Knowing what type of lung cancer a patient has is critical to determine which drug will work best and which therapies are safest in the era of personalised medicine. Key to making that judgement is an adequate tumour specimen for the pathologist to determine the tumour’s histology, a molecular description of a tumour based on the appearance of cells under a microscope. But not all specimens are perfect, and are sometimes so complex that a definitive diagnosis presents a challenge.
Scientists at the Universities of North Carolina and Utah have developed a histology expression predictor for the most common types of lung cancer: adenocarcinoma, carcinoid, small cell carcinoma and squamous cell carcinoma. This predictor can confirm histologic diagnosis in routinely collected paraffin samples of patients’ tumours and can complement and corroborate pathologists’ findings.
Neil Hayes, MD, MPH, associate professor of medicine and corresponding author of the study says, ‘As we learn more about the genetics of lung cancer, we can use that understanding to tailor therapies to the individual’s tumour. Gene expression profiling has great potential for improving the accuracy of the histologic diagnosis. Historically, gene expression analysis has required fresh tumour tissue that is usually not possible in routine clinical care. We desperately needed to extend the analysis of genes (aka RNA) to paraffin samples that are routinely generated in clinical care, rather than fresh frozen tissue. That is the major accomplishment of the current study and one of the first large scale endeavours in lung cancer to show this is possible.
‘Our predictor identifies the major histologic types of lung cancer in paraffin-embedded tissue specimens which is immediately useful in confirming the histologic diagnosis in difficult tissue biopsy specimens.’ Dr. Hayes is a member of UNC Lineberger Comprehensive Cancer Center.
The scientists used 442 samples of formalin-fixed paraffin-embedded specimens from lung cancer patients at UNC and the University of Utah Health Sciences Center as they developed their predictor.
First author Matthew Wilkerson, PhD, explains, ‘Our question was, ‘Can histology be predicted accurately by gene expression?’ We had lung cancer genes we already knew were differentially expressed in the different tumour types, so we measured them in tumour paraffin specimens. Next we developed a predictor in an independent set of tumour samples. We then compared the predictor to the actual clinical diagnosis and had additional pathologists review the samples. We showed accuracy as least as good as the pathologist. Our predictor exhibited a mean accuracy of 84 percent, and when compared with pathologist diagnoses, yielded similar accuracy and precision as the pathologists.’

Dr. Hayes summarizes, ‘Going beyond meeting a current need of increasing the accuracy of histologic diagnosis is expected to be the ultimate benefit of this technology. There are many additional characteristics of tumours that could be leveraged for clinical purposes once the world of gene expression analysis from paraffin is efficient from clinical samples. We anticipate additional uses such as predicting responses to additional therapies and prognostication as near term additions.’ University of North Carolina Health Care

Researchers have developed a reliable way to use a finger-stick blood sample to detect fibromyalgia syndrome, a complicated pain disorder that often is difficult to diagnose.
If it were someday made available to primary care physicians, the test could knock up to five years off of the wait for a diagnosis, researchers predict.
In a pilot study, the scientists used a high-powered and specialized microscope to detect the presence of small molecules in blood-spot samples from patients known to have fibromyalgia.
By ‘training’ the equipment to recognise that molecular pattern, the researchers then showed that the microscope could tell the difference between fibromyalgia and two types of arthritis that share some of the same symptoms.
Though more analysis is needed to identify exactly which molecules are related to development of the disorder itself, the researchers say their pilot data are promising.
‘We’ve got really good evidence of a test that could be an important aid in the diagnosis of fibromyalgia patients,’ said Tony Buffington, professor of veterinary clinical sciences at The Ohio State University and senior author of the study. ‘We would like this to lead to an objective test for primary care doctors to use, which could produce a diagnosis as much as five years before it usually occurs.’
Patients with fibromyalgia are often desperate by the time they receive treatment because of the lengthy process required to make a diagnosis. The main symptoms, persistent pain and fatigue, mimic many other conditions, so physicians tend to rule out other potential causes before diagnosing fibromyalgia. Additional symptoms include disrupted sleep and memory or thought problems. An estimated 5 million American adults have the disorder, according to the National Institute of Arthritis and Musculoskeletal and Skin Diseases.
‘The importance of producing a faster diagnosis cannot be overstated, because patients experience tremendous stress during the diagnostic process. Just getting the diagnosis actually makes patients feel better and lowers costs because of reductions in anxiety,’ said Kevin Hackshaw, associate professor of medicine, division of rheumatology and immunology, at Ohio State’s Wexner Medical Center and lead author of the study.
The technology used in this work is infrared microspectroscopy, which identifies the biochemical content of a blood sample based on where peaks of molecules appear in the infrared spectrum. The technology offers hints at the molecules present in the samples based on how molecular bonds vibrate when they are struck by light.
The spectroscopy works on dried blood, so just a few drops from a finger stick produce enough blood to run this test.
Researchers first obtained blood samples from patients diagnosed with fibromyalgia (14), rheumatoid arthritis (15) and osteoarthritis (12). These other conditions were chosen for comparison because they produce similar symptoms as fibromyalgia, but are easier to diagnose.
