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CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Cell Biology, Signaling & Cell Circuits, Conference Coverage with Social Media, CRISPR/Cas9 & Gene Editing, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Drug Toxicity, Gene Regulation, Gene Regulation and Evolution, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Metabolism, Metabolomics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Drug Discovery, Pharmacodynamics and Pharmacokinetics, Pharmacogenomics, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged anti-cancer drugs, Cancer - General, CRISPR-Cas9, Duodna, FDA, gene editing, gene expression, genetics, genome, Genome engineering, health, MIT, mouse model, research, science, tumor model on September 2, 2015| Leave a Comment »

CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

  UPDATED 6/11/2021

CRISPR Diagnostics: CRISPR-dx Comes of Age: Tool in Drug Development

The past five years has seen a rapid expansion of the ability of CRISPR based tools toward diagnostic testing. Recently, CRISPR has been used to detect SARS-CoV-2 in patients. An article in the journal Science describes the different classes of CRISPR diagnostics in use today .

Update near end of post

UPDATED 8/08/2020

Association to Causation: Using GWAS to Identify Druggable Targets

A Gen Webinar Thursday, August 6, 2020; 11:00am – 12:30pm EST

See at end of post

Curator: Stephen J. Williams, Ph.D.

• 

 

CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.

CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner. Although CRISPR arrays were first identified in the Escherichia coli genome in 1987 (Ishino et al., 1987), their biological function was not understood until 2005, when it was shown that the spacers were homologous to viral and plasmid sequences suggesting a role in adaptive immunity (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007). The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008).

Jennifer Doudna, PhD Professor of Molecular and Cell Biology and Chemistry, University of California, Berkeley Investigator, Howard Hughes Medical Institute has recently received numerous awards and accolades for the discovery of CRISPR/Cas9 as a tool for mammalian genetic manipulation as well as her primary intended research target to understand bacterial resistance to viral infection.

A good post on the matter and Dr. Doudna can be seen below:

http://pharmaceuticalintelligence.com/2014/06/13/215-245-6132014-jennifer-doudna-the-biology-of-crisprs-from-genome-defense-to-genetic-engineering/

In Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting inheritable metabolic disorders in which may benefit from a CRISPR-Cas9 mediated therapy is discussed. However this curation is meant to focus on CRISPR/CAS9 AS A TOOL IN PRECLINICAL DRUG DEVELOPMENT.

 

Three Areas of Importance of CRISPR/Cas9 as a TOOL in Preclinical Drug Discovery Include:

1. Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE
2. CRISPR/CAS9 Use in Developing Models of Disease
3. CRISPR/CAS9 Use as a Diagnostic Tool

• Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

I.     Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE

The advent of the first tools for manipulating genetic material (cloning, PCR, transgenic technology, and before microarray and other’omic methods) allowed scientists to probe novel, individual gene functions as well as their variants and mutants in a “one-gene-at-a time” process. In essence, a gene (or mutant gene) was sequenced, cloned into expression vectors and transfected into recipient cells where function was evaluated.

However, some of the experimental issues with this methodology involved

• Lack of knowledge of insertion site of the transgene – this leads to off-target effects usually due to insertion of a transgene in front of unwanted promoters or insertion at a site resulting in gene disruption or even mutagenesis. In an extreme case, such as transposon-induced mutagenesis may lead to transformation as described in an earlier post on this site How Mobile Elements in “Junk” DNA Promote Cancer – Part 1: Transposon-mediated Tumorigenesis

• Most transfections experiments result in NON ISOGENIC cell lines – by definition the insertion of a transgene alters the genetic makeup of a cell line. Simple transfection experiments with one transgene compared to a “null” transfectant compares non-isogenic lines, possibly confusing the interpretation of gene-function studies. Therefore a common technique is to develop cell lines with inducible gene expression, thereby allowing the investigator to compare a gene’s effect in ISOGENIC cell lines.

1. Use of CRSPR in Highthrough-put Screening of Genetic Function

A very nice presentation and summary of CRSPR’s use in determining gene function in a high-throughput manner can be found below

www.rna.uzh.ch/events/journalclub/20140429JCCaihong.pdf

1. Determining Off-target Effects of Gene Therapy Simplified with CRSPR

In GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases (from This Journal’s series on Live Meeting Coverage) at a 2014 Koch lecture

Shengdar Q Tsai and J Keith Joung describe

“

an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint ‘hotspots’.

SOURCE http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3117.html

“

II. CRISPR/Cas9 Use in Developing Models of Disease

 

1. Developing Animal Tumor Models

In a post this year I discussed a talk at the recent 2015 AACR National Meeting on a laboratories ability to use CRISPR gene editing in-vivo to produce a hepatocarcinoma using viral delivery. The post can be seen here: Notes from Opening Plenary Session – The Genome and Beyond from the 2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015

1) In this talk Dr. Tyler Jacks discussed his use of CRSPR to generate a mouse model of liver tumor in an immunocompetent mouse. Some notes from this talk are given below

1. B) Engineering Cancer Genomes: Tyler Jacks, Ph.D.; Director, Koch Institute for Integrative Cancer Research

• Cancer GEM’s (genetically engineered mouse models of cancer) had moved from transgenics to defined oncogenes
• Observation that p53 -/- mice develop spontaneous tumors (lymphomas)
• then GEMs moved to Cre/Lox systems to generate mice with deletions however these tumor models require lots of animals, much time to create, expensive to keep;
• figured can use CRSPR/Cas9 as rapid, inexpensive way to generate engineered mice and tumor models
• he used CRSPR/Cas9 vectors targeting PTEN to introduce PTEN mutations in-vivo to hepatocytes; when they also introduced p53 mutations produced hemangiosarcomas; took ONLY THREE months to produce detectable tumors
• also produced liver tumors by using CRSPR/Cas9 to introduce gain of function mutation in β-catenin

See an article describing this study by MIT News “A New Way To Model Cancer: New gene-editing technique allows scientists to more rapidly study the role of mutations in tumor development.”

The original research article can be found in the August 6, 2014 issue of Nature[1]

And see also on the Jacks Lab site under Research

2)     In the Upcoming Meeting New Frontiers in Gene Editing multiple uses of CRISPR technology is discussed in relation to gene knockout/function studies, tumor model development and

New Frontiers in Gene Editing

Session Spotlight:
BUILDING IN VIVO MODELS FOR DRUG DISCOVERY

Genome Editing Animal Models in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Biology-Discovery, Genetics and Pharmacogenomics, Merck Research Laboratories

Recent advances in genome editing have greatly accelerated and expanded the ability to generate animal models. These tools allow generating mouse models in condensed timeline compared to that of conventional gene-targeting knock-out/knock-in strategies. Moreover, the genome editing methods have expanded the ability to generate animal models beyond mice. In this talk, we will discuss the application of ZFN and CRISPR to generate various animal models for drug discovery programs.

