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That is the question…

Anyone who follows healthcare news, as I do , cannot help being impressed with the number of scientific and non-scientific items that mention the applicability of Magnetic Resonance Imaging (‘MRI’) to medical procedures.

A very important aspect that is worthwhile noting is that the promise MRI bears to improve patients’ screening – pre-clinical diagnosis, better treatment choice, treatment guidance and outcome follow-up – is based on new techniques that enables MRI-based tissue characterisation.

Magnetic resonance imaging (MRI) is an imaging device that relies on the well-known physical phenomena named “Nuclear Magnetic Resonance”. It so happens that, due to its short relaxation time, the 1H isotope (spin ½ nucleus) has a very distinctive response to changes in the surrounding magnetic field. This serves MRI imaging of the human body well as, basically, we are 90% water. The MRI device makes use of strong magnetic fields changing at radio frequency to produce cross-sectional images of organs and internal structures in the body. Because the signal detected by an MRI machine varies depending on the water content and local magnetic properties of a particular area of the body, different tissues or substances can be distinguished from one another in the scan’s resulting image.

The main advantages of MRI in comparison to X-ray-based devices such as CT scanners and mammography systems are that the energy it uses is non-ionizing and it can differentiate soft tissues very well based on differences in their water content.

In the last decade, the basic imaging capabilities of MRI have been augmented for the purpose of cancer patient management, by using magnetically active materials (called contrast agents) and adding functional measurements such as tissue temperature to show internal structures or abnormalities more clearly.

 

In order to increase the specificity and sensitivity of MRI imaging in cancer detection, various imaging strategies have been developed. The most discussed in MRI related literature are:

  • T2 weighted imaging: The measured response of the 1H isotope in a resolution cell of a T2-weighted image is related to the extent of random tumbling and the rotational motion of the water molecules within that resolution cell. The faster the rotation of the water molecule, the higher the measured value of the T2 weighted response in that resolution cell. For example, prostate cancer is characterized by a low T2 response relative to the values typical to normal prostatic tissue [5].

T2 MRI pelvis with Endo Rectal Coil ( DATA of Dr. Lance Mynders, MAYO Clinic)

  • Dynamic Contrast Enhanced (DCE) MRI involves a series of rapid MRI scans in the presence of a contrast agent. In the case of scanning the prostate, the most commonly used material is gadolinium [4].

Axial MRI  Lava DCE with Endo Rectal ( DATA of Dr. Lance Mynders, MAYO Clinic)

  • Diffusion weighted (DW) imaging: Provides an image intensity that is related to the microscopic motion of water molecules [5].

DW image of the left parietal glioblastoma multiforme (WHO grade IV) in a 59-year-old woman, Al-Okaili R N et al. Radiographics 2006;26:S173-S189

  • Multifunctional MRI: MRI image overlaid with combined information from T2-weighted scans, dynamic contrast-enhancement (DCE), and diffusion weighting (DW) [5].

Source AJR: http://www.ajronline.org/content/196/6/W715/F3

  • Blood oxygen level-dependent (BOLD) MRI: Assessing tissue oxygenation. Tumors are characterized by a higher density of micro blood vessels. The images that are acquired follow changes in the concentration of paramagnetic deoxyhaemoglobin [5].

In the last couple of years, medical opinion leaders are offering to use MRI to solve almost every weakness of the cancer patients’ pathway. Such proposals are not always supported by any evidence of feasibility. For example, a couple of weeks ago, the British Medical Journal published a study [1] concluding that women carrying a mutation in the BRCA1 or BRCA2 genes who have undergone a mammogram or chest x-ray before the age of 30 are more likely to develop breast cancer than those who carry the gene mutation but who have not been exposed to mammography. What is published over the internet and media to patients and lay medical practitioners is: “The results of this study support the use of non-ionising radiation imaging techniques (such as magnetic resonance imaging) as the main tool for surveillance in young women with BRCA1/2 mutations.”.

Why is ultrasound not mentioned as a potential “non-ionising radiation imaging technique”?

Another illustration is the following advert:

An MRI scan takes between 30 to 45 minutes to perform (not including the time of waiting for the interpretation by the radiologist). It requires the support of around 4 well-trained team members. It costs between $400 and $3500 (depending on the scan).

The important question, therefore, is: Are there, in the USA, enough MRI  systems to meet the demand of 40 million scans a year addressing women with radiographically dense  breasts? Toda there are approximately 10,000 MRI systems in the USA. Only a small percentage (~2%) of the examinations are related to breast cancer. A

A rough calculation reveals that around 10000 additional MRI centers would need to be financed and operated to meet that demand alone.

References

  1. Exposure to diagnostic radiation and risk of breast cancer among carriers of BRCA1/2 mutations: retrospective cohort study (GENE-RAD-RISK), BMJ 2012; 345 doi: 10.1136/bmj.e5660 (Published 6 September 2012), Cite this as: BMJ 2012;345:e5660 – http://www.bmj.com/content/345/bmj.e5660
  1. http://www.auntminnieeurope.com/index.aspx?sec=sup&sub=wom&pag=dis&itemId=607075
  1. Ahmed HU, Kirkham A, Arya M, Illing R, Freeman A, Allen C, Emberton M. Is it time to consider a role for MRI before prostate biopsy? Nat Rev Clin Oncol. 2009;6(4):197-206.
  1. Puech P, Potiron E, Lemaitre L, Leroy X, Haber GP, Crouzet S, Kamoi K, Villers A. Dynamic contrast-enhanced-magnetic resonance imaging evaluation of intraprostatic prostate cancer: correlation with radical prostatectomy specimens. Urology. 2009;74(5):1094-9.
  1. Advanced MR Imaging Techniques in the Diagnosis of Intraaxial Brain Tumors in Adults, Al-Okaili R N et al. Radiographics 2006;26:S173-S189 ,

http://radiographics.rsna.org/content/26/suppl_1/S173.full

  1. Ahmed HU. The Index Lesion and the Origin of Prostate Cancer. N Engl J Med. 2009 Oct; 361(17): 1704-6
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Pull at Cancer’s Levers

Curator: Larry H. Bernstein, MD, FCAP

 

Driving Cancer Immunotherapy 

The Stakes in Immuno-Oncology Are Too High for Researchers to Pull at Cancer’s Levers Blindly. Researchers Need a System.

  • Within the past decade or so, a revolutionary idea has emerged in the minds of scientists, physicians, and medical experts. Instead of using man-made chemicals to treat cancer, let us instead unleash the power of our own bodies upon the malignancy.

    This idea is the inspiration behind cancer immunotherapy, which is, according to most experts, a therapeutic approach that involves training the immune system to fight off cancer. In the words of one expert, cancer immunotherapy means “taking the immune system’s inherent properties and turbo charging those to fight cancer.”

    Cancer immunotherapy technologies are being developed to accomplish
    several tasks:

    • Enhance the molecular targeting of cancer cells
    • Report the rate of killing by specific immune agents
    • Direct immune cells toward tumor destruction.

