Scientists at The University of Nottingham have demonstrated for the first time that it is possible to selectively sequence fragments of DNA in real time, greatly reducing the time needed to analyse biological samples.

A paper describes a novel technique for highly selective DNA sequencing, called ‘Read Until’.  The method, used with real-time nanopore sequencing, enables the user to analyse only DNA strands that contain pre-determined signatures of interest.

Dr Matt Loose, of the Cell and Developmental Biology Research Group in the University’s School of Life Sciences, has been working with the MinION, a new portable DNA sequencing technology produced by biotech company Oxford Nanopore Technologies. All sequencing was carried out at The University of Nottingham Next Generation Sequencing Facility, DeepSeq.

“This is the first time that direct selection of specific DNA molecules has been shown on any device,” said Dr Loose. “We hope that it will enable many future novel applications, especially for portable sequencing. This makes sequencing as efficient as possible and will provide a viable, informatics based alternative to traditional wet lab enrichment techniques. The application of this approach to a wide number of problems from pathogen detection to sequencing targeted regions of the human genome is now within reach.”

The pocket-sized MinION device – the same technology which NASA recently sent to the International Space Station in an effort to investigate whether DNA sequencing is possible in microgravity – employs tiny molecular pores in a membrane that ‘sense’ the sequence of DNA fragments passing through these nanopores, producing minute fluctuations in a current trace. These current traces, termed ‘squiggles’ then need to be converted to DNA bases using base caller software, often located in the cloud. The University of Nottingham team used signal processing techniques to map these squiggles to reference sequences, by passing this step.

In the paper, the Nottingham team go further, showing that this squiggle matching technique can be performed at a rate that enables decisions to be made about the fragment of DNA that is being sequenced before it has completely passed through the nanopore. Depending on the sequence, individual nanopores within the MinION can then be instructed to continue sequencing or to eject the current DNA fragment and start sequencing another. The Nottingham team show that this ‘real-time selective sequencing’, or as some have called it ‘DNA tasting’, can reduce the time needed to sequence key DNA fragments or enable the analysis of pathogen samples where there is host and other DNA present in the sample.

The Read Until method/technique was developed by applying dynamic time warping to match short query current traces to references, demonstrating selection of specific regions of small genomes, individual amplicons from a group of targets, or normalisation of amplicons in a set.

Nottingham University www.nottingham.ac.uk/news/pressreleases/2016/july/nottingham-researchers-show-novel-technique-that-can-‘taste’-dna.aspx

Researchers have identified a new pathway and with it a protein, BRD4, necessary for breast cancer cells to spread.

The findings may provide a new target to suppress breast cancer metastasis.

Triple-negative breast cancer is considered the worst subgroup of breast cancer. It is highly aggressive and responds poorly to the current therapeutic tools resulting in a dismal prognosis for patients. Furthermore, the lack of identified targets has limited the development of new drug strategies.

Researchers from Boston University School of Medicine (BUSM) used breast cancer cell lines that present the clinical characteristics of an aggressive breast cancer subtype (clinically described as a triple-negative breast cancer). They then used an experimental design to model cancer cell metastasis. By suppressing the expression of the protein BRD4 in these cell lines, they observed that their dissemination capabilities were blocked, indicating that BRD4 drives breast cancer dissemination. In addition, they conducted a screening analysis of human breast tumours and found that tumours with a high expression of BRD4 were more likely to metastasize.

“The current treatment options for a triple-negative cancer are unacceptably limited. It is crucial to identify new therapeutic targets to tackle challenging cancer types, including triple negative breast cancer. BDR4 targeting represents an innovative strategy to ablate breast cancer metastasis,” explained lead investigator Guillaume Andrieu, PhD, a post-doctoral research associate at Boston University School of Medicine.

Although obesity per se is not thought of as a carcinogen, the abnormal, inflamed microenvironments found in obesity are critical for progression, invasion and metastasis of triple negative breast cancer. “Bromodomain and ExtraTerminal domain (BET) proteins, which include BRD2, BRD3 and BRD4, are known to regulate production of inflammatory mediators. Our study proposes that BRD4 couples inflammation to breast cancer dissemination. Thus, small molecules that block BET proteins possess anti-inflammatory properties that can be useful for therapy,” he added.

Although these findings primarily focus on breast cancer and metastasis, the researchers plan to expand their results to the treatment of prostate cancer, which they believe has similar pathways involved in its metastasis.

Boston University Medial Center www.bu.edu/news/2016/11/15/researchers-identify-protein-required-for-breast-cancer-metastasis/

Ludwig researchers working in collaboration with colleagues in Australia and the US have shown that fragments of tumour DNA circulating in the blood can be used to gauge the risk of colorectal cancer recurrence and the efficacy of chemotherapy following surgery. The finding is an important step toward the development of a non-invasive and more effective test for the detection, monitoring and treatment of cancer.

‘Prior studies, including ones from our group, have shown that this technique is sensitive enough to detect tumour DNA fragments in patients with advanced cancer,” Bert Vogelstein, co-director of the Ludwig Center at Johns Hopkins and one of the study leaders. “But this new study gets us one major step closer to the real goal, because it suggests that it can detect residual disease in early stage patients well before conventional clinical or radiologic criteria can.’

The decision of whether a stage II colon cancer patient should be treated with adjuvant, or post-operation, chemotherapy remains one of the most challenging areas in colorectal oncology. Such assessments are currently made by combining a number of clinical and pathologic features—such as the tumour’s appearance under the microscope, including how far it has spread through the bowel wall—or looking for the presence of cancer-specific genetic markers that have prognostic significance.

However, current methods are imprecise, and as a result doctors tend to err on the side of caution. Currently, up to 40 percent of stage II patients undergo the rigors and risks of adjuvant chemotherapy even though only a small fraction of them are destined to experience a cancer relapse.

‘The routine procedure is to give six months of chemotherapy, but we don’t have any way of knowing if the treatment is effective,’ said Jeanne Tie, a Ludwig investigator at the Walter and Eliza Hall Institute of Medical Research (WEHI) in Victoria, Australia, and lead author of the study.

Cancer cells often shed their DNA into the blood when they die, and recent advances in technology have made it possible to capture and profile these relatively rare fragments of DNA. Mutations in such circulating tumour DNA (ctDNA) can serve as extremely specific cancer biomarkers.

‘We have to be able to pick out a single tumour DNA among ten thousand normal DNA fragments,’ Tie said. ‘That’s the level of sensitivity that we needed to get down to, and that wasn’t possible until now.’

For the current study, Tie and her colleagues collected tumour samples from 230 patients with stage II colorectal cancer. They analysed the DNA of the tumour specimens and then designed personalized assays to target each patient’s particular genetic mutations.

The assays were applied to blood samples taken from the patients four to 10 weeks after surgery to remove tumours. Twenty of the 230 patients tested positive for ctDNA, and of