serous tumors. For example, PIK3R1, which was previously found to be somatically mutated in 8% of serous endometrial tumors, was not somatically mutated in the tumors that formed the discovery screen. It was estimated that, for genes that are mutated in 8% of all serous endometrial cancers, a discovery screen of 12 tumors has 25% power to detect 2 mutated tumors and 63% power to detect 1 mutated tumor; for genes that are mutated in 20% of all serous endometrial cancers, the discovery screen had an estimated 72.5% power to detect 2 mutated tumors and 93% power to detect 1 mutated tumor. Massively parallel sequencing of additional cases will undoubtedly yield deeper insights into the mutational landscape of serous endometrial cancer.

Thus, it was reported as one of the first exome sequencing analyses of serous endometrial cancers, which are clinically aggressive tumors that have been poorly characterized genomically. These findings implicate the disruption of chromatin-remodeling and ubiquitin ligase complex genes in 50% of serous endometrial tumors and 35% of clear-cell endometrial tumors. High frequencies of somatic mutations in CHD4 (17%), EP300 (8%), ARID1A (6%), TSPYL2 (6%), FBXW7 (29%), SPOP (8%), MAP3K4 (6%) and ABCC9 (6%) were identified. The high frequency and specific distributions of mutations in CHD4, FBXW7 and SPOP strongly suggest that these are likely to be driver events in serous endometrial cancer. Overall, 36.5% of serous tumors had a mutated chromatin-remodeling gene, and 35% had a mutated ubiquitin ligase complex gene, implicating frequent mutational disruption of these processes in the molecular pathogenesis of one of the deadliest forms of endometrial cancer.

Source References:

http://www.nature.com/ng/journal/v44/n12/full/ng.2455.html

Read Full Post »

Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Chemical Genetics, Genome Biology, Population Health Management, Genetics & Pharmaceutical, tagged Cancer - General, chromatin remodelling, Endometrial cancer, Exome sequencing, National Human Genome Research Institute, National Institutes of Health, serous endometrial tumors, somatic mutation, SPOP, Ubiquitin on November 19, 2012| 6 Comments »

Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Endometrial cancer is the sixth most commonly diagnosed cancer in women worldwide, causing ~74,000 deaths annually1. Serous endometrial cancers are a clinically aggressive subtype with a poorly defined genetic etiology2–4.

Whole-exome sequencing was used to comprehensively search for somatic mutations within ~22,000 protein-encoding genes in 1 13 primary serous endometrial tumors. Subsequently 18 genes were resequenced, which were mutated in more than 1 1 1 tumor and/or were components of an enriched functional grouping, from 40 additional serous tumors. High frequencies of somatic mutations in CHD4 (17%), EP300 (8%), ARID1A (6%), TSPYL2 (6%), FBXW7 (29%), SPOP (8%), MAP3K4 (6%) and ABCC9 (6%) were identified. Overall, 36.5% of serous tumors had a mutated chromatin-remodeling gene, and 35% had a mutated ubiquitin ligase complex gene, implicating frequent mutational disruption of these processes in the molecular pathogenesis of one of the deadliest forms of endometrial cancer.

The study provides new insights into the somatic mutations present in serous endometrial cancer exomes. However, it is important to acknowledge that this discovery screen is underpowered to detect all somatically mutated genes that drive serous tumors. For example, PIK3R1, which was previously found to be somatically mutated in 8% of serous endometrial tumors58, was not somatically mutated in the tumors that formed this discovery screen.

It was estimated that, for genes that are mutated in 8% of all serous endometrial cancers, a discovery screen of 12 tumors has 25% power to detect 2 mutated tumors and 63% power to detect 1 mutated tumor; for genes that are mutated in 20% of all serous endometrial cancers, the discovery screen had an estimated 72.5% power to detect 2 mutated tumors and 93% power to detect 1 mutated tumor.

Massively parallel sequencing of additional cases will undoubtedly yield deeper insights into the mutational landscape of serous endometrial cancer. Here, it was reported one of the first exome sequencing analyses of serous endometrial cancers, which are clinically aggressive tumors that have been poorly characterized genomically.

The findings implicate the disruption of chromatin-remodeling and ubiquitin ligase complex genes in

50% of serous endometrial tumors and
35% of clear-cell endometrial tumors.

The high frequency and specific distributions of mutations in CHD4, FBXW7 and SPOP strongly suggest that these are likely to be driver events in serous endometrial cancer.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed?term=Exome%20sequencing%20of%20serous%20endometrial%20tumors%20identifies%20recurrent%20somatic%20mutations%20in%20chromatin-remodeling%20and%20ubiquitin%20ligase%20complex%20genes