    Since its inception, the field has evolved, and it continues to do so. It began with in vivo investigations of tumor growth and development, and it progressed through laboratory investigations of cellular morphology and survival curves. And now it is adopting pathway analysis to guide therapeutic development and improve patient care.

    To begin to understand cancer immunotherapy, one must understand how the immune system targets tumor cells. One of the prominent adaptive components of the immune system is the T cell, which responds to perceived threats through the massive increase in clonal T cells targeted in some way toward the diseased cell or pathogen.

  • The T-Cell Repertoire

    Adaptive Biotechnologies’ immunoSEQ Assay, a high-throughput research platform for immune system profiling, is designed to generate sequencer-ready libraries using highly optimized primer sets in a multiplex PCR format that targets T- and B-cell receptor genes. This image depicts how the assay’s two-step PCR process can be used to quantify the clonal diversity of immune cells.

    Immunologists call this process VJD rearrangement. It happens during T-lymphocyte development and affects three gene regions, the variable (V), the diversity (D), and the joining (J) regions. This rearrangement of the genetic code allow for the structural diversity in T-cell receptors responsible for antigenic specificity including antigenic targets on tumor cells. In the case of cancer, specificity is complicated because the tumor is actually part of the body itself, one of the reasons cancers naturally evade detection.

    The specificity problem would always hinder attempts to goad the immune system into attacking cancer, scientists realized, unless technologies emerged that could efficiently track the clonal diversity of T cells inside patients. Existing technologies, such as spectratyping, were inadequate.  In 2007, when Dr. Robins and his collaborators began developing the technology, only 10,000 T-cell receptor sequences had been reported in all the literature using older methodologies.

    “The immunology field of the time had no connection with high-throughput sequencing,” notes Dr. Robins, recalling his days as a computational biologist for the Fred Hutchinson Cancer Research Center. “It became clear that instead of using this old technology to look at T-cell receptors, we could just directly sequence them—if we could amplify them correctly.”

    With its first experiment, Dr. Robins’ team ended up with six million T-cell receptor sequences. “Our approach,” Dr. Robins modestly suggests, “kind of changed the scale of what we were able to do.” The team went on to develop advanced multiplex sequencing technology, doing work that essentially started the field of immune sequencing. “Previously,” maintains Dr. Robins, “no one had ever been able to quantitatively do a multiplex PCR.”

    Adaptive Biotechnologies’ product, the ImmunoSEQ® assay, uses several hundred primer pairs to quantify the clonal diversity of T cells. Using this technology, researchers and clinicians can focus on T-cell clones that are expanded specifically in or near a tumor or that are circulating in the blood stream.

    “You obviously can’t get a serial sample of the tumor,” explains Dr. Robins, “but you can get serial samples of blood,” allowing for immune cell repertoire tracking during the progression of a disease. The technology is already being used to assess leukemias in the clinic, directly tracking the leukemia itself based on the massive clonal expansion of a single cancerous B or T cell.

    Eventually, Dr. Robins’ team hopes to monitor serial changes in T cell clones before, during, and after therapeutic intervention. The team has even developed a tumor infiltrating lymphocyte (TIL) assay to examine clones that are attracted to tumors.

     

Circulating Tumor Cells

“Years ago, they were just interested in what was happening in the tumor,” says Daniel Adams, senior research scientist at Creatv MicroTech. “Now people have realized that the immune system is reacting to the tumor.”

Scientists such as Adams have been tracking tumor cells and tumor-modified stromal cells, as well as components of the non-adaptive immune system, directly within the bloodstream to examine changes that occur over time.

“We can’t go back in to re-biopsy the patient every year, or every time there is a recurrence,” says Adams, “It’s just not feasible.”

That is why Creatv MicroTech, with locations in Maryland and New Jersey, has developed the CellSieve, a mechanical cell filter. The CellSieve, which improves on older technology through better polymers and engineering, isolates circulating tumor cells (CTCs) and stromal cells in order to capture them for further clinical analysis.

Isolation, culture and expansion of cells isolated on CellSieve™. (A) MCF-7 cells spiked into vacutainers, isolated by filtration and cultured on CellSieve for 2-3 weeks. A 3 dimensional cluster attributed to this cell line is seen on the filter. (green=anti-cytokeratin, blue=DAPI) (B) PANC-1 cells spiked into vacutainers, isolated by filtration and grown on CellSieve for 2-3 weeks. PANC-1 is seen growing as a monolayer on the filter. (C) SKBR3 cells are spiked into blood, filtered by CellSieve. The CTCs are identified by presence of anti-cytokeratin and anti-EpCAM, and absence of anti-CD45. After CTCs are counted, cells are subtyped by HER2 FISH. (D) SKBR3 cells are spiked into vacutainers, isolated by filtration and grown on CellSieve for 2-3 weeks. Expanded colonies were directly analyzed as a whole colony and as individual cells, molecularly by HER2/CR17 FISH probes. (E) Circulating stromal cell, e.g. a 70 µm giant cancer associated macrophage can be identified for clinical use, myeloid marker in red. (F) A cell of interest can be identified and restained with immunotherapeutic biomarkers, e.g. PD-L1 (green) and PD-1 (purple). (G) After filtration, cells were identified with histopathological stains (e.g. H&E) for cytological analysis. (H) After H&E, external cell structures were analyzed by SEM. [Creatv MicroTech].

 

“As a patient goes through therapy, the patient’s resistance builds, and the cancer recurs in different subpopulations,” states Adams. “And after a few years, the original tumor mass is no longer applicable to what is growing in the patient farther down the road.”

Although CTCs are exceedingly rare in the bloodstream, with just one or two in every 5 to 10 mL of blood, and although these cells have a very low viability, the surviving CTCs have a high prognostic value.

“We looked at 30 to 40 breast cancer patients over two years,” reports Adams. “And we showed that if you have a dividing CTC, you have a 90% chance of dying in two years and a 100% chance of dying within two and half years.”

Furthermore, the immune system response can be tracked, says Adams, by examining stromal cells, which can also be collected with the CellSieve filtration device. That is, these cells can be collected serially. Much recent evidence supports the conclusion that stromal cells in the tumor environment co-evolve with the tumor, suggesting that stromal marker changes reflect tumor changes.

“There is this plethora of stromal cells and tumor cells out there in the circulation for you to look at,” declares Adams. “Once the cells are isolated, you can subject them to pathological approaches, biomarker approaches, or molecular approaches—or all of the above.”

A MicroTech Creatv study published in the Royal Society of Chemistry showed the efficacy of following up CTC isolation with techniques such as fluorescence in situ hybridization (FISH), histopathological analysis, and cell culture.