In vivo Cancer Modeling and Genetic Screening Using CRISPR/Cas9
Sidi Chen, Ph.D., Postdoctoral Fellow, Laboratories of Dr. Phillip A. Sharp and Dr. Feng Zhang, Koch Institute for Integrative Cancer Research at MIT and Broad Institute of Harvard and MIT

Here we describe a genome-wide CRISPR-Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library. The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late stage primary tumors were found to target a small set of genes, suggesting specific loss-of-function mutations drive tumor growth and metastasis.

FEATURED PRESENTATION: In vivo Chromosome Engineering Using CRISPR-Cas9
Andrea Ventura, M.D., Ph.D., Assistant Member, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center

We will discuss our experience using somatic genome editing to engineer oncogenic chromosomal rearrangements in vivo. More specifically, we will present the results of our ongoing efforts aimed at modeling cancers driven by chromosomal rearrangements using viral mediated delivery of Crispr-Cas9 to adult animals.

RNAi and CRISPR/Cas9-Based in vivo Models for Drug Discovery
Christof Fellmann, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Jennifer Doudna, Department of Molecular and Cell Biology, The University of California, Berkeley

Genetically engineered mouse models (GEMMs) are a powerful tool to study disease initiation, treatment response and relapse. By combining CRISPR/Cas9 and “Sensor” validated, tetracycline-regulated “miR-E” shRNA technology, we have developed a fast and scalable platform to generate RNAi GEMMs with reversible gene silencing capability. The synergy of CRISPR/Cas9 and RNAi enabled us to not only model disease pathogenesis, but also mimic drug therapy in mice, providing us capability to perform preclinical studies in vivo.

In vivo Genome Editing Using Staphylococcus aureus Cas9
Fei Ann Ran, Ph.D., Post-doctoral Fellow, Laboratory of Dr. Feng Zhang, Broad Institute and Junior Fellow, Harvard Society of Fellows

The RNA-guided Cas9 nuclease from the bacterial CRISPR/Cas system has been adapted as a powerful tool for facilitating targeted genome editing in eukaryotes. Recently, we have identified an additional small Cas9 nuclease from Staphylococcus aureus that can be packaged with its guide RNA into a single adeno-associated virus (AAV) vector for in vivo applications. We demonstrate the use of this system for effective gene modification in adult animals and further expand the Cas9 toolbox for in vivo genome editing.

OriGene, Making the Right Tools for CRISPR Research
Xuan Liu, Ph.D., Senior Director, Marketing, OriGene

CRISPR technology has quickly revolutionized the scientific community. Its simplicity has democratized the genome editing technology and enabled every lab to consider its utility in gene function research. As the largest tool box for gene functional research, OriGene created a large collection of CRISPR-related tools, including various all-in-one vectors for gRNA cloning, donor vector backbones, genome-wide knockout kits, AAVS1 insertion vectors, etc. OriGene’s high quality products will accelerate CRISPR research.

1. Transgenic Animals : Custom Mouse and Rat Model Generation Service Using CRISPR/Cas9 by AppliedStem Cell Inc. (http://www.appliedstemcell.com/)

A critical component of producing transgenic animals is the ability of each successive generations to pass on the transgene. In her post on this site, A NEW ERA OF GENETIC MANIPULATION  Dr. Demet Sag discusses the molecular biology of Cas9 systems and their efficiency to cause point mutations which can be passed on to subsequent generations

“

This group developed a new technology for editing genes that can be transferable change to the next generation by combining microbial immune defense mechanism, CRISPR/Cas9 that is the latest ground breaking technology for translational genomics with gene therapy-like approach.

• In short, this so-called “mutagenic chain reaction” (MCR) introduces a recessive mutation defined by CRISPR/Cas9 that lead into a high rate of transferable information to the next generation. They reported that when they crossed the female MCR offspring to wild type flies, the yellow phenotype observed more than 95 percent efficiency.

“

The advantage of CRISPR/Cas9 over ZFNs or TALENs is its scalability and multiplexibility in that multiple sites within the mammalian genome can be simultaneously modified, providing a robust, high-throughput approach for gene editing in mammalian cells.

Applied StemCell, Inc. offers various services related to animal models including conventional transgenic rats, and phenotype analysis using knock-in, knock-out strategies.

Further explanation of their use of CRSPR can be found at the site below:

http://pharmaceuticalintelligence.com/2014/10/29/gene-editing-at-crispr-speed-services-and-tools/

In addition, ReproCELL Inc., a Tokyo based stem cell company, uses CRSPR to develop

· Tailored disease model cells (hiPSC-Disease Model Cells)

• 2 types of services

• ReproUNUS™-g：human iPS cell derived functional cells involving gene editing by CRISPR/Cas9 system
• eproUNUS™-p：patient derived iPS cell derived functional cells

III. Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

As of now it is unclear as to the strategy of pharma in how to use this technology for toxicology testing however a few companies have licensed the technology to use across their R&D platforms including

• AstraZeneca
• Thermo
• Novartis
• GE Healthcare

A recent paper used a sister technique TALEN to generate knock-in pigs which suggest that it would be possible to generate pigs with human transgenes, especially in human liver isozymes in orer to study hepatotoxicity of drugs.

Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs

Jing Yao, Jiaojiao Huang, Tang Hai, Xianlong Wang, Guosong Qin, Hongyong Zhang, Rong Wu, Chunwei Cao, Jianzhong Jeff Xi, Zengqiang Yuan, Jianguo Zhao

Sci Rep. 2014; 4: 6926. Published online 2014 November 5. doi: 10.1038/srep06926

UPDATED 8/08/2020

Association to Causation: Using GWAS to Identify Druggable Targets

A Gen Webinar Thursday, August 6⋅11:00am – 12:30pm

This webinar is available at https://www.genengnews.com/resources/webinars/association-to-causation-using-gwas-to-identify-druggable-target/

Speakers:

Martin Kampmann, PhD

Associate Professor
UCSF
Investigator
Chan Zuckerberg Biohub

Kevin Holden, PhD

Head of Science
Synthego

Abhi Saharia, PhD

VP, Commercial Development
Synthego

Human genetics provides perhaps the single best opportunity to innovate and improve clinical success rates, through the identification of novel drug targets for complex disease. Even as correlation identifies multiple genetic variants associated with disease, it is challenging to conduct requisite functional studies to identify the causal variants, especially since most association signals map to non-coding regions of the genome.

Genetic editing technologies, such as CRISPR, have enabled the modeling of associated variants at their native loci, including non-coding loci, empowering the identification of underlying biological mechanisms of disease with potential causal genes. However, genome editing is largely manual today severely limiting scale, and forcing the use of rational filters to prioritize which variants to investigate functionally.

In this GEN webinar, we will discuss several strategies enabling large-scale functional investigation of disease-associated variants in a cost- and time-effective manner, including different types of pooled CRISPR-based screens and the development of a fully automated genome engineering platform. We will also review how optimization of genome engineering on this platform enables the engineering of disease-associated variants at scale in pluripotent cells.

