Genomic testing of biopsies from patients with deadly, treatment-resistant cancerous blood syndromes called histiocytoses allowed doctors to identify genes fuelling the ailments and use targeted molecular drugs to successfully treat them.

Researchers from the Cincinnati Children’s Cancer and Blood Diseases Institute have recently report their data.  They recommend the regular use of comprehensive genomic profiling at diagnosis to positively impact clinical care, as well as rigorous clinical trials to verify and extend the diagnostic and treatment conclusions in their study.

Histiocytoses are a group of disorders in which abnormal accumulations of white blood cells form tumours on vital organs, leading to systemic organ damage or death. About half of the patients can be treated successfully with chemotherapy, but others are treatment resistant.

Study authors conducted genomic profiling of biopsies from 72 child and adult patients with a variety of treatment-resistant histiocytoses, including the most common one in children, Langerhans cell histiocytosis (LCH), according to the lead investigator, Ashish Kumar, MD, PhD.

Twenty-six patients with treatment-resistant disease had gene mutations involving either BRAF or MAP2K1 that directly activate the MAP-kinase cancer pathway. Researchers determined such patients would benefit from the targeted molecular therapies dabrafenib or trametinib, which block the MAP kinase pathway. The approved cancer drugs were prescribed off label to the histiocytosis patients.

‘In the last year, three patients we treated were infants with disease that was resistant to several rounds of intense chemotherapy. In the past, these children either would have suffered serious complications including death or would have had to endure more intensive treatments and the ensuing toxicities, including the risk of death,” Kumar said. “All three are thriving now on one oral medication that put their disease into remission.”

In one case a 22-month-old child was referred to Cincinnati Children’s for treatment-resistant LCH that was complicated by a secondary diagnosis of HLH (hemophagocytic lymphohistiocytosis). HLH is a difficult-to-treat and often-fatal autoimmune disorder in which an overheated immune system causes uncontrolled inflammation and organ damage. The little girl, whose condition was worsening with organ failure, had a mutation in the BRAF gene. 

Two days after starting targeted treatment with oral dabrafenib (which blocks the MAP-kinase pathway) the little girl’s fever disappeared and a week later her organ function returned to normal, according to study authors.

Previous studies, future directions
For their JCI Insight research project, in addition to their own laboratory tests, study authors drew from data in previous research papers by a number of institutions, which examined genetic and molecular processes affecting white blood cell expansion in different types of histiocytosis.

As Kumar and his colleagues continue their research, they plan to test methodologies that could expand the use of genomic profiling of patient biopsies and targeted molecular therapies in more patients with recurrent, treatment-resistant disease.

Cincinnati Children’s www.cincinnatichildrens.org/news/release/2017/treating-deadly-disorders

Massachusetts General Hospital (MGH) researchers have identified a mechanism that controls the expression of genes regulating the growth of the most aggressive form of medulloblastoma, the most common paediatric brain tumour. The team also identifies potential targets for future treatments.

“We set out to find the most important regulators of gene expression programs in medulloblastoma,” says senior author Miguel Rivera, MD, of the MGH Department of Pathology and the Center for Cancer Research. “To do that we used a powerful genomic technology called chromatin profiling to map all the genomic elements contributing to transcription regulation in Group 3 medulloblastoma – the most aggressive subtype. This goes beyond measuring gene expression because it tells you how genes are turned on and off.”

Medulloblastoma is a fast-growing tumour that arises in the developing brain and most commonly affects children under the age of 10. Four molecular variants, each with different patterns of DNA alteration and gene expression, have been identified. Subtypes WNT and SSH are the best understood; the other two – Group 3 and Group 4 – are poorly understood and account for 60 percent of tumours.

Cells regulate whether specific genes are transcribed into RNA through the action of transcription factors, proteins that bind to DNA and either stimulate or suppress the expression of their target genes. Rivera’s team used advanced genomic technologies to identify key DNA elements called enhancers that were active in primary Group 3 medulloblastoma samples and cell lines. The transcription factor OTX2, which plays a role in normal brain development and is known to be highly expressed in Group 3 medulloblastomas, was present at the majority of active enhancer sites in tumours, suggesting it may have a role in promoting the expression of tumour-associated genes.

Subsequent experiments revealed that OTX2 can function as a “pioneer factor,” opening up chromatin – which consists of DNA wound around proteins called histones – to activate enhancers and that its function is amplified by a second transcription factor called NEUROD1. The investigators then identified a set of genes the expression of which was significantly reduced when OTX2 was suppressed. Among these genes, they found that expression of the kinase NEK2 responded to OTX2 levels and that its depletion or pharmacologic inhibition strongly reduced the growth and survival of medulloblastoma cells.

“Overall, our findings show that OTX2 is a critical factor in regulating gene expression programs in Group 3 medulloblastoma and possibly in the WNT and Group 4 subtypes, where it is also expressed,” says Rivera, who is an assistant professor of Pathology at Harvard Medical School. “This work points to OTX2 itself and its target genes – including NEK2 – as potential therapeutic targets. Disruption of the relationship between OTX2 and NEUROD1 may also be a potential treatment strategy.

Massachusetts General Hospitalwww.massgeneral.org/about/pressrelease.aspx?id=2063

“Mutations are part of life. They are mistakes in a gene like typos in a text message,” said Watanabe-Smith, a postdoctoral fellow with the OHSU Knight Cancer Institute. “But which mutations cause cancer? That’s the real question. And this problem is impossible to understand without a strong model system to test those mutations.”

Watanabe-Smith’s research sought to better understand one “typo” in a standard leukaemia assay, or test. While studying cancer biology and completing his doctorate in the lab of Brian Druker, M.D., at the OHSU Knight Cancer Institute, Watanabe-Smith encountered a new problem: an issue with the model system itself.

“When I was sequencing the patient’s DNA to make sure the original, known mutation is there, I was finding additional, unexpected mutations in the gene that I didn’t put there. And I was getting different mutations every time,” said Watanabe-Smith.

He decided to formally study this phenomenon with his lab advisers, who included Druker; Cristina Tognon, Ph.D., scientific director, Druker lab; and Anupriya Agarwal, Ph.D., assistant professor of hematology & medical oncology, OHSU School of Medicine; researcher with the OHSU Knight Cancer Institute, all co-authors on the paper.  

His initial research, identifying and characterizing a growth-activating mutation in a patient with T-cell leukaemia and was first published last April. This research published was focused on better understanding the lab’s model system, to ensure that future researchers trying to identify cancer-causing mutations are using accurate and reproducible methods.

Their research investigates a common cell line assay, used since the 1980’s, to detect which mutations are important in driving leukaemia and other cancers. They found this assay is prone to a previously unreported flaw, where the cells, called Ba/F3 cells, can acquire additional mutations.

“The potential impact is that a non-functional mutation could appear functional, and a researcher could publish results that would not be reproducible,” Watanabe-Smith said. “Then we had the question: ‘Did the cells transform because of a mutation the patient had, or did they transform because these new mutations they managed to pick up somewhere?’”

Ultimately, he says, the research team recommends an additional step in the Ba/F3 assay (sequencing outgrown cell lines) to improve reproducibility of future results. While the results urge further research, the message to scientific community is clear: There seems to be more potential for problems than previously anticipated in this standard assay.

OHSU Knight Cancer Institutenews.ohsu.edu/2017/02/21/gene-mutations-cause-leukemia-but-which-ones

Gene discovery research is uncovering new information about similarities and differences underlying various neurodevelopmental disorders.  These are a wide-ranging collection of conditions that affect the brain.  They include autism, intellectual impairments, developmental delays, attention deficits, tic disorders and language difficulties.

To better understand how gene-disrupting mutations contribute to the biology of neurodevelopmental disorders, researchers recently conducted a large, international, multi-institutional study.   

More than 11,700 affected individuals and nearly 2,800 control subjects underwent targeted DNA sequencing of 208 suspected disease-risk genes. The candidate genes were chosen based on previously published studies.  By looking at greater numbers of cases and using a reliable yet inexpensive molecular inversion probe, the project team wanted to measure the statistical significance of individual, implicated genes.

The study leaders were Holly A. F. Stessman, Bo Xiong and Bradley P. Coe, of the genome sciences laboratory of Evan Eichler at the University of Washington School of Medicine and the Howard Hughes Medical Institute.  Stessman is now at Creighton University.

Their samples were collected through the Autism Spectrum/Intellectual Disability 15-center network spanning seven countries and four continents.  An advantage of this collection, the researchers said, is the ability to check back on a large fraction of cases to try to relate genetic results to clinical findings.  

In their study population, the researchers associated 91 genes with the risk of a neurodevelopmental disorder. These included 38 genes not previously suspected of playing a role.  Based on some of the family studies, however, mutations even in two or more of the risk genes may not be necessary or sufficient to cause disease.

Of the 91 genes, 25 were linked with forms of autism without intellectual disability. The scientists also described a gene network that appeared to be related to high-functioning autism.  Individuals with this form of autism have average to above average intelligence, but may struggle in learning to talk, interact socially, or manage anxiety and sensory overload.  
While observing that some genes were more closely associated with autism and others with intellectual or developmental impairments, the researchers found that most of the genes implicated were mutated in both conditions.  This result reinforces the substantial overlap among these conditions in their underlying genetics and observable characteristics.

“Most of these genes are clearly risk factors for neurodevelopmental disorders in a broad sense,” the researchers explained.  “But analysis of both the genetic and subsequent patient follow-up data did single out some genes with a statistical bias towards autism spectrum disorder, rather than an intellectual disability or developmental delay.”

Additional findings suggest that less severe mutations may be behind autism that is not accompanied by intellectual disability.

University of Washington Health Systemhsnewsbeat.uw.edu/story/sorting-out-risk-genes-brain-development-disorders

Following a three-year study of the Arizona State University football program, researchers at the Translational Genomics Research Institute (TGen) have created the largest dataset to date of extracellular small RNAs, which are potential biomarkers for diagnosing medical conditions, including concussions.
 
The study amassed a collection of biomarkers from the ASU student-athletes’ biofluids: blood, urine and saliva. A portion of that information will be used with data from helmet sensors that recorded the number, intensity and direction of head impacts during games and practices from the 2013-16 football teams. TGen researchers are using that combined data to potentially develop new diagnostic and therapeutic tools.
 
"Large datasets – examining different biofluids, isolation methods, detection platforms and analysis tools – are important to further our understanding of the extent and types of extracellular materials present when someone is injured or develops disease," said Dr. Kendall Van Keuren-Jensen, TGen Associate Professor of Neurogenomics and Co-Director of TGen’s Center for Noninvasive Diagnostics, and one of the study’s senior authors.
 
"Concussion safety, protocol and diagnostics are key components of Sun Devil Athletics’ student-athlete welfare program," said Ray Anderson, ASU Vice President for University Athletics. "Our partnership with TGen and the research conducted with these biomarkers will ideally provide doctors, trainers and administrators with a mechanism to proactively safeguard the health of our student-athletes. We are proud and excited to be a part of this ground-breaking study that will significantly expand research in this important area of scientific discovery."
 
Because the data is being published in an open access journal, they are available to aid other researchers studying how to develop tests for the detection and extent of injuries involving everything from automobile accidents to battlefield explosions.
 
Sensors in the ASU student-athlete football helmets were wirelessly connected to a field-level computer as part of the Sideline Response System – a head impact monitoring and research tool developed and deployed by Riddell, a leading provider of helmets to the NFL and major college football teams. 
 
TGen researchers used advanced genomic sequencing to identify the biomarkers of extracellular RNA (exRNA), strands of genetic material that are released from cells, and which can be detected in biofluids. TGen sequenced these biomarkers from among 183 blood samples, 204 urine samples and 46 saliva samples derived from among 55 consenting student-athletes, ages 18-25.
 
"The small RNA profile of each biofluid is distinct," the study said. "These data significantly contribute to the current number of sequenced exRNA samples from young healthy individuals."  
 
By identifying biofluids associated with healthy individuals, researchers hope to use these as standards for assessing disease and injury: "Establishing a baseline for individuals when they are healthy may provide the most meaningful comparisons when exploring early indicators of disease, severity or outcome," the study said.

TGen
www.tgen.org/home/news/2017-media-releases/tgen-asu-riddell-concussion-study-results.aspx#.WM1FeGTyv5Y