The scientists analysed each sample with the infrared microspectroscopy to identify the molecular patterns associated with each disease. This functioned as a ‘training’ phase of the study.
When the researchers then entered blinded blood samples into the same machinery, each condition was accurately identified based on its molecular patterns.
‘It separated them completely, with no misclassifications,’ Buffington said. ‘That’s very important. It never mistook a patient with fibromyalgia for a patient with arthritis. Clearly we need more numbers, but this showed the technique is quite effective.’
The researchers also analyzed some of the potential chemicals that could someday function as biomarkers in the fibromyalgia blood samples, but further studies are needed to identify the molecules responsible for the spectral patterns, he said. EurekAlert

A gene long presumed dead comes to life under the full moon of inflammation, Stanford University School of Medicine scientists have found.
The discovery may help explain how anti-inflammatory steroid drugs work. It also could someday lead to entirely new classes of anti-inflammatory treatments without some of steroids’ damaging side effects.
Chronic inflammation plays a role in cancer and in autoimmune, cardiovascular and neurodegenerative diseases, among others. Anti-inflammatory steroid drugs are widely prescribed for treating the inflammatory states that underlie or exacerbate these conditions.
‘Inflammation tells your body something is wrong,’ said the study’s senior author, Howard Chang, MD, PhD, professor of dermatology at Stanford and the recipient of an early career scientist award from the Howard Hughes Medical Institute. ‘But after it does its job of alerting immune cells to a viral or bacterial infection or spurring them to remove debris from a wound site, it has to get turned off before it causes harm to healthy tissue.’
That appears to be what the ‘undead’ gene does. Chang’s team, which identified it, has named it Lethe, after the stream in Greek mythology that makes the deceased who cross it forget their pasts.
　
The master regulator of inflammation inside cells — a bulky complex of several proteins, collectively called NF-kappa-B — is a transcription factor: It can switch on hundreds or even thousands of genes in a cell’s nucleus. When aroused by signals at the cell surface (typically delivered by circulating proteins or microbial components), NF-kappa-B activates pro-inflammatory genes, gearing that cell up to combat viral or bacterial assaults and respond to an injury.
Lethe, which the investigators found is activated by NF-kappa-B, subdues the master regulator’s massive influence on the genome, curtailing the inflammatory response.
NF-kappa-B also plays a key role in ageing. In a study published in 2007 in Genes and Development, Chang and his colleagues showed that old skin cells in which NF-kappa-B was temporarily inactivated began to act young. This finding has since been confirmed in other tissues and by other researchers.
To learn more about NF-kappa-B, Chang’s group decided to activate it and see which genes get turned on or off. But rather than ‘normal’ genes, which are essentially recipes for making proteins, they were curious about DNA sequences that generate long non-coding RNA molecules, or lncRNAs, which Chang helped to discover during the past decade.
RNA is best known as the intermediate material in classic protein production. Gene-reading machines in cells produce RNA transcripts, or copies, of protein-coding genes. These transcripts, known as messenger RNAs, are free to leave the cell nucleus for the cytoplasm, where they can transmit genes’ instructions to the protein-making machines situated there.
But lately RNA has been shown to play an increasing number of additional roles that have nothing to do with making proteins. The lncRNAs Chang studied are made by the same molecular machinery that protein-coding genes use to make a messenger RNA. Instead of heading for the cytoplasm to make proteins, though, lncRNAs can remain in the nucleus and directly regulate genes. More than 10,000 lncRNAs have now been discovered, although scientists are only beginning to understand what they do.
To see which lncRNAs were induced during inflammation, Chang and his colleagues exposed cultured fibroblasts from embryonic mice to TNF-alpha, an immune-signalling protein known to trigger NF-kappa-B. They found that levels of hundreds of lncRNAs inside the cells were driven either up or down by TNF-alpha stimulation.
Of those lncRNAs, a total of 54 were copied from so-called pseudogenes: DNA sequences that, while they closely resemble genes, don’t code for proteins. More than 11,000 pseudogenes — one for every two protein-coding genes — have been identified in the human genome. Scientists believe pseudogenes are copies of actual genes that, during the replication of some ancestral organism’s germ cell, were accidentally inserted into the genome and, redundant but harmless, came along for the evolutionary ride. Over the intervening eons, these genetic doppelgangers have roamed along the genome, mutated and decayed to the point where, it is believed, they no longer do anything at all.
‘Pseudogenes have been considered to be completely silent, ignored by cells’ DNA-reading machinery,’ Chang said. ‘But we got a real surprise. When a cell is subjected to an inflammatory stress signal, it’s like Night of the Living Dead.’
Equally surprising, Chang said, is that different signalling chemicals or microbial components (such as bits of bacterial cell walls or of viral DNA) wake up different groups of lncRNA-encoding DNA sequences, including pseudogenes. ‘They’re not really dead, after all. They just need very specific signals to set them in motion.’
Lethe was one such pseudogene tripped off by stimulation of NF-kappa-B. Lethe directly interfered with the complex’s ability to seat itself on appropriate DNA sequences, shutting down the pro-inflammatory genes the transcription factor ordinarily activates.
