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Old 01-07-2013, 10:27 AM
gdpawel gdpawel is offline
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Default Simply finding a mutation

The idea of simply finding a mutation and then pick an appropriately targeted drug seems like a nice idea. However, not every key that looks like it will fit a lock will actually turn it. The same is likely to be the case with targeted drugs. There are numerous common mutations in various tumor types, but they don't know that all those mutations are going to turn out to be relevant, as many of them are essentially bystanders.

Gene mutations are changes in a genomic sequence, the DNA sequence of a cell's genome. These random sequences can be defined as sudden and spontaneous changes in the cell. Sequencing is a laboratory process that determines the nucleotide sequence of DNA (can involve the whole genome or whole exome or be targeted to as little as one coding sequence). A genome is the complete set of genetic material. An exome is part of the genome formed by genes that code for proteins and other functional gene products (known as exons).

Gene rearrangement is a structural alteration of a chromosome that causes a change in the order of its loci. The carbon skeleton of a molecule is rearranged to give a structural isomer of the original molecule. DNA rearrangements are known to occur only in cancer cells and not in non-cancerous cells, making them fit to be tumor markers for cancer. The incidence of ALK gene rearrangement in patients with NSCLC is in the range of 2-4 percent, ROS1 gene rearrangement in the range of 1-2 percent, while EGFR mutations are found in approximately 15 percent. These are largely mutually exclusive events.

Why don't all the mutation positive patients respond and why do some mutation negative patients respond? Cancer biology is complex. Molecular biologists can only seek and identify that which they know about from earlier (a priori). What happens if you have MEK, ERK, SHH, LKB1, FGFR, PI3K, etc.? There are numerous mutations, insertions and deletions. A gene mutation, deletion, translocation or amplification could disrupt many cell functions, leading to drug resistance, or could inactivate or damage the doors through which a drug enters a cell.

Lung cancer arises not only from gene mutations but also from small fragments of RNA that can up- or down-regulate normal genes in abnormal ways. The fact that normal genes can function abnormally under the control of these small RNA sequences is just one more example of the genotype-phenotype dichotomy that cannot be adequately examined on static contemporary genomic platforms.

It isn't just molecular analysis, it is whether the capacity to judge phenotypes will be easily achieved at the genotype level. Systems biology suggests that the simple knowledge of a gene's presence or absence does not confer a biological behavior. Biology is not linear.

The real question that should be asked is whether Tarceva would work on your individual cancer cells, not whether you have the ALK gene or the EGFR gene or some other rearrangement. The answer is that Tarceva can work for any lung cancer patient, or not. It's a question of whether your individual cancer cells are "sensitive" to Tarceva or are they "resistant" to Tarceva, regardless of the gene your cancer cells "hang" with.

To learn more about this:

Chasing Gene Mutations

[url]http://cancerfocus.org/forum/showthread.php?t=3620

Tarceva Delivers Benefits Across Broad Range of NSCLC Patients

[url]http://cancerfocus.org/forum/showthread.php?t=1132

The Role of Genomic Testing for Predicting Response to Targeted Therapy

[url]http://cancerfocus.org/forum/showthread.php?t=3652

The Problem with KRAS Mutation Studies

[url]http://cancerfocus.org/forum/showthread.php?t=3583

Gene Expression Signatures Not Ready for Prime Time

[url]http://cancerfocus.org/forum/showthread.php?t=3533

Anti-Angiogenic Activity and VEGF Pathway Inhibition of Tarceva

[url]http://cancerfocus.org/forum/showthread.php?t=3738

Gene Mutation vs Chromosomal Theory of Cancer

[url]http://cancerfocus.org/forum/showthread.php?t=3764

Targeted Therapy is Still Trial-And-Error Treatment

[url]http://cancerfocus.org/forum/showthread.php?t=3522

Functional Profiling to Select Chemotherapy in Advanced NSCLC

[url]http://cancerfocus.org/forum/showthread.php?t=3775
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Last edited by gdpawel : 01-07-2013 at 10:46 AM. Reason: hyperlink url addresses
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Old 01-17-2013, 10:04 AM
gdpawel gdpawel is offline
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Default There's More to Tumor Drug Resistance Than Just Gene Mutations

(Chemotherapy Advisor) - The appearance of new genetic mutations during cancer therapy has long been correlated with drug resistance, treatment failure, and ultimately, relapse. But using single-cell genome sequencing, researchers at the Ontario Cancer Institute and Princess Margaret Cancer Center in Toronto, Canada have shown that gene mutations alone cannot explain drug-resistant cancer.