• They will be presenting on use of wide scale CRSPR screens to validate druggable targets
• The presenters will also discuss new platforms for these wide scale screens

Martin Kampmann, PhD UCSF

• Multiple genetic variants associated with disease
• Big gap between accumulation of genetic variant information and functions of these variants
• CRSPRi or CRSPa (siRNA coupled or enhancer coupled CRSPR guides)
• Arrayed screens: multiplate guide RNAs and phenotype measured (phenotype can be morphology, complex biological systems like organoids or non autonomous functions
• Using pooled screens and use of suitable cell model critical for this strategy
• For example in iPSC vs. neurons has different expression patterns upon same CRSPR of UBA1
• Advantage is using CRSPR to take iPSC from diseased variant patient to make a corrected isogenic control then introduce gRNAs and use modifier screens to determine phenotypes
• Generated a platform called CRISPRbrain.org to do bioinformatics on various experiments with different guide RNAs (CRSPRs)

Abhi Saharia, PhD Syntheco

• Target identification with CSRSPR at Scale
• Nature medicine paper did GWAS and found 27 SNV associated with high risk disease and a rational filter focused on 1 SNV in noncoding region but why study a single variant and if studied all 27 would they have been able to identify a more representative druggable set?
• Goal is to reduce or eliminate these rational filters
• HALO (scalable RNA guide), ECLIPSE platform (automated generation of modified cell lines, BIOINFORMATIC platform (integrated informatics)
• Syntheco uses an electroporation with ribonucleic proteins (RNP) to give highest efficiency and minimizes off target as complex is only in cells for a short period of time
• They confirm they are doing single cell cloning by using automated microscopy to confirm single cell growth in each cloning well

Kevin Holden, Head of Science at Syntheco

• Engineering iPSc genetically modified cells at scale
• The closer you get to your target site the more efficient your CRSPR so a big factor when making guides, especially for knock-in CRSPR
• Adding a small molecule non homologous end joining inhibitor increases efficiency to 95%
• Cold shocking the cells also assists in homologous repair
• Use cleavage resistant templates

III. CRISPR/CAS9 AS A DIAGNOSTIC TOOL

     In the journal Science, Omar Abudayyeh and Jonathan Gootenberg discuss how CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA).  In addition they discuss the work by Jiao et al. in combining this technique to develop a rapid and sensitive SARS-CoV2 diagnostic test.

Omar O. Abudayyeh, Jonathan S. Gootenberg. Science  28 May 2021: CRISPR Diagnostics
Vol. 372, Issue 6545, pp. 914-915; DOI: 10.1126/science.abi9335

Summary

Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.

Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.

There are multiple types of CRISPR systems comprising basic components of a single protein or protein complex, which cuts a specific DNA or RNA target programmed by a complementary guide sequence in a CRISPR-associated RNA (crRNA). The type V and VI systems and the CRISPR-associated endonucleases Cas12 (2, 3) and Cas13 (4, 5) bind and cut DNA or RNA, respectively. Furthermore, upon recognizing a target DNA or RNA sequence, Cas12 and Cas13 proteins exhibit “collateral activity” whereby any DNA or RNA, respectively, in the sample is cleaved regardless of its nucleic acid sequence (4, 6). Thus, reporter DNAs or RNAs, which allow for visual or fluorescent detection upon cleavage, can be added to a sample to infer the presence or absence of specific DNA or RNA species (4–8).

Initial versions of CRISPR-dx utilizing Cas13 alone were sensitive to the low picomolar range, corresponding to a limit of detection of millions of molecules in a microliter sample. To improve sensitivity, preamplification methods, such as recombinase polymerase amplification (RPA), PCR, loop-mediated isothermal amplification (LAMP), or nucleic acid sequence–based amplification (NASBA), can be used with Cas12 or Cas13 to enable a limit of detection down to a single molecule (8). This preamplification approach, applicable to both Cas12 and Cas13 (6, 7), enabled a suite of detection methods and multiplexing up to four orthogonal targets (7). Additional developments expanded CRISPR-dx readouts beyond fluorescence, including lateral flow (7), colorimetric (9), and electronic or material responsive readouts (10), allowing for instrument-free approaches. In addition, post–collateral-cleavage amplification methods, such as the use of the CRISPR-associated enzyme Csm6, have been combined with Cas13 to further increase the speed of CRISPR-dx tests (7). As an alternative to collateral-cleavage–based detection, type III CRISPR systems, which involve large multiprotein complexes capable of targeting both DNA and RNA, have been used for SARS-CoV-2 detection through production of colorimetric or fluorometric readouts (11).

FDA-authorized CRISPR-dx tests are currently only for use in centralized labs, because the most common CRISPR detection protocols require fluid handling steps and two different incubations, precluding their immediate use at the point of care. Single-step formulations have been developed to overcome this limitation, and these “one-pot” versions of CRISPR-dx are simple to run, operate at a single temperature, and run without complex equipment, producing either fluorescence or lateral flow readouts. The programmability of CRISPR makes new diagnostic tests easier to develop, and within months of the release of the SARS-CoV-2 genome, many COVID-19–specific CRISPR tests were reported and distributed around the world.

The broader capability for Cas enzyme–enhanced nucleic acid binding or cleavage has led to several other detection modalities. Cas9-based methods for cleaving nucleic acids in solution for diagnostic purposes have been combined with other detection platforms, such as destruction of undesired amplicons for preparation of next-generation sequencing libraries (12), or selective removal of alleles for nucleotide-specific detection (13). Alternatively, the programmable cleavage event from the Cas nuclease can be used to initiate an amplification reaction (14). Cas9-based DNA targeting has also been used for nucleotide detection in combination with solid-state electronics, promising an amplification-free platform for detection. In this platform, called CRISPR-Chip, the Cas9 protein binds nucleotide targets of interest (often in the context of the native genome) to graphene transistors, where the presence of these targets alters either current or voltage (15). By utilizing additional Cas9 orthologs and specific guide designs, CRISPR-Chip approaches have been tuned for single–base-pair sensitivity (15). Because they are integrated with electronic readers, CRISPR-Chip platforms may allow facile point-of-care detection with handheld devices.