Cancer-Killing Assays

Diverse mechanisms are at play in cancer biology. Our understanding of these mechanisms contributes to a couple of virtuous cycles. It strengthens and is strengthened by diagnostic approaches, such as immune- and tumor-cell monitoring. The same could be said of therapeutic approaches. Cancer biology will inform and be informed by cancer immunotherapies such as adoptive cell transfer. To maintain the virtuous cycle, however, it will be necessary to conduct in vitro testing.

“There is no doubt that immunotherapy is going to play a major role in the treatment of cancer,” says Brandon Lamarche, Ph.D., technical communicator and scientist at ACEA Biosciences. “Regardless of what the route is, what is going to have to happen in terms of the research area is that you need an effective cell-killing assay.”

ACEA Biosciences, a San Diego-based company, has developed a microtiter plate that is coated with gold electrodes across 75% of the well bottoms. When the microtiter plates are placed in the company’s xCELLigence plate reader, the electrodes enable the detection of changes in cell morphology and viability through electrical impedance.

“The instrument provides a weak electric potential to the electrodes on the plate, so you get electrons flowing between these electrodes,” explains Dr. Lamarche. Researchers can then apply reagents or non-adherent immune cell suspensions to adherent cancer cells and examine the effect.

Dr. Lamarche asserts that the xCELLigence system overcomes problems that bedevil competing cell-killing assays. These problems include leaky and radioactive labels, such as chromium 51, and assays that can only provide users with an endpoint for cell killing. “With xCELLigence,” he insists, “you’re getting the full spectrum of what’s happening, and there’s all kinds of subtleties in the cell-killing curves that are very informative in terms of the biology.”

ACEA would like to see the xCELLigence system become the new standard in cell-killing assays from standard research to clinical testing on patient tumors. Dr. Lamarche envisions a day when patient tumor cells are quickly screened with therapeutic scenarios to determine the most efficacious killing option. “xCELLigence technology,” he suggests, “enables you to quickly sample a broad spectrum of conditions with a very simple workflow.”

Bioinformatics of Immuno-Oncology

From monitoring to treatment modalities, the field of cancer immunotherapy is aided by bioinformatics-minded data-mining experts, such as the analysts at Thompson-Reuters who are compiling data archives and applying advanced analytics to find new targets. “Essentially,” says Richard Harrison Ph.D., the company’s chief scientific officer for the life science division, “for every stage within pharmaceutical drug development, we have a database associated with that.”

The analysts at Thompson-Reuters curate and compile databases such as MedaCore and Cortellis, which they provide to their clients to help them with their research and clinical studies. “We can take customer data, and using our tools and our pathway maps, we can help them understand what their data is telling them,” explains Dr. Harrison.

Matt Wampole, Ph.D., a solutions scientist at Thompson-Reuters, spends his days reaching out and working with customers to help them understand and better use the company’s products. “Bench researchers,” he points out, “don’t necessarily know what is upstream of whatever expression change might be leading to a particular change in regulation.” Dr. Wampole indicates that he is part of a “solution team” that aids clients in determining important signaling cascades, regulators, and so on.

“We have a group of individuals who are very ‘skilling’ experts in the field,” Dr. Wampole continues, “including experts in the areas such as biostatistics, data curation, and data analytics. These experts help clients identify models, stratify patients, understand mechanisms, and look into disease mechanisms.”

Dr. Harrison sums up the Thompson-Reuters approach as follows: “We look for master regulators that can serve as both targets and biomarkers.” By examining the gene signatures from both the patient and from curated datasets, in the case of cancer immunotherapy, they hope to segregate patients according to what drugs will work best for them.

  • “We are working with a number of pharmaceutical companies to put our approach into practice for clinical trials,” informs Dr. Harrison. The approach has already been applied in several studies, including one that used data analysis of cell lines to help predict drug response in patients. Another study helped stratify glioblastoma patients.

  • Tumor-Targeted Delivery Platform

    PsiOxus Therapeutics, which is focused on immune therapeutics in oncology, has developed a patented platform for tumor-targeted delivery based on its oncolytic vaccine, Enadenotucirev (EnAd), which can be delivered systemically via intravenous administration.

    According to company officials, EnAd’s anti-cancer scope can be expanded by adding new genes, thereby enabling the creation of a broad range of unique immuno-oncology therapeutics. In a recent study conducted at the University of Oxford, researchers led by Philip G. Jakeman, Ph.D., sought to improve the models for evaluating cancer therapeutics by introducing ex vivo methodologies for research into colorectal cancer.

    The ex vivo approach utilized was able to exploit a major advantage by preserving the three-dimensional architecture of the tumor and its associated compartments, including immune cells. The study, which was presented at the International Summit on Oncolytic Viral Therapeutics in Quebec, showed the tissue slice model can provide a novel means to assessing an oncolytic vaccine in a system that more accurately recapitulates human tumors, provide a more stringent test for oncolytic viruses, such as EnAd, and allow study of the human immune cells within the tumor 3D context.

    By maintaining the components of the tumor immune microenvironment, this new methodology could become useful in analyzing anti-viral responses within tumors, or even in evaluating therapeutics that target immunosuppressive tumor micro-environments, noted the Oxford team.

     

 

Deciphering the Cancer Transcriptome

A Rogue’s Gallery of Malignant Outliers May Hide in Transcriptome Profiles That Emphasize Averages

http://www.genengnews.com/gen-articles/deciphering-the-cancer-transcriptome/5729/

 

The key link between genomic instability and cancer progression is transcriptome dynamics. The shifts in transcriptome dynamics that contribute to cancer evolution may come down to statistical outliers. [iStock/zmeel]

  • In recent years, scientists have adopted a gene-centric view of cancer, a tendency to see each malignant transformation as the consequence of alterations in a discrete number of genes or pathways. These alterations are, fortunately, absent from healthy cells, but they pervert malignant cells.

    The gene-centric view takes in molecular landscapes illuminated by genomic and transcriptomic technologies. For example, genomes can be cost-effectively sequenced within hours. Such capabilities have made it possible to interrogate associations between genotypes and phenotypes for increasing numbers of conditions, and to collect data from progressively larger patient groups.

    As genomic and transcriptomic technologies rise, they reveal much—but much remains hidden, too. Perhaps these technologies are less like the sun and more like the proverbial streetlight, the one that narrows our searches because we’re inclined to stay in the light, even though what we hope to find may lie in the shadows.

    “Each individual study that looks at the cancer transcriptome is impressive and tells a convincing story, but if we put several high-quality papers together, there are very few genes that overlap,” says Henry H. Heng, Ph.D., professor of molecular medicine, genetics, and pathology at Wayne State University. “This shows that something is wrong.”

  • Distinct Karyotypes

    One of the major observations in Dr. Heng’s lab is that the intra- and intertumor cellular heterogeneity results in nearly every cancer cell having a unique, distinct karyotype, that is, an important but often ignored genotype. “Biological systems need a lot of heterogeneity,” notes Dr. Heng. “People like to think that this is noise, but heterogeneity is a fundamental buffer system for biological function to be achievable. Moreover, it is the key agent for cellular adaptation.”

    To capture the degree of genomic heterogeneity at the genome level and its impact on cancer cell growth, Dr. Heng and colleagues performed serial dilutions to isolate single mouse ovarian surface epithelial cells that had undergone spontaneous transformation. Spectral karyotyping revealed that within a short timeframe each of these unstable cells exhibited a very distinct karyotype. In these unstable cells, cloning at the level of the karyotype was not possible.

    Stable cells exhibited a normal growth distribution, i.e., no subset of stable cells contributed disproportionately to the overall growth of the cell population. In contrast, unstable cell populations showed a non-normal growth distribution, with few cells contributing most to the cell population’s growth. For example, a single unstable colony contributed more than 70% to the cell population’s growth. This finding suggests that although average profiles can be used to describe non-transformed cells, they cannot be taken to represent the biology of malignant cells.

    “Most people who study the transcriptome want to get rid of the noise, but the noise is in fact the strategy that cancer uses to be successful,” explains Dr. Heng. “Each individual cancer cell is very weak but together the entity becomes very robust.”

    In a recent model that Dr. Heng and colleagues proposed, system inheritance visualizes chromosomes not merely as the vehicle for transmitting genetic information, but as the genetic network organizer that shapes the physical interactions between genes in the three-dimensional space. Based on this model, individual genes represent parts of the system. The same genes can be reorganized to form different systems, and chromosomal instability becomes more important than the contribution of individual genes and pathways to cancer biology.

    The vital link between genomic instability and cancer progression is transcriptome dynamics, and the shifts in those dynamics that contribute to cancer evolution may come down to statistical outliers.

    “Transcriptome studies rarely focus on single-cell analyses, which means important outliers are frequently ignored,” declares Dr. Heng. “This preoccupation with uninformative averages explains why we have learned so little despite having examined so many transcriptomes.”

  • Chimeras and Fusion Genes

    “Our focus is on chimeric RNA molecules,” says Laising Yen, Ph.D, assistant professor of pathology at Baylor College of Medicine. “This category of RNAs is very special because their sequences come from different genes.”

    In a study that was designed to capture chimeric RNAs in prostate cancer, Dr. Yen and his colleagues performed high-throughput sequencing of the transcriptomes from human prostate cancer samples. “We found far more chimeric RNAs, in terms of abundance, and a number of species that are not seen in normal tissue,” reports Dr. Yen. This approach identified over 2,300 different chimeric RNA species. Some of these chimeras were present in prostate cancer cell lines, but not in primary human prostate epithelium cells, which points toward their relevance in cancer.

    “Most of these chimeric RNAs do not have a genomic counterpart, which means that they could be produced by trans-splicing,” explains Dr. Yen. During trans-splicing, individual RNAs are generated and trans-spliced together as a single RNA, which provides a mechanism for generating a chimera.

    “The other possibility is that in cancer cells, where gene–gene boundaries are known to become broken, chimeras can be formed by cis-splicing from a very long transcript that encodes several neighboring genes located on the same chromosome,” informs Dr. Yen. Chimeric RNAs formed by either of these two mechanisms can potentially translate into fusion proteins, and these aberrant proteins may have oncogenic consequences.

    Another effort in Dr. Yen’s laboratory focuses on chromosomal aberrations in ovarian cancer. One of the hallmarks of ovarian cancer is the high degree of genomic rearrangement and the increased genomic instability.

    “When we looked at ovarian cancers, we did not find as many chimeric RNAs,” notes Dr. Yen. “But we found many fusion genes.” Gene fusions, similarly to chimeric RNAs, increase the diversity of the cellular proteome, which could be used selectively by cancer cells to increase their rates of proliferation, survival, and migration.

    A recent study in Dr. Yen’s lab identified BCAM-AKT2, a recurrent fusion gene that is specific and unique to high-grade serous ovarian cancer. BCAM-AKT2 is the only fusion gene in this malignancy that was proved to be translated into a fusion kinase in patients, which points toward its functional significance and potential therapeutic value.

    “Recurrent fusion genes, which are repeatedly found in many patients in precisely made forms, indicate that there is a reason that they are present,” concludes Dr. Yen. “This might have important therapeutic implications.”

  • Context-Specific Patterns

    “We contributed to a study of tumor gene expresssion that we are currently revisiting because so much more data has become available,” says Barbara Stranger, Ph.D., assistant professor, Institute for Genomics and Systems Biology, University of Chicago. “The data is being processed in homogenized analytic pipelines, and we can look at many more tumor types across the Cancer Genome Atlas than a few years ago.”

    Previously, Dr. Stranger and colleagues performed expression quantitative trait loci (eQTL) analyses to examine mRNA and miRNA expression in breast, colon, kidney, lung, and prostate cancer samples. This approach identified 149 known cancer risk loci, 42 of which were significantly associated with expression of at least one transcript.

    Causal alleles are being prioritized using a fine-mapping strategy that integrated the eQTL analysis with genome-wide DNAseI hypersensitivity profiles obtained from ENCODE data. These analyses are focusing on capturing differences across tumors and on performing comparisons with normal tissue, and one of the challenges is the lack of normal tissue from the same patients.

    “But still there is a lot of power in these analyses because they are based on large-scale genomic datasets. Also, these tumor datasets can be compared with large-scale normal tissue genomics datasets, such as the NIH’s Genotype-Tissue Expression (GTEx) project,” clarifies Dr. Stranger. “This helps us characterize differences between those tumors and normal tissue in terms of the genetics of gene regulation.”

    An ongoing effort in Dr. Stranger’s laboratory involves elucidating how the effect of genetic polymorphisms is shaped by context. Stimulated cellular states, cell-type differences, cellular senescence, and disease are some of the contexts that are known to impact genetic polymorphisms.

    “We have seen a lot of context specificity,” states Dr. Stranger. “Our observations suggest that a genetic polymorphism can have a specific effect in regulating a particular gene or transcript in one context, and another effect in another context.”

    Another example of cellular context is sex, and an active area of investigation in Dr. Stranger’s lab proposes to dissect the manner in which sex differences shape the regulatory effects of genetic polymorphisms.

    “Thinking about sex-specific differences is not very different from thinking about a different cellular environment,” notes Dr. Stranger.

    The expression of specific transcription factors can be determined by sex; consequently, a polymorphism that interacts with a transcription factor may have functional outcomes that can be seen in only one of the sexes.

    “There are gene-level and gene-splicing differences that we see in normal tissues between males and females, and we want to take the same approach and look at the cancer context to see whether the genetic regulation of gene expression and transcript splicing is different between individuals and whether it has a sex bias,” concludes Dr. Stranger. “Finally, we want to see how that differs in cancer relative to normal tissues.”

    Early Clinical Impact

An increasing number of clinicians are adding the cancer transcriptome to their precision medicine program. They have found that the transcriptome is important in identifying clinically impactful results. [iStock/DeoSum]

“Over the last two years,” says Andrew Kung, M.D., Ph.D., chief of the Division of Pediatric Hematology, Oncology, and Stem Cell Transplantation at Columbia University Medical Center, “we have included the cancer transcriptome as part of our precision medicine program.” Dr. Kung and colleagues developed a clinical genomics test that includes whole-exome sequencing of tumors and normal tissue and RNA-seq of the tumor.

“Our results show that the transcriptome is very important in identifying clinically impactful results,” asserts Dr. Kung. “The technology has really moved from a research tool to real clinical application.” In fact, the test has been approved by New York State for use in cancer patients.

The data from transcriptome profiling has enabled identification of translocations, verification of somatic alterations, and assessment of expression levels of cancer genes.  Dr. Kung and his colleagues are using genomic information for initial diagnosis and prognostic decisions, as well as the investigation of potentially actionable alterations and the monitoring of disease response.

To gain insight into gene-expression changes, transcriptome analysis usually compares two different types of tissues or cells. For example, analyses may attempt to identify differentially expressed genes in cancer cells and normal cells.

“In patients with cancer, we usually do not have access to the normal cell of origin, making it harder to identify the genes that are over- or under-expressed,” explains Dr. Kung. “Fortunately, the vast amounts of existing gene-expression data allow us to identify genes whose expression are most changed relative to models built on the expression data aggregated across large existing datasets.”

These genomic technologies were first used to augment the care of pediatric patients at Columbia. The technologies were so successful that they attracted philanthropic funding, which is being used to expand access to genomic testing to all children with high-risk cancer across New York City.

Other related articles published in this Open Access Online Scientific Journal include the following:

Immunotherapy in Combination, 2016 MassBio Annual Meeting  03/31/2016 8:00 AM – 04/01/2016 3:00 PM Royal Sonesta Hotel, Cambridge, MA

Live Press Coverage: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2016/04/01/plenary-session-immunotherapy-in-combination-2016-massbio-annual-meeting-03312016-800-am-04012016-300-pm-royal-sonesta-hotel-cambridge-ma/

 

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How do we address medical diagnostic errors?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

It is my observation over the course of 50 years in medicine that there is no simple resolution to a frequently identified problem, which has grown more serious with the implementation of hospital-wide and system-wide health information technology.  There has been reduction of hospital staff, nurses and physicians, with an increased patient load even while the technology has advanced significantly.  Over this investment in technology and manpower the hospital staff time has become dependent on the medical record and a large portion of their time is drawn away from patient care.  This is not a substantial improvement in the last decade.  The problem has been a lack of focus on the correct design of these systems.  They should be interactive and they should have incorporated anticipatory logic.  Instead, they are not that much more than large electronic filing cabinets. There are large sources of information in laboratory, radiology and pharmacy and are siloed.  When one drives a car, one does not need to look under the hood, a sentiment that has been expressed by a real expert.

 

Diagnostic Errors Get the Attention of the Institute of Medicine, Reinforcing Efforts by Nation’s Clinical Pathology Laboratory Scientists to Improve Patient Safety

March 25, 2016         DARK DAILY info@darkreport.com via cmail2.com

 

Along with its assessment of the rate of errors in diagnosis, the IOM has a plan to improve, but will doctors accept the IOM’s advice, or continue business as usual?

Diagnostic errors in the American healthcare system is a problem that is now on the radar screen of policymakers at the Institute of Medicine (IOM). Pathologists andclinical laboratory professionals will welcome this development, because recommendations from the IOM carry weight with Congress.

Thus, should the IOM develop specific actions items intended to reduce medical errors, not only are these suggestions likely to involve more effective use of medical laboratory tests by physicians, but there is a strong probability that Congress might eventually write these recommendations into future healthcare legislation.

The Institute of Medicine is a division of the National Academies of Sciences, Engineering, and Medicine. The IOM recently convened a committee that released a list of recommendations to address the problem of diagnostic errors in medicine. Those recommendations, however, are running up against ingrained mindsets and overconfidence on the part of physicians who are reluctant to include decision-support technology in the diagnostic process.

Misdiagnoses in healthcare has led to tragic consequences. Over the years, the IOM has worked to increase the public’s awareness of the problem of misdiagnosis. It has also encouraged health information technology (HIT) developers to provide clinicians with the decision-support systems they need to prevent misdiagnosis from happening in the first place. But getting physicians to use the new tools has become a challenge.

IOM Dedicated to Improving Diagnostic Accuracy 

Preventing diagnostic errors has been a primary target in medical reform for many years. A 1999 report by the U.S Institute of Medicine (IOM) titled: “To Err Is Human: Building a Safer Health System” first drew attention to the rate of preventable medical errors in the U.S.

Since then, much has been written about the problem of diagnostic inaccuracy. Although many provider organizations are working to improve it, progress has been slow.

Click here to see image

Edward Hoffer, MD (above), FACP, FACC, FACMI, is a faculty member at the Massachusetts General Hospital Laboratory of Computer Science. He told Modern Healthcare that getting doctors to use diagnostic software is a hard sell. “The main problem we face is trying to convince physicians that they actually need to use it,” he concluded. (Image copyright: Massachusetts General Hospital, Laboratory of Computer Science.)

Solutions for Preventing Errors Face Many Challenges

From the first evaluation of the patient, to the clinical laboratory, to physician follow-up; errors can occur throughout the chain of care. And a culture that discourages reporting of medical errors can make it very difficult for healthcare organizations to resolve these issues.

For example, an investigation by the Milwaukee Journal Sentinel uncovered what they say is a “secretive system [that] hides [medical] lab errors from the public and puts patients at risk.”

To make matters worse, according to the Journal Sentinel article, federal agencies that are tasked with oversight of the more than 35,000 clinical laboratories in the U.S. (such as the Joint Commission) are overloaded and thus may be missing many violations that would lead to sanctions or outright loss of accreditation.

The Milwaukee Journal Sentinel stated that, “even when serious violations are identified, offending labs are rarely sanctioned except in the most extreme cases.” And that “in 2013, just 90 sanctions were issued—accounting for not even 1% of the 35,000 labs that do high-level lab testing in the United States.”

Additionally, there are different types of misdiagnoses, which also complicates matters. A study by three professors at the Dartmouth Center for Healthcare Deliver Science investigated the cost of what they termed “silent misdiagnoses.”

According to a Dartmouth Now article outlining the study, the disparity between “the treatments patients want and what doctors think they want” accounts for a great number of misdiagnoses. The study’s authors call these misdiagnoses “silent” because they are rarely reported or recorded.

Click here to see image

Albert G. Mulley, Jr., MD, MPP (above), is Managing Director, Global Health Care Delivery Science, The Dartmouth Institute for Health Policy and Clinical Practice; Professor of Medicine, Geisel School of Medicine at Dartmouth; and the lead author of the Dartmouth study. Mulley says that listening to the patient is more important than ever. “Today, the rise in treatment options makes this even more critical, not only to reach a correct medical diagnosis but also to understand fully the patients’ preferences, and reduce the huge waste in time and money that comes from delivery of services that patients often neither want nor need.” (Photo copyright: Dartmouth Now.)

IOM Committee on Diagnostic Error in Healthcare Releases Recommendations

In September, 2015, the IOM released its latest report in the series of reports about medical errors and diagnostic problems that goes back 17 years. The series started in 1999 with “To Err is Human: Building A Safer Health System” in 1999. This was followed in 2001 by “Crossing the Quality Chasm: A New Health System for the 21st Century.”

This newest report is titled, “Improving Diagnosis in HealthCare.” It comes from the IOM’s National Academies of Medicine’s Committee on Diagnostic Error in Healthcare. The study lists recommendations that the IOM says will help improve the rate of diagnostic errors. Included are:

• Facilitate more effective teamwork in the diagnostic process;

• Enhance education in the healthcare community on the topic of improving diagnostics;

• Incorporate diagnostic support into electronic health records (EHRs), and make EHRs fully interoperable;

• Set up systems to identify, learn from, and reduce diagnostic errors;

• Establish a culture that encourages investigation of mistakes;

• Develop a collaborative reporting system that involves clinicians, government agencies, and liability insurers that allows everyone to learn from diagnostic errors and near misses;

• Design a payment system that supports correct diagnosis; and

• Provide funding for research into how to improve the diagnostic process.

Click here to see video

This video (above) is of the IOM’s public release in September, 2015, of the Committee on Diagnostic Error in Healthcare’s report, “Improving Diagnosis in Health Care.” A full text of the report can be downloaded by clicking here, or by visiting http://iom.nationalacademies.org/reports/2015/improving-diagnosis-in-healthcare.

Technology Exists to Support Accurate Diagnoses

One area of development that offers real hope for lowering the rate of misdiagnosis is medical decision-support software—specifically in clinical informatics, where the integration of health data with health information technology (HIT) supports physician decision making at the point of care.

Whether that software is part of an organization’s EHR system, or is a standalone program dedicated to reducing diagnostic errors, can make a difference. For example, a research study published in the Journal of Clinical Bioinformatics) suggests that health information technology has an important role to play in improving diagnostic accuracy.

The challenge is that, although the technology currently exists, diagnostic error rates remain high. A research study published in the Journal of the American Medical Association Internal Medicine (JAMA) in 2013 may explain why. It states that overconfidence could prevent some physicians from re-evaluating questionable diagnoses.

In a Modern Healthcare article, Edward Hoffer, MD, FACP, FACC, FACMI, a faculty member at the Massachusetts General Hospital Laboratory of Computer Science, said that getting doctors to use diagnostic software is a hard sell. “The main problem we face is trying to convince physicians that they actually need to use it,” he concluded.

All of the building blocks for improving the diagnostic process are either in place, or as in the case of EHR interoperability, in the works. Clinical laboratory personnel are uniquely positioned to assist physicians in improving through communication and the careful use of technology.

—Dava Stewart

Related Information:

To Err Is Human

Weak Oversight Allows Lab Failures to Put Patients at Risk  

Dartmouth Study: ‘Silent’ Misdiagnoses by Doctors Are Common, and Come at Great Cost 

Press Release: Doctors ‘Silent’ Misdiagnoses Cost Patients Dearly . . . And US Health Care Billions

Improving Diagnosis in Health Care

Clinical Decision Support Systems for Improving Diagnostic Accuracy and Achieving Precision Medicine 

Using Software To Avoid Misdiagnoses 

In Conversation with…Mark L. Graber, MD 

 

All of the building blocks for improving the diagnostic process are either in place, or as in the case of EHR interoperability, in the works.   Nothing could be further from reality than what has been stated.   There is no minimization of keystrokes.  There is no anticipatory logic.  There is no automated comparison of medication and diagnoses electronically.  There is no check to determine the consistency of the laboratory results with a probability of diagnosis, or automated identification of tests that would clarify the situation.

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North Star Imaging and Instron is in Southern California for Helping the Industry, Academia, and Research

Reporter: Danut Dragoi, PhD

Today I visited North Star Imaging and Instron companies for their Open House in Irvine, California. I believe this is the first in California. Knowing some of their products very well I realized, after professional presentations and live demos that these machines can help Californian industry, Department of Homeland Security, as well as the research in many aspects of it, from nondestructive evaluation of key industrial products, medical devices inspections, aeronautic composites, as well as new applications in actual research such as Laser Metal Printing, Mechanical Engineering, bio-implants, etc. I found very interesting the presentation on imaging a 3D Printed object with an internal complex topology. For the quality of their pictures, cross sections and resolution I congratulate the team that put these breakthroughs together. The picture below is an X5000 x-ray machine capable  of producing high quality computed tomography pictures (CT) of various objects from many domains of industry and research.

NSI-X5000CT

Image SOURCE: http://4nsi.com/events/open-house

The famous STL files for 3D printing are produced here in a X5000 machine. A definition of STL file is given here. The STL (STereoLithography) is a file format native to the stereolithography CAD software created by 3D Systems. STL has several after-the-fact backronyms such as “Standard Triangle Language” and “Standard Tessellation Language”. This file format is supported by many other software packages; it is widely used for rapid prototyping, 3D printing and computer-aided manufacturing. STL files describe only the surface geometry of a three-dimensional object without any representation of color, texture or other common CAD model attributes. The STL format specifies both ASCII and binary representations. Binary files are more common, since they are more compact. An STL file describes a raw unstructured triangulated surface by the unit normal and vertices (ordered by the right-hand rule) of the triangles using a three-dimensional Cartesian coordinate system. STL coordinates must be positive numbers, there is no scale information, and the units are arbitrary. Once the 3D object is scanned, the STL file can be downloaded to the 3D printer machine and a copy of the original can be produced. For people working on Medical 3D Printing this machine is extremely useful.

Before a product is tested with Instron, the 3D pictures are important to be produced. In this way a complete analysis can be done in a very convenient time-machine and time-operator way. The next Figure below shows an Instron machine taken from here 

Instron

Image SOURCE: http://www.renishaw.com/en/instron-equips-its-new-electropuls-linear-torsion-tester-with-advanced-renishaw-encoders–29004

According with the information released, link in here, Instron, headquartered in Massachusetts, USA, is a global market leader in the materials testing industry. It manufactures and services a comprehensive range of materials testing equipment and accessories for the research, industrial and academic sectors. A variety of Instron systems test samples ranging from components for jet engines to medical syringes.

Instron has just launched an advanced bi-axial variant of the ElectroPuls E3000 All-Electric test instrument. The E3000 is a compact table-top instrument comprising: a load frame, crosshead with combined linear/torsion actuator, Dynacell load cell and T-slot table for fixing samples.

The state-of-the-art ElectroPuls series includes the E1000, E3000 and E10000 fatigue test systems. These are suited for biomedical / biomechanical research applications and feature a wide dynamic performance range and low force characteristics. ElectroPuls is all-electric and utilises linear motor technology, which eliminates the need for ball / lead-screws and enables slow-speed static tests through to high-frequency dynamic tests at over 100 Hz.

The new E3000 linear-torsion is a smaller-scale equivalent of the E10000 linear-torsion system and includes a rotation axis with a standard range of ±135° as well as optional multi-turn capability for applications such as orthopaedic bone-screw testing. An ElectroPuls bi-axial linear-torsion test can be conducted on most materials and has found applications in testing inter-vertebral disc prostheses, various bio-materials, athletic footwear and elastomeric components.

The Open House of the two companies, NSI and Instron, was very well organized and it was a great success for Californian industry, Academia, Medical testing, and Research.

Source

http://4nsi.com/events/open-house

http://www.renishaw.com/en/instron-equips-its-new-electropuls-linear-torsion-tester-with-advanced-renishaw-encoders–29004

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brain implants without wires

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Powering brain implants without wires with thin-film wireless power transmission system

Avoids risk of infections through skull opening and leakage of cerebrospinal fluid, and allows for free-moving subjects and more flexible uses of brain-computer interfaces
February 8, 2016

http://www.kurzweilai.net/powering-brain-implants-without-wires-with-thin-film-wireless-power-transmission-system

 

Schematic of proposed architecture of an implantable wireless-powered neural interface system that can provide power to implanted devices. Adding a transmitter chip could allow for neural signals to be transmitted via the antenna for external processing. (credit: Toyohashi University Of Technology)

 

A research team at Toyohashi University of Technology in Japan has fabricated an implanted wireless power transmission (WPT) device to deliver power to an implanted neural interface system, such as a brain-computer interface (BCI) device.

Described in an open-access paper in Sensors journal, the system avoids having to connect an implanted device to an external power source via wires through a hole in the skull, which can cause infections through the opening and risk of infection and leakage of the cerebrospinal fluid during long-term measurement. The system also allows for free-moving subjects, allowing for more natural behavior in experiments.

 

Photographs of fabricated flexible antenna and bonded CMOS rectifier chip with RF transformer (credit: Kenji Okabe et al./Sensors)

 

The researchers used a wafer-level packaging technique to integrate a silicon large-scale integration (LSI) chip in a thin (5 micrometers), flexible parylene film, using flip-chip (face-down) bonding to the film. The system includes a thin-film antenna and a rectifier to convert a radio-frequency signal to DC voltage (similar to how an RFID chip works). The entire system measures 27 mm × 5 mm, and the flexible film can conform to the surface of the brain.

 

http://www.kurzweilai.net/images/Warwick-turns-on-light.jpg

Coventry University prof. Kevin Warwick turns on a light with a double-click of his finger, which triggers an implant in his arm (wired to a computer connected to the light). Adding an RF transmitter chip (and associated processing) to the Toyohashi system could similarly allow for controlling devices, but without wires. (credit: Kevin Warwick/element14)

 

The researchers plan to integrate additional functions, including amplifiers, analog-to-digital converters, signal processors, and  a radio frequency circuit for transmitting (and receiving) data.

Tethered Braingate brain-computer interface for paralyzed patients (credit: Brown University)

 

Such a system could perform some of the functions of the Braingate system, which allows paralyzed patients to communicate (see “People with paralysis control robotic arms using brain-computer interface“).

This work is partially supported by Grants-in-Aid for Scientific Research, Young Scientists, and the Japan Society for the Promotion of Science.

https://youtu.be/LW6tcuBJ6-w

element14 | Kevin Warwick’s BrainGate Implant

 

Abstract of Co-Design Method and Wafer-Level Packaging Technique of Thin-Film Flexible Antenna and Silicon CMOS Rectifier Chips for Wireless-Powered Neural Interface Systems

In this paper, a co-design method and a wafer-level packaging technique of a flexible antenna and a CMOS rectifier chip for use in a small-sized implantable system on the brain surface are proposed. The proposed co-design method optimizes the system architecture, and can help avoid the use of external matching components, resulting in the realization of a small-size system. In addition, the technique employed to assemble a silicon large-scale integration (LSI) chip on the very thin parylene film (5 μm) enables the integration of the rectifier circuits and the flexible antenna (rectenna). In the demonstration of wireless power transmission (WPT), the fabricated flexible rectenna achieved a maximum efficiency of 0.497% with a distance of 3 cm between antennas. In addition, WPT with radio waves allows a misalignment of 185% against antenna size, implying that the misalignment has a less effect on the WPT characteristics compared with electromagnetic induction.

 

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Controlling CAR-T cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New discovery – Remote control of CAR-T cells

T cells, Cancer immune therapy, autoimmune

CAR-T cells have been emerging as an effective approach to treat cancer and autoimmune diseases. A problem with CAR-T cells is that once they are infused, they are on their own exerting autonomous activities, which can lead to severe side effects due to the extensive lysis of tumor cells. Researchers have been seeking ways to control CAR-T cells after they are infused to balance the desired therapeutic effect and the side effect. Recently, a group of researchers at UCSF found a way to control CAR-T cells after they are put into patients, through a rapamycine analogue gated chimeric receptor.

CAR-T cell system has two components: one is the recognition domain that binds to CD19 to target B cells; the other is the functional domain to activate cellular pathways to killing the targeted cells. Those two domains are typically preassembled. What this group of researchers did is to separate those two domains and make them come together only in the presence of the activating molecule, a rapamycine analogue. They showed that in the absence of the activating molecule, CAR-T cells still bound to CD19. But, they didn’t kill the targeted cells unless the activating molecule was present. In addition, by adjusting the dose of the activating molecule, the strength of CAR-T cells activities can be titrated as well.

Chia-Yung Wu, etc. (October 2015) Remote control of therapeutic T cells through a small molecule–gated chimeric receptor.Science

 

Remote control of therapeutic T cells through a small molecule–gated chimeric receptor

 

 

https://pharmaceuticalintelligence.com/2016/02/06/reengineering-therapeutics/

Reengineering Therapeutics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

The synNotch solution: UCSF scientists engineer a next-gen T-cell immunotherapy

Sunday, January 31, 2016 | By John Carroll

CAR-T has been all the rage in cancer R&D for several years now as a slate of biotech upstarts pursue highly promising work reengineering T cells into attack weapons by adding a chimeric antigen receptor that can zero in on particular cancer cells. The approach has been highly effective in acute lymphoblastic leukemia, triggering an attack on B cells by homing in on the CD19 antigen, a breakthrough that has inspired a race to the regulatory finish line with the first CAR-Ts.

 

https://pharmaceuticalintelligence.com/2016/02/11/regulatory-dna-engineered/

Regulatory DNA engineered

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New Type of CRISPR Screen Probes the Regulatory Genome

Aaron Krol    http://www.bio-itworld.com/2016/2/8/new-type-crispr-screen-probes-regulatory-genome.html

February 8, 2016 | When a geneticist stares down the 3 billion DNA base pairs of the human genome, searching for a clue to what’s gone awry in a single patient, it helps to narrow the field. One of the most popular places to look is the exome, the tiny fraction of our DNA―less than 2%―that actually codes for proteins. For patients with rare genetic diseases, which might be fully explained by one key mutation, many studies sequence the whole exome and leave all the noncoding DNA out. Similarly, personalized cancer tests, which can help bring to light unexpected treatment options, often sequence the tumor exome, or a smaller panel of protein-coding genes.

 

sjwilliamspa commented on Controlling CAR-T cells

Controlling CAR-T cells Larry H. Bernstein, MD, FCAP, Curator LPBI New discovery – Remote control of CAR-T cells CAR-T …

Interesting method to use a chimeric heterodimer receptor to control CD19 activity however it would be ineresting to see if cancer replapses occur more frequently. Originally it was thought the CART would act, after initial treatment, eventually to patrol the body for any recurring tumor cells. Using rapamycin would be interesting although there had been some immunotoxic concerns with chronic use (although long term use of rapamycin and other mtor inhibitors seemed to prolong lifespan in immunodeficient animals)

Temsirolimus, an Inhibitor of Mammalian Target of Rapamycin athttp://clincancerres.aacrjournals.org/content/14/5/1286.short

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p53 tumor drug resistance mechanism target

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

Biologists unravel drug-resistance mechanism in tumor cells

Targeting the RNA-binding protein that promotes resistance could lead to better cancer therapies.

 

P53, which helps healthy cells prevent genetic mutations, is missing from about half of all tumors. Researchers have found that a backup system takes over when p53 is disabled and encourages cancer cells to continue dividing. In the background of this illustration are crystal structures of p53 DNA-binding domains.

http://news.mit.edu/sites/mit.edu.newsoffice/files/styles/news_article_image_top_slideshow/public/images/2015/MIT-Cancer-Drug-Resistance_0.jpg

 

P53, which helps healthy cells prevent genetic mutations, is missing from about half of all tumors. Researchers have found that a backup system takes over when p53 is disabled and encourages cancer cells to continue dividing. In the background of this illustration are crystal structures of p53 DNA-binding domains.

Image: Jose-Luis Olivares/MIT (p53 illustration by Richard Wheeler/Wikimedia Commons)

About half of all tumors are missing a gene called p53, which helps healthy cells prevent genetic mutations. Many of these tumors develop resistance to chemotherapy drugs that kill cells by damaging their DNA.

MIT cancer biologists have now discovered how this happens: A backup system that takes over when p53 is disabled encourages cancer cells to continue dividing even when they have suffered extensive DNA damage. The researchers also discovered that an RNA-binding protein called hnRNPA0 is a key player in this pathway.

“I would argue that this particular RNA-binding protein is really what makes tumor cells resistant to being killed by chemotherapy when p53 is not around,” says Michael Yaffe, the David H. Koch Professor in Science, a member of the Koch Institute for Integrative Cancer Research, and the senior author of the study, which appears in the Oct. 22 issue of Cancer Cell.

The findings suggest that shutting off this backup system could make p53-deficient tumors much more susceptible to chemotherapy. It may also be possible to predict which patients are most likely to benefit from chemotherapy and which will not, by measuring how active this system is in patients’ tumors.

Rewired for resistance

In healthy cells, p53 oversees the cell division process, halting division if necessary to repair damaged DNA. If the damage is too great, p53 induces the cell to undergo programmed cell death.

In many cancer cells, if p53 is lost, cells undergo a rewiring process in which a backup system, known as the MK2 pathway, takes over part of p53’s function. The MK2 pathway allows cells to repair DNA damage and continue dividing, but does not force cells to undergo cell suicide if the damage is too great. This allows cancer cells to continue growing unchecked after chemotherapy treatment.

“It only rescues the bad parts of p53’s function, but it doesn’t rescue the part of p53’s function that you would want, which is killing the tumor cells,” says Yaffe, who first discovered this backup system in 2013.

In the new study, the researchers delved further into the pathway and found that the MK2 protein exerts control by activating the hnRNPA0 RNA-binding protein.

RNA-binding proteins are proteins that bind to RNA and help control many aspects of gene expression. For example, some RNA-binding proteins bind to messenger RNA (mRNA), which carries genetic information copied from DNA. This binding stabilizes the mRNA and helps it stick around longer so the protein it codes for will be produced in larger quantities.

“RNA-binding proteins, as a class, are becoming more appreciated as something that’s important for response to cancer therapy. But the mechanistic details of how those function at the molecular level are not known at all, apart from this one,” says Ian Cannell, a research scientist at the Koch Institute and the lead author of the Cancer Cell paper.

In this paper, Cannell found that hnRNPA0 takes charge at two different checkpoints in the cell division process. In healthy cells, these checkpoints allow the cell to pause to repair genetic abnormalities that may have been introduced during the copying of chromosomes.

One of these checkpoints, known as G2/M, is controlled by a protein called Gadd45, which is normally activated by p53. In lung cancer cells without p53, hnRNPA0 stabilizes mRNA coding for Gadd45. At another checkpoint called G1/S, p53 normally turns on a protein called p21. When p53 is missing, hnRNPA0 stabilizes mRNA for a protein called p27, a backup to p21. Together, Gadd45 and p27 help cancer cells to pause the cell cycle and repair DNA so they can continue dividing.

Personalized medicine

The researchers also found that measuring the levels of mRNA for Gadd45 and p27 could help predict patients’ response to chemotherapy. In a clinical trial of patients with stage 2 lung tumors, they found that patients who responded best had low levels of both of those mRNAs. Those with high levels did not benefit from chemotherapy.

“You could measure the RNAs that this pathway controls, in patient samples, and use that as a surrogate for the presence or absence of this pathway,” Yaffe says. “In this trial, it was very good at predicting which patients responded to chemotherapy and which patients didn’t.”

“The most exciting thing about this study is that it not only fills in gaps in our understanding of how p53-deficient lung cancer cells become resistant to chemotherapy, it also identifies actionable events to target and could help us to identify which patients will respond best to cisplatin, which is a very toxic and harsh drug,” says Daniel Durocher, a senior investigator at the Samuel Lunenfeld Research Institute of Mount Sinai Hospital in Toronto, who was not part of the research team.

The MK2 pathway could also be a good target for new drugs that could make tumors more susceptible to DNA-damaging chemotherapy drugs. Yaffe’s lab is now testing potential drugs in mice, including nanoparticle-based sponges that would soak up all of the RNA binding protein so it could no longer promote cell survival.

This work was supported in part by the Charles and Marjorie Holloway Foundation.

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