Tracking individual human colorectal tumor subclone cells that had been xenografted into mice, and sequencing their exomes (the gene-encoding regions of subclone genomes), the team discovered dramatic functional heterogeneity even among genetically identical clones; these included different tumor propagation patterns and different susceptibilities to oxaliplatin, which was reported by the the researchers in Science.1

The findings represent “a major conceptual advance in understanding tumor growth and treatment response,” said senior author John Dick, PhD, a pioneer in cancer stem cell research. “The data show that gene sequencing of tumors to find the spectrum of their mutations is definitely not the whole story when it comes to determining which therapies will be most effective.”

While some cells of a subclone contributed to tumor growth, others quickly became dormant, even though they harbored the same mutations as the more active cells, Dr. Dick's team found—and these dormant cells survived oxaliplatin therapy.

“This is a paradigm shift that shows research also needs to focus on the biological properties of cells,” Dr. Dick said. Treatments that force dormant cells back into growth cycles could make them more sensitive to chemotherapies, he theorizes.

“Targeting the biology and growth properties of cancer cells could expand the repertoire of usable therapeutic agents and provide better outcomes for patients,” he added.

The study is “avante garde in its documentation that types of subcloning can happen against a stability of genetic changes—a clone within a given set of genetic changes can evolve into subpopulations within that clone without changing their genetic background, their mutations,” said Stephen Baylin, MD, Professor of Oncology and Deputy Director of the Cancer Center at the Johns Hopkins University School of Medicine.

Epigenetic factors, such as different DNA-methylation patterns that can silence gene expression, might help explain behavioral heterogeneity among genetically identical subclones, Dr. Baylin postulates.

“If you have got such genetic stability, then it's likely that the other facets of the subclones that emerged could have an epigenetic basis—long-term changes in gene expression,” he told ChemotherapyAdvisor.com. “Things like epigenetic abnormalities could be contributing to the emergence of new subclones with distinct properties.”

The new study “emphasizes those possibilities,” he said. When dormant tumor cells “come out and replenish the tumor,” other studies have shown that they do so with a “different epigenetic state” that appears to contribute to their drug resistance, he noted.

The traditional paradigm, with new mutations causing some tumor cells' drug resistance, is not completely wrong, Dr. Baylin is quick to point out. “That can happen,” he said of mutation-driven resistance. However, Baylin believes,the new findings reported by Dr. Dick and his colleagues strongly suggest epigenetics is another “big player” in drug resistance.

Epigenetics-targeting drugs already exist, he notes. For example, azacitidine (5-azacytidine) and decitabine (5-aza-2′deoxycytidine) inhibit DNA methylation and are approved by the FDA for myelodysplastic syndrome. Histone deacetylase inhibitors might also target epigenetic pathways in tumors.

At low doses, ongoing studies in Dr. Baylin's lab suggest that azacitadine “sensitizes patients to subsequent chemotherapies or a new form of immunotherapy,” he said.

The findings reported by Dr. Dick's team indeed suggest that nongenetic targets for personalized anticancer agents are waiting to be identified, agrees Charis Eng, MD, PhD, FACP. Candidate targets include both epigenetic alterations and tumor microenvironments (healthy cells adjacent to tumors), she said.

Dr. Eng is the Hardis and American Cancer Society Professor and founding Chair of the Genomic Medicine Institute and directs the Institute's clinical component, the Center for Personalized Genetic Healthcare at the Cleveland Clinic. She believes that even though tumors' subclone gene mutations were identical from cell to cell, their functional genomics—“the ways they interact with each other, the ways they signal and make transcripts”—might still be quite variable.

Gene mutations, in other words, are just one part of a larger puzzle. Epigenetics, proteomics, even microbiomes, or the genomes of bacteria living on epithelial tissues in which tumors emerge, may all help explain why some subclone cells go dormant and evade chemotherapeutic attacks, while others succumb to treatment, she believes.

“Genomic changes are like the skeleton. The genome is the skeleton and everything else—the methylation, microenvironment, the microbiome—will be the flesh, the meat, the muscle and the skin,” Dr. Eng explained to ChemotherapyAdvisor.com. “So a look at everything, a snapshot profile of all the –omes, or what I call ‘integrated –omics,' which is the strength of my lab, integrates all the –omic platforms to see whether we can come up with an integrated view of what a cancer looks like.”

Even the Cancer Genome Atlas Project has added RNA and epigenetic assays to its profiling of tumor genomes, she notes.

The “sum total of the integration of all the ‘-omes' from all the cancer cells,” rather than any one component, may dictate chemotherapy responses in many cancers, she suspects. If that's the case, gene mutation-targeting drugs could one day represent just one part of clinical oncology's personalized-therapy arsenal.

“I have a funny feeling that even the three-dimensional positioning of the cells (within tumors) and how they talk to each other—whether by message or protein or exchanging genes—also matters,” Dr. Eng said.