 

Other related articles on CRISPR/Cas9 were published in this Open Access Online Scientific Journal, include the following:

Search Results for ‘CRISPR’

Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

CRISPR/Cas9 genome editing tool for Staphylococcus aureus Cas9 complex (SaCas9) @ MIT’s Broad Institute

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

Using CRISPR to investigate pancreatic cancer

Simple technology makes CRISPR gene editing cheaper

RNAi, CRISPR, and Gene Editing: Discussions on How To’s and Best Practices @14th Annual World Preclinical Congress June 10-12, 2015 | Westin Boston Waterfront | Boston, MA

CRISPR/Cas9: Contributions on Endoribonuclease Structure and Function, Role in Immunity and Applications in Genome Engineering

CRISPR-CAS editing brings cloning of woolly mammoth one step closer to reality

GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases

The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century

CRISPR: Applications for Autoimmune Diseases @UCSF

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Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Posted in Biological Networks, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Prevention: Research & Programs, Computational Biology/Systems and Bioinformatics, Gene Regulation and Evolution, Genome Biology, Health Economics and Outcomes Research, Personalized and Precision Medicine & Genomic Research, tagged antitumor therapy, Bioinformatics, Cancer - General, Cell Biology, Computational Biology/Systems and Bioinformatics, DNA, DNA Sequencing, driver mutations, gene expression, genetics, genome, genomics, informatics, Lung cancer, mutation, mutational analysis, National Institutes of Health, Non-small cell lung cancer, Personalized medicine, Proceedings of the National Academy of Sciences of the United States of America, Prostate cancer, research, smoking, The Cancer Genome Atlas (TCGA), The Clinical Lung Cancer Genome Project (CLCGP), whole exome sequencing on September 5, 2014| Leave a Comment »

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Curator, Writer: Stephen J. Williams, Ph.D.

UPDATED 10/10/2021

(photo credit: cancer.gov)

A report Lung Cancer Genome Surveys Find Many Potential Drug Targets, in the NCI Bulletin,

http://www.cancer.gov/ncicancerbulletin/091812/page2

summarizes the clinical importance of five new lung cancer genome sequencing projects. These studies have identified genetic and epigenetic alterations in hundreds of lung tumors, of which some alterations could be taken advantage of using currently approved medications.

The reports, all published this month, included genomic information on more than 400 lung tumors. In addition to confirming genetic alterations previously tied to lung cancer, the studies identified other changes that may play a role in the disease.

Collectively, the studies covered the main forms of the disease—lung adenocarcinomas, squamous cell cancers of the lung, and small cell lung cancers.

“All of these studies say that lung cancers are genomically complex and genomically diverse,” said Dr. Matthew Meyerson of Harvard Medical School and the Dana-Farber Cancer Institute, who co-led several of the studies, including a large-scale analysis of squamous cell lung cancer by The Cancer Genome Atlas (TCGA) Research Network.

Some genes, Dr. Meyerson noted, were inactivated through different mechanisms in different tumors. He cautioned that little is known about alterations in DNA sequences that do not encode genes, which is most of the human genome.

Four of the papers are summarized below, with the first described in detail, as the Nature paper used a multi-‘omics strategy to evaluate expression, mutation, and signaling pathway activation in a large cohort of lung tumors. A literature informatics analysis is given for one of the papers.  Please note that links on GENE names usually refer to the GeneCard entry.

Paper 1. Comprehensive genomic characterization of squamous cell lung cancers[1]

The Cancer Genome Atlas Research Network Project just reported, in the journal Nature, the results of their comprehensive profiling of 230 resected lung adenocarcinomas. The multi-center teams employed analyses of

• microRNA
• Whole Exome Sequencing including
  • Exome mutation analysis
  • Gene copy number
  • Splicing alteration
• Methylation
• Proteomic analysis

Summary:

Some very interesting overall findings came out of this analysis including:

• High rates of somatic mutations including activating mutations in common oncogenes
• Newly described loss of function MGA mutations
• Sex differences in EGFR and RBM10 mutations
• driver roles for NF1, MET, ERBB2 and RITI identified in certain tumors
• differential mutational pattern based on smoking history
• splicing alterations driven by somatic genomic changes
• MAPK and PI3K pathway activation identified by proteomics not explained by mutational analysis = UNEXPLAINED MECHANISM of PATHWAY ACTIVATION

however, given the plethora of data, and in light of a similar study results recently released, there appears to be a great need for additional mining of this CGAP dataset. Therefore I attempted to curate some of the findings along with some other recent news relevant to the surprising findings with relation to biomarker analysis.

Makeup of tumor samples

230 lung adenocarcinomas specimens were categorized by:

Subtype

33% acinar

25% solid

14% micro-papillary

9% papillary

8% unclassified

5% lepidic

4% invasive mucinous
Gender

Smoking status

81% of patients reported past of present smoking

The authors note that TCGA samples were combined with previous data for analysis purpose.

A detailed description of Methodology and the location of deposited data are given at the following addresses:

Publication TCGA Web Page: https://tcga-data.nci.nih.gov/docs/publications/luad_2014/

Sequence files: https://cghub.ucsc.edu

Results:

Gender and Smoking Habits Show different mutational patterns

 

WES mutational analysis

1. a) smoking status

– there was a strong correlations of cytosine to adenine nucleotide transversions with past or present smoking. In fact smoking history separated into transversion high (past and previous smokers) and transversion low (never smokers) groups, corroborating previous results.

→ mutations in groups              Transversion High                   Transversion Low

TP53, KRAS, STK11,                 EGFR, RB1, PI3CA

     KEAP1, SMARCA4 RBM10

 

1. b) Gender

Although gender differences in mutational profiles have been reported, the study found minimal number of significantly mutated genes correlated with gender. Notably:

• EGFR mutations enriched in female cohort
• RBM10 loss of function mutations enriched in male cohort

Although the study did not analyze the gender differences with smoking patterns, it was noted that RBM10 mutations among males were more prevalent in the transversion high group.

Whole exome Sequencing and copy number analysis reveal Unique, Candidate Driver Genes

Whole exome sequencing revealed that 62% of tumors contained mutations (either point or indel) in known cancer driver genes such as:

KRAS, EGFR, BRMF, ERBB2

However, authors looked at the WES data from the oncogene-negative tumors and found unique mutations not seen in the tumors containing canonical oncogenic mutations.

Unique potential driver mutations were found in

TP53, KEAP1, NF1, and RIT1

The genomics and expression data were backed up by a proteomics analysis of three pathways:

1. MAPK pathway
2. mTOR
3. PI3K pathway

…. showing significant activation of all three pathways HOWEVER the analysis suggested that activation of signaling pathways COULD NOT be deduced from DNA sequencing alone. Phospho-proteomic analysis was required to determine the full extent of pathway modification.

For example, many tumors lacked an obvious mutation which could explain mTOR or MAPK activation.

 

Altered cell signaling pathways included:

• Increased MAPK signaling due to activating KRAS
• Higher mTOR due to inactivating STK11 leading to increased proliferation, translation

Pathway analysis of mutations revealed alterations in multiple cellular pathways including:

• Reduced oxidative stress response
• Nucleosome remodeling
• RNA splicing
• Cell cycle progression
• Histone methylation

Summary:

Authors noted some interesting conclusions including:

1. MET and ERBB2 amplification and mutations in NF1 and RIT1 may be unique driver events in lung adenocarcinoma
2. Possible new drug development could be targeted to the RTK/RAS/RAF pathway
3. MYC pathway as another important target
4. Cluster analysis using multimodal omics approach identifies tumors based on single-gene driver events while other tumor have multiple driver mutational events (TUMOR HETEROGENEITY)

Paper 2. A Genomics-Based Classification of Human Lung Tumors[2]

The paper can be found at

http://stm.sciencemag.org/content/5/209/209ra153

by The Clinical Lung Cancer Genome Project (CLCGP) and Network Genomic Medicine (NGM),*,†

Paper Summary

This sequencing project revealed discrepancies between histologic and genomic classification of lung tumors.

Methodology

– mutational analysis by whole exome sequencing of 1255 lung tumors of histologically

defined subtypes

– immunohistochemistry performed to verify reclassification of subtypes based on sequencing data

Results

• 55% of all cases had at least one oncogenic alteration amenable to current personalized treatment approaches
• Marked differences existed between cluster analysis within and between preclassified histo-subtypes
• Reassignment based on genomic data eliminated large cell carcinomas
• Prospective classification of 5145 lung cancers allowed for genomic classification in 75% of patients
• Identification of EGFR and ALK mutations led to improved outcomes

Conclusions:

It is feasible to successfully classify and diagnose lung tumors based on whole exome sequencing data.

Paper 3. Genomic Landscape of Non-Small Cell Lung Cancer in Smokers and Never-Smokers[3]

A link to the paper can be found here with Graphic Summary: http://www.cell.com/cell/abstract/S0092-8674%2812%2901022-7?cc=y?cc=y

Methodology

• Whole genome sequencing and transcriptome sequencing of cancerous and adjacent normal tissues from 17 patients with NSCLC
• Integrated RNASeq with WES for analysis of
  • Variant analysis
  • Clonality by variant allele frequency anlaysis
  • Fusion genes
• Bioinformatic analysis
  • PathScan, KEGG for pathway analysis
  • COSMIC for reported mutations
  • ChimeraScan, defuse, BreakFusion for fusion protein analysis

Results

• 3,726 point mutations and more than 90 indels in the coding sequence
• Smokers with lung cancer show 10× the number of point mutations than never-smokers
• Novel lung cancer genes, including DACH1, CFTR, RELN, ABCB5, and HGF were identified

• Tumor samples from males showed high frequency of MYCBP2 MYCBP2 involved in transcriptional regulation of MYC.

• Variant allele frequency analysis revealed 10/17 tumors were at least biclonal while 7/17 tumors were monoclonal revealing majority of tumors displayed tumor heterogeneity
• Novel pathway alterations in lung cancer include cell-cycle and JAK-STAT pathways
• 14 fusion proteins found, including ROS1-ALK fusion. ROS1-ALK fusions have been frequently found in lung cancer and is indicative of poor prognosis[4].
• Novel metabolic enzyme fusions
• Alterations were identified in 54 genes for which targeted drugs are available.           Drug-gable mutant targets include: AURKC, BRAF, HGF, EGFR, ERBB4, FGFR1, MET, JAK2, JAK3, HDAC2, HDAC6, HDAC9, BIRC6, ITGB1, ITGB3, MMP2, PRKCB, PIK3CG, TERT, KRAS, MMP14

Table. Validated Gene-Fusions Obtained from Ref-Seq Data

Note: Gene columns contain links for GeneCard while Gene function links are to the    gene’s GO (Gene Ontology) function.

GeneA (5′)	GeneB (3′)	GeneA function (link to Gene Ontology)	GeneB function (link to Gene Ontology)		known function (refs)
GRIP1	TNIP1	glutamate receptor IP	transcriptional repressor
SGMS1	STK10	sphingolipid synthesis	ser/thr kinase
RASSF3	TTYH2	GTP-binding protein	chloride anion channel
KDELR2	ROS1, GOPC	ER retention seq. binding	proto-oncogenic tyr kinase
ACSL4	DCAF6	fatty acid synthesis	?
MARCH8	PRKG1	ubiquitin ligase	cGMP dependent protein kinase
APAF1	UNC13B, TLN1	caspase activation	cytoskeletal
EML4	ALK	microtubule protein	tyrosine kinase		♦
EDR3,PHC3	LOC441601	polycomb pr/DNA binding	?
DKFZp761L1918,RHPN2	ANKRD27	Rhophilin (GTP binding pr	ankyrin like
VANGL1	HAO2	tetraspanin family	oxidase
CACNA2D3	FLNB	VOC Ca++ channel	filamin (actin binding)

† Author’s Note:

There has been a recent literature on the importance of the EML4-ALK fusion protein in lung cancer. EML4-ALK positive lung tumors were found to be les chemo sensitive to cytotoxic therapy[5] and these tumor cells may exhibit an epitope rendering these tumors amenable to immunotherapy[6]. In addition, inhibition of the PI3K pathway has sensitized EMl4-ALK fusion positive tumors to ALK-targeted therapy[7]. EML4-ALK fusion positive tumors show dependence on the HSP90 chaperone, suggesting this cohort of patients might benefit from the new HSP90 inhibitors recently being developed[8].

Table. Significantly mutated genes (point mutations, insertions/deletions) with associated function.

Gene	Function
TP53	tumor suppressor
KRAS	oncogene
ZFHX4	zinc finger DNA binding
DACH1	transcription factor
EGFR	epidermal growth factor receptor
EPHA3	receptor tyrosine kinase
ENSG00000205044
RELN	cell matrix protein
ABCB5	ABC Drug Transporter

Table. Literature Analysis of pathways containing significantly altered genes in NSCLC reveal putative targets and risk factors, linkage between other tumor types, and research areas for further investigation.

Note: Significantly mutated genes, obtained from WES, were subjected to pathway analysis (KEGG Pathway Analysis) in order to see which pathways contained signicantly altered gene networks. This pathway term was then used for PubMed literature search together with terms “lung cancer”, “gene”, and “NOT review” to determine frequency of literature coverage for each pathway in lung cancer. Links are to the PubMEd search results.

KEGG pathway Name	# of PUBMed entries containing Pathway Name, Gene ANDLung Cancer
Cell cycle	1237
Cell adhesion molecules (CAMs)	372
Glioma	294
Melanoma	219
Colorectal cancer	207
Calcium signaling pathway	175
Prostate cancer	166
MAPK signaling pathway	162
Pancreatic cancer	88
Bladder cancer	74
Renal cell carcinoma	68
Focal adhesion	63
Regulation of actin cytoskeleton	34
Thyroid cancer	32
Salivary secretion	19
Jak-STAT signaling pathway	16
Natural killer cell mediated cytotoxicity	11
Gap junction	11
Endometrial cancer	11
Long-term depression	9
Axon guidance	8
Cytokine-cytokine receptor interaction	8
Chronic myeloid leukemia	7
ErbB signaling pathway	7
Arginine and proline metabolism	6
Maturity onset diabetes of the young	6
Neuroactive ligand-receptor interaction	4
Aldosterone-regulated sodium reabsorption	2
Systemic lupus erythematosus	2
Olfactory transduction	1
Huntington’s disease	1
Chemokine signaling pathway	1
Cardiac muscle contraction	1
Amyotrophic lateral sclerosis (ALS)	1

A few interesting genetic risk factors and possible additional targets for NSCLC were deduced from analysis of the above table of literature including HIF1-α, mIR-31, UBQLN1, ACE, mIR-193a, SRSF1. In addition, glioma, melanoma, colorectal, and prostate and lung cancer share many validated mutations, and possibly similar tumor driver mutations.

 please click on graph for larger view

Paper 4. Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing[9]

For full paper and graphical summary please follow the link: http://www.cell.com/cell/abstract/S0092-8674%2812%2901061-6

Highlights

• Exome and genome characterization of somatic alterations in 183 lung adenocarcinomas
• 12 somatic mutations/megabase
• U2AF1, RBM10, and ARID1A are among newly identified recurrently mutated genes
• Structural variants include activating in-frame fusion of EGFR
• Epigenetic and RNA deregulation proposed as a potential lung adenocarcinoma hallmark

Summary

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Paper 5. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer[10]

Highlights

• Whole exome and transcriptome (RNASeq) sequencing 29 small-cell lung carcinomas
• High mutation rate 7.4 protein-changing mutations/million base pairs
• Inactivating mutations in TP53 and RB1
• Functional mutations in CREBBP, EP300, MLL, PTEN, SLIT2, EPHA7, FGFR1 (determined by literature and database mining)
• The mutational spectrum seen in human data also present in a Tp53-/- Rb1-/- mouse lung tumor model

 

Curator Graphical Summary of Interesting Findings From the Above Studies

The above figure (please click on figure) represents themes and findings resulting from the aforementioned studies including

questions which will be addressed in Future Posts on this site.

UPDATED 10/10/2021

The following article uses RNASeq to screen lung adenocarcinomas for fusion proteins in patients with either low or high tumor mutational burden. Findings included presence of MET fusion proteins in addition to other fusion proteins irrespective if tumors were driver negative by DNASeq screening.

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Source:

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Ryma Benayed, Michael Offin, Kerry Mullaney, Purvil Sukhadia, Kelly Rios, Patrice Desmeules, Ryan Ptashkin, Helen Won, Jason Chang, Darragh Halpenny, Alison M. Schram, Charles M. Rudin, David M. Hyman, Maria E. Arcila, Michael F. Berger, Ahmet Zehir, Mark G. Kris, Alexander Drilon and Marc Ladanyi

Clin Cancer Res August 1 2019 (25) (15) 4712-4722; DOI: 10.1158/1078-0432.CCR-19-0225

Abstract

Purpose: Targeted next-generation sequencing of DNA has become more widely used in the management of patients with lung adenocarcinoma; however, no clear mitogenic driver alteration is found in some cases. We evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions and MET exon 14 (METex14) alterations in DNA sequencing (DNAseq) driver–negative lung cancers.

Experimental Design: Lung cancers driver negative by MSK-IMPACT underwent further analysis using a custom RNAseq panel (MSK-Fusion). Tumor mutation burden (TMB) was assessed as a potential prioritization criterion for targeted RNAseq.

Results: As part of prospective clinical genomic testing, we profiled 2,522 lung adenocarcinomas using MSK-IMPACT, which identified 195 (7.7%) fusions and 119 (4.7%) METex14 alterations. Among 275 driver-negative cases with available tissue, 254 (92%) had sufficient material for RNAseq. A previously undetected alteration was identified in 14% (36/254) of cases, 33 of which were actionable (27 in-frame fusions, 6 METex14). Of these 33 patients, 10 then received matched targeted therapy, which achieved clinical benefit in 8 (80%). In the 32% (81/254) of DNAseq driver–negative cases with low TMB [0–5 mutations/Megabase (mut/Mb)], 25 (31%) were positive for previously undetected gene fusions on RNAseq, whereas, in 151 cases with TMB >5 mut/Mb, only 7% were positive for fusions (P < 0.0001).

Conclusions: Targeted RNAseq assays should be used in all cases that appear driver negative by DNAseq assays to ensure comprehensive detection of actionable gene rearrangements. Furthermore, we observed a significant enrichment for fusions in DNAseq driver–negative samples with low TMB, supporting the prioritization of such cases for additional RNAseq.

Translational Relevance

Inhibitors targeting kinase fusions have shown dramatic and durable responses in lung cancer patients, making their comprehensive detection critical. Here, we evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions in patients where no clear mitogenic driver alteration is found by DNA sequencing (DNAseq)–based panel testing. We found actionable alterations (kinase fusions or MET exon 14 skipping) in 13% of cases apparently driver negative by previous DNAseq testing. Among the driver-negative samples tested by RNAseq, those with low tumor mutation burden (TMB) were significantly enriched for gene fusions when compared with the ones with higher TMB. In a clinical setting, such patients should be prioritized for RNAseq. Thus, a rational, algorithmic approach to the use of targeted RNA-based next-generation sequencing (NGS) to complement large panel DNA-based NGS testing can be highly effective in comprehensively uncovering targetable gene fusions or oncogenic isoforms not just in lung cancer but also more generally across different tumor types.

A Commentary is in the same issue at https://clincancerres.aacrjournals.org/content/25/15/4586?iss=15

Wake Up and Smell the Fusions: Single-Modality Molecular Testing Misses Drivers

by Kurtis D. Davies and Dara L. Aisner

Abstract

Multitarget assays have become common in clinical molecular diagnostic laboratories. However, all assays, no matter how well designed, have inherent gaps due to technical and biological limitations. In some clinical cases, testing by multiple methodologies is needed to address these gaps and ensure the most accurate molecular diagnoses.

See related article by Benayed et al., p. 4712

In this issue of Clinical Cancer Research, Benayed and colleagues illustrate the growing need to consider multiple molecular testing methodologies for certain clinical specimens (1). The rapidly expanding list of actionable molecular alterations across cancer types has resulted in the wide adoption of multitarget testing approaches, particularly those based on next-generation sequencing (NGS). NGS-based assays are commonly viewed as “one-stop shops” to detect a vast array of molecular variants. However, as Benayed and colleagues discuss, even well-designed and highly vetted NGS assays have inherent gaps that, under certain circumstances, are ideally addressed by analyzing the sample using an alternative approach.

In the article, the authors examined a cohort of lung adenocarcinoma patient samples that had been deemed “driver- negative” via MSK-IMPACT, an FDA-cleared test that is widely considered by experts in the field to be one of the best examples of a DNA-based large gene panel NGS assay (2). Of 589 driver-negative cases, 254 had additional material amenable for a different approach: RNA-based NGS designed specifically for gene fusion and oncogenic gene isoform detection. After accounting for quality control failures, 232 samples were successfully sequenced, and, among these, 36 samples (representing an astonishing 15.5% of tested cases) were found to be positive for a driver gene fusion or oncogenic isoform that had not been detected by DNA-based NGS. The real-world value derived from this orthogonal testing schema was more than theoretical, with 8 of 10 (80%) patients demonstrating clinical benefit when treated according to the alteration identified via the RNA-based approach.

To detect gene rearrangements that lead to oncogenic gene fusions (and to detect mutations and insertions/deletions that lead to MET exon 14 skipping), MSK-IMPACT employs hybrid capture-based enrichment of selected intronic regions from genomic DNA. While this approach has proven to be successful in a variety of settings, there are associated limitations that were determined in this study to underlie the discrepancies between MSK-IMPACT and the RNA-based assay. First, some introns that are involved in clinically actionable rearrangement events are very large, thus requiring substantial sequencing capital that can represent a disproportionate fraction of the assay. Despite the ability via NGS to perform sequencing at a large scale, this sequencing capacity is still finite, and thus decisions must be made to sacrifice coverage of certain large genomic regions to ensure sufficient sequencing depth for other desired genomic targets. In the case of MSK-IMPACT (and most other DNA-based NGS assays), certain important introns in NTRK3 and NRG1 are not included in covered content, simply because they are too large (>90 Kb each). The second primary problem with DNA-based analysis of introns is that they often contain highly repetitive elements that are extremely difficult to assess via NGS due to their recurring presence across the genome. Attempts to sequence these regions are largely unfruitful because any sequencing data obtained cannot be specifically aligned/mapped to the desired targeted region of the genome (3). This is particularly true for intron 31 of ROS1, because it contains two repetitive long interspersed nuclear elements, and many DNA-based assays, including MSK-IMPACT, poorly cover this intron (4). In this study by Benayed and colleagues, the most common discrepant alteration was fusion involving ROS1, which accounted for 10 of 36 (28%) cases. At least six of these, those that demonstrated fusion to ROS1 exon 32, were likely directly explained by incomplete intron 31 sequencing. RNA-based analysis is able to overcome the above described limitations owing to the simple fact that sequencing is focused on exons post-splicing and the need to sequence introns is entirely avoided (Fig. 1).

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Figure 1.

Schematic representation of underlying genomic complexities that can lead to false-negative gene fusion results in DNA-based NGS analysis. In some cases, RNA-based approaches may overcome the limitations of DNA-based testing.

Lack of sufficient intronic coverage could not account for all of the discrepancies between DNA-based and RNA-based analysis however. Six samples in the cohort were found to be positive for MET exon 14 skipping based on RNA. In five of these, genomic alterations in MET introns 13 or 14 were observed, however they did not conform to canonical splice site alterations and thus were not initially called (although this was addressed by bioinformatics updates). In RNA-based testing, however, determination of exon skipping is simplified such that, regardless of the specific genomic alteration that interferes with splicing, absence of the exon in the transcript is directly observed (5). In another two of the discrepant cases, tumor purity was observed to be low in the sample, meaning that the expected variant allele frequency (VAF) for a genomic event would also likely be low, potentially below detectable levels. However, overexpression of the fusions at the transcript level was theorized to compensate for low VAF (Fig. 1). Additional explanations for discordant findings between the assays included sample-specific poor sequencing in selected introns and complex rearrangements that hindered proper capture (Fig. 1).

The take home message from Benayed and colleagues is simply this: there is no perfect assay that will detect 100% of the potential actionable alterations in patient samples. Even an extremely well designed, thoroughly vetted, and FDA-cleared assay such as MSK-IMPACT will have inherent and unavoidable “holes” due to intrinsic limitations. The solution to this dilemma, as adeptly described by Benayed and colleagues, is additional testing using a different approach. While in an ideal world every clinical tumor sample would be tested by multiple modalities to ensure the most comprehensive clinical assessment, the reality is that these samples are often scant and testing is fiscally burdensome (and often not reimbursed). Therefore, algorithms to determine which samples should be reflexed to secondary assays after testing with a primary assay are critical for maximizing benefit. In this study, the first algorithmic step was lack of an identified driver (because activated oncogenic drivers tend to exist exclusively of each other), which amounted to 23% of samples tested with the primary assay. In addition, the authors found a significantly higher rate of actionable gene fusions in samples with a low (<5 mut/Mb) tumor mutational burden, meaning that this metric, which was derived from the primary assay, could also be used to help inform decision making regarding additional testing. While this scenario is somewhat specific to lung cancer, similar approaches could be prescribed on a cancer type–specific basis.

These findings should be considered a “wake-up call” for oncologists in regard to the ordering and interpretation of molecular testing. It is clear from these and other published findings that advanced molecular analysis has limitations that require nuanced technical understanding. As this arena evolves, it is critical for oncologists (and trainees) to gain an increased comprehension of how to identify when the “gaps” in a test might be most clinically relevant. This requires a level of technical cognizance that has been previously unexpected of clinical practitioners, yet is underscored by the reality that opportunities for effective targeted therapy can and will be missed if the treating oncologist is unaware of how to best identify patients for whom additional testing is warranted. This study also highlights the mantra of “no test is perfect” regardless of prestige of the testing institution, number of past tests performed, or regulatory status. NGS, despite its benefits, does not mean all-encompassing. It is only through the adaptability of laboratories to utilize knowledge such as is provided by Benayed and colleagues that advances in laboratory medicine can be quickly deployed to maximize benefits for oncology patients.

References:

1. Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012, 489(7417):519-525.
2. A genomics-based classification of human lung tumors. Science translational medicine 2013, 5(209):209ra153.
3. Govindan R, Ding L, Griffith M, Subramanian J, Dees ND, Kanchi KL, Maher CA, Fulton R, Fulton L, Wallis J et al: Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 2012, 150(6):1121-1134.
4. Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, Asaka R, Hamanaka W, Ninomiya H, Uehara H et al: RET, ROS1 and ALK fusions in lung cancer. Nature medicine 2012, 18(3):378-381.
5. Morodomi Y, Takenoyama M, Inamasu E, Toyozawa R, Kojo M, Toyokawa G, Shiraishi Y, Takenaka T, Hirai F, Yamaguchi M et al: Non-small cell lung cancer patients with EML4-ALK fusion gene are insensitive to cytotoxic chemotherapy. Anticancer research 2014, 34(7):3825-3830.
6. Yoshimura M, Tada Y, Ofuzi K, Yamamoto M, Nakatsura T: Identification of a novel HLA-A 02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene. Oncology reports 2014, 32(1):33-39.
7. Yang L, Li G, Zhao L, Pan F, Qiang J, Han S: Blocking the PI3K pathway enhances the efficacy of ALK-targeted therapy in EML4-ALK-positive nonsmall-cell lung cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2014.
8. Workman P, van Montfort R: EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence. Cancer discovery 2014, 4(6):642-645.
9. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, Cho J, Suh J, Capelletti M, Sivachenko A et al: Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 2012, 150(6):1107-1120.
10. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, Plenker D, Leenders F, Sun R, Zander T et al: Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nature genetics 2012, 44(10):1104-1110.

Other posts on this site which refer to Lung Cancer and Cancer Genome Sequencing include:

Multi-drug, Multi-arm, Biomarker-driven Clinical Trial for patients with Squamous Cell Carcinoma called the Lung Cancer Master Protocol, or Lung-MAP launched by NCI, Foundation Medicine, and Five Pharma Firms

US Personalized Cancer Genome Sequencing Market Outlook 2018 –

Comprehensive Genomic Characterization of Squamous Cell Lung Cancers

International Cancer Genome Consortium Website has 71 Committed Cancer Genome Projects Ongoing

Non-small Cell Lung Cancer drugs – where does the Future lie?

Lung cancer breathalyzer trialed in the UK

Diagnosing Lung Cancer in Exhaled Breath using Gold Nanoparticles

Multi-drug, Multi-arm, Biomarker-driven Clinical Trial for patients with Squamous Cell Carcinoma called the Lung Cancer Master Protocol, or Lung-MAP launched by NCI, Foundation Medicine, and Five Pharma Firms

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Second Annual Single-Cell Sequencing of Cancer Rountable August 20,21, 2014 Washington DC

Posted in Bio Instrumentation in Experimental Life Sciences Research, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Personalized and Precision Medicine & Genomic Research, RNA Biology, Cancer and Therapeutics, Scientific & Biotech Conferences: Press Coverage, tagged Biology, Cancer - General, circulating tumor ce;;s, Circulating tumor cell, computational genomics, DNA Sequencing, Food and Drug Administration, genetics, genome, genomics, Harvard Medical School, National Institutes of Health, Personalized medicine, single cell on June 19, 2014| Leave a Comment »

Second Annual Single-Cell Sequencing of Cancer Rountable August 20,21, 2014 Washington DC

Reporter:  Stephen J. Williams, PhD

CSHL, UCLA & Einstein to Lead Roundtable Discussions on Single-Cell Sequencing Interactive discussions on three of the key questions researchers are facing when considering single-cell analysis will be held on the second day of the Single-Cell Sequencing Conference at Next Generation Dx Summit, taking place August 20-21, 2014 in Washington, DC. For full program details and to register, please visit NextGenerationDx.com/Single-Cell-Sequencing.Making Single-Cell Analysis Cost Effective for Clinical Use Moderator: James Hicks, Ph.D., Research Professor, Cancer Genomics, Cold Spring Harbor Laboratory Methods for capture: What are the tradeoffs? Combining RNA, DNA and protein analysis What genomic assays are most informative? Can assays be certifiable? Finding a Needle in a Haystack: Towards Diagnosing Rare Soft Tissue Cancer Stem Cells (CSCs) Moderator: Michael Masterman-Smith, Ph.D., Entrepreneurial Scientist, UCLA California NanoSystems Institute Rethinking companion diagnostics for cancer to incorporate analysis of CSCs Current direct methodologies of CSC detection/isolation Current proxy methodologies of CSC detection/isolation The hope and promise of single-cell assay tools and technologies Why Single-Cell Sequencing? Moderator: Jan Vijg, Ph.D., Professor and Chairman, Genetics, Albert Einstein College of Medicine Sample limitations, e.g., prenatal diagnostics and CTCs Sample limitations, e.g., prenatal diagnostics and CTCs To study cell-to-cell variation, e.g., in tumors as well as normal tissues To overcome technological constraints, e.g., detecting somatic mutations Cell-to-cell fluctuations in gene expression can easily impair function, yet can be undetectable by measuring averages How many different cell types are there?

View Brochure    |   Register (Advance Registration Ends July 18)  |   NextGenerationDx.com/Single-Cell-Sequencing About the Conference Sequencing data from bulk DNA or RNA from multiple cells provide global information on average states of cell populations. But with whole-genome amplification and NGS, researchers can detect variation in individual cancer cells and dissect tumor evolution. Such cancer genome sequencing will improve oncology by detecting rare tumor cells early, measuring intra-/intertumor heterogeneity, guiding chemotherapy and controlling drug resistance. The Single-Cell Sequencing conference explores the latest strategies, data analyses and clinical considerations that influence and aid cancer diagnosis, prognosis and prediction and will lead to individualized cancer therapy. Sessions include presentations spanning the opportunities of clinical single-cell analysis from: Sunney Xie, Ph.D., Mallinckrodt Professor. Chemistry and Chemical Biology, Harvard University Maximilian Diehn, M.D., Ph.D., Assistant Professor, Radiation Oncology, Stanford Cancer Institute, Institute for Stem Cell Biology & Regenerative Medicine, Stanford University Denis Smirnov, Associate Scientific Director, US Biomarker Oncology, Janssen R&D US James Hicks, Ph.D., Research Professor, Cancer Genomics, Cold Spring Harbor Laboratory Jan Vijg, Ph.D., Professor and Chairman, Genetics, Albert Einstein College of Medicine John F. Zhong, Ph.D., Associate Professor, Pathology, University of Southern California School of Medicine Mark Hills, Ph.D., Research Scientist, Peter M. Lansdorp Laboratory, BC Cancer Research Centre Michael Masterman-Smith, Ph.D., Entrepreneurial Scientist, UCLA California NanoSystems Institute Parveen Kumar, Research Scientist, Thierry Voet Laboratory, Human Genetics, University of Leuven Peter Nemes, Ph.D., Assistant Professor, Chemistry, George Washington University Theresa Zhang, Ph.D., Vice President, Research Services, Personal Genome Diagnostics Yong Wang, Ph.D., Senior Postdoctoral Fellow, Nicholas E. Navin Laboratory, Genetics, Bioinformatics, MD Anderson Cancer Center Zivana Tezak, Ph.D., Associate Director, Science and Technology, Personalized Medicine, Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD), Center for Devices and Radiological Health (CDRH), FDA   Recommended Pre-Conference Courses NGS Data Analysis – Determining Clinical Utility of Genome Variants Monday, August 18 | 9:00am – 12:00pm This course will explore the strategies of genomic data analysis and interpretation, an emergent discipline that seeks to deliver better answers from NGS data so that patients and their physicians can determine informed healthcare decisions. View Details NGS as a Diagnostics Platform Monday, August 18 | 2:00pm – 5:00pm The focus of this short course will be on understanding the use of NGS in clinical diagnosis, practical implementation of NGS in clinical laboratories and analysis of large data sets by using bioinformatics tools to parse and interpret data in relation to the clinical phenotype. The concluding presentation will be dedicated to quality and standardization of NGS assays. View Details Register   |   View Agenda   | NextGenerationDx.com   #NGDx14

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