Several pseudogenes were activated in a selective manner. For example, TNF-alpha and another circulating signalling protein — but not microbial parts — activated Lethe.
Because some pseudogenes sit near protein-coding genes, some scientists have argued that the generation of RNA transcripts from the pseudogenes is simply an artifact of normal transcription of full-fledged protein-coding genes. ‘There’s a tendency to assume it’s some protein-coding gene that NF-kappa-B is really targeting, and to downplay the activation of a lncRNA as noise, a ‘ripple effect’ like the one you see when a boat goes by,’ Chang said.
But TNF-alpha failed to activate two nearby protein-coding genes on either side of Lethe. Reciprocally, stimuli that turned these two other genes on didn’t affect Lethe. Meanwhile, two other pseudogenes that very closely resemble Lethe were not activated by TNF-alpha, as Lethe was.
Another surprising finding was that dexamethasone, a commonly prescribed anti-inflammatory steroid drug, activates Lethe. Various other steroid hormones that are not anti-inflammatory in nature, such as vitamin D or oestrogen or a male steroid hormone, failed to boost Lethe levels.
‘We’re wondering whether there might be ways to artificially raise Lethe levels without steroids. These drugs have potentially deleterious side effects such as elevated blood pressure and blood sugar, thinning of bones and general suppression of the immune system,’ Chang said.
The study results suggest that not only Lethe but other pseudogenes undergo similarly selective awakenings to generate lncRNAs in response to different external inflammatory stimuli. ‘From the pattern of activated lncRNAs, you can tell what the cell has encountered — a virus, a bacteria or something else,’ Chang said. ‘These patterns of activation may be able to serve as an indicator of what kind of inflammatory situation or pathogenic invasion is responsible.’ Stanford University Medical Center

Massachusetts General Hospital (MGH) researchers and their colleagues have used digital versions of a standard molecular biology tool to detect a common tumour-associated mutation in the cerebrospinal fluid (CSF) of patients with brain tumours. In their report the investigators describe using advanced forms of the gene-amplification technology polymerase chain reaction (PCR) to analyse bits of RNA carried in membrane-covered sacs called extracellular vesicles for the presence of a tumour-associated mutation in a gene called IDH1.
‘Reliable detection of tumour-associated mutations in cerebrospinal fluid with digital PCR would provide a biomarker for monitoring and tracking tumours without invasive neurosurgery,’ says Xandra Breakefield, PhD, of the MGH Molecular Neurogenetics Unit, corresponding author of the paper. ‘Knowing the IDH1 mutation status of these tumours could help guide treatment decisions, since a number of companies are developing drugs that specifically target that mutant enzyme.’
Both normal and tumour cells regularly release extracellular vesicles, which contain segments of RNA, DNA or proteins and can be found in blood, CSF and other body fluids. A 2008 study from the MGH team was able to identify a relatively large tumour-associated mutation in extracellular vesicles from the blood of brain tumour patients, but most current diagnostic technologies that analyse CSF do not capture molecular or genetic information from central nervous system tumours.
In addition, explains Leonora Balaj, PhD, of MGH Neurology, co-lead author of the current report, ‘Tumour-specific EVs make up only a small percentage of the total number of EVs found in either blood or cerebrospinal fluid, so finding rare, single-nucleotide mutations in a sample of blood or CSF is very challenging. These digital PCR techniques allow the amplification of such hard-to-find molecules, dramatically improving the ability to identify tumour-specific changes without the need for biopsy.’
The current study used two forms of digital PCR – BEAMing and Droplet Digital PCR – to analyse extracellular vesicles in the blood and CSF of brain tumour patients and healthy controls for the presence of a single-nucleotide IDH1 mutation known to be associated with several types of cancer. Both forms of PCR were able to detect both the presence and abundance of mutant IDH1 in the CSF of 5 of the 8 patients known to have IDH1-mutant tumours. Two of the three mutation-positive tumours that had false negative results were low grade and the third was quite small, suggesting a need for future studies of more samples to determine how the grade and size of the tumours affect the ability to detect mutations. The failure to detect tumour-associated mutations in blood samples with this technology may indicate that CSF is a better source for extracellular vesicles from brain tumours.
The ability to non-invasively determine the genetic makeup of brain tumours could have a significant effect on patient care, explains study co-author Fred Hochberg, MD, MGH Neurology. ‘The current approach for patients who may have a brain tumour is first to have a brain scan and then a biopsy to determine whether a growth is malignant. Patients may have a second operation to remove the tumour prior to beginning radiation therapy and chemotherapy, but none of these treatments are targeted to the specific molecular nature of the tumour.
‘Having this sort of molecular diagnostic assay – whether in spinal fluid or blood – would allow us to immediately initiate treatment that is personalised for that patient without the need for surgical biopsy,’ he adds. ‘For some patients, the treatment could shrink a tumour before surgical removal, for others it may control tumour growth to the point that surgery is not necessary, which in addition to keeping patients from undergoing an unnecessary procedure, could save costs. We still have a long way to go to improve survival of these malignancies, so every improvement we can make is valuable.’ Massachusetts General Hospital