But she is quick to point out that the Science study involved xenografting human tumors into mice. Even though that model worked elegantly to show that subclone heterogeneity is not attributable to genetic mutations alone, it may not be the best way to find out exactly what else is responsible, she cautioned—especially if tumor microenvironments are involved.

“When the microenvironment is not represented well, alterations in the microenvironment might be missed,” she explained. “We have to ask: what is the interaction of each of these subclones with the mouse environment?”

Taking human tumors out of their human microenvironmental context might itself “have effects on different expressions of genes in the cancer,” Dr. Eng noted.

“We'd been assuming the enemy was simple-minded,” she said. “The enemy is complex. We need a multidisciplinary approach to look at the DNA and to understand the microenvironment, how it turns genes off and on in different contexts and even in different (cell) positions within tumors.”

Dr. Baylin agrees that the new findings will open doors to new avenues of research.

“We need to work on extending these observations to other tumors and to really keep studying the mechanisms that account for how these subclones emerge,” he said. “And then we need to correlate those changes really carefully with drug resistance so we can understand the molecular underpinnings in resistance patterns, and learn how to tailor therapeutic approaches to those molecular mechanisms.”

Reference

1. Kreso A, O'Brien CA, van Galen P, et al. Variable clonal repopulation dynamics influence chemotherapy responses in colorectal cancer. Science.

[url]http://www.nature.com/polopoly_fs/1.11710%21/menu/main/topColumns/topLeftColumn/pdf/491027a.pdf

[url]http://www.sciencemag.org/content/early/2012/12/12/science.1227670.abstract?sid=63872db4-97e2-4580-a9eb-46531083d53e
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  #3  
Old 01-27-2013, 12:30 AM
gdpawel gdpawel is offline
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Default Genomics vs Proteonomics vs Functionality

Scott L. Shreeve, M.D.

Anyone familiar with cellular biology knows that having the genetic sequence of a known gene (genotype) does not equate to having the disease state (phenotype) represented by that gene. It requires specific cellular triggers and specialized cellular mechanisms to literally translate the code into the work horse of the cellular world - proteins.

As a result, while genomics provides a methodology to understand the sheet music of life, there is more interest in the proteomic symphony that results by literally bringing the dead notes to life. Since the actual performance of the sheet music can vary, based on the whims of the composer, there is the individual variation or free-lancing of the orchestral performers.

So while the sheet music is absolutely essential to even contemplate a symphonic performance, no one goes to listen to the sheet music. It is the individual performances, the living-breathing-audible-panoramic-splended-view of the whole chamber coming to life that ultimately stirs the soul.

In an analogous fashion, the proteins are the cellular accessories that literally add life to an otherwise staid genetic makeup. Infinitely more variable, infinitely more possibilities, and as a result, infinitely more interesting.

[url]http://crossoverhealth.com/2007/11/genomics-vs-proteonomics-accessorizing-your-genes/

I cannot agree more. The people wowed by genomics perhaps do not see the complexities and redundancies of human biology are beyond the ken of genomics. In cancer medicine, getting better drug development tools from advances in genomics and proteomics is one thing, but harnessing those tools for drug testing and for delivery to patients is another matter entirely.

While genomics focuses on how genes direct activities within the cell, proteomics studies how proteins carry out those directions. Genes create the blueprints for the production of proteins within the cell. A protein is a molecule that makes a cell behave in a certain way. It does so by interacting with other proteins in a complex series of steps.

In chemotherapy selection, gene and protein testing examine a single process within the cell or a relatively small number of processes. The aim is to tell if there is a theoretical predisposition to drug response. Whole cell "functional profiling" tests not only for the presence of genes and proteins but also for their functionality, for their interaction with other genes, proteins and processes occurring within the cell, and for their response to anti-cancer drugs.

Genomics is only important insofar as it influences proteonomics, which is only important insofar as it influences protein function analysis (are proteins active or inactive), which is only important insofar as it influences cell function analysis (cell culture testing). There is an inverse hierachy between relevance and ease of measurement. So genomics and proteonomics are not the only potential key to genetic disease.

Cancer is a complex disease and needs to be attacked on many fronts. The best thing to do is to combine these different tests in ways which make the most sense.

[url]http://cancerfocus.org/forum/showthread.php?t=3666

Lecture on issues related to cell culture testing

A 33 minute lecture on functional profiling of human cancer, using cell culture drug resistance testing on fresh human tumor specimens obtained by surgical biopsy. This lecture was given at Charite University Medical Center in Berlin, Germany on March 19, 2009 by Larry Weisenthal, M.D., PhD. The audience consisted primarily of oncologists and surgeons working in the field of gynecologic oncology. Thanks to Dr. Frank Kischkel, TherapySelect GmbH & Co. KG, Heidelberg, Germany [url]http://vimeo.com/7577309
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Last edited by gdpawel : 03-01-2013 at 02:10 AM. Reason: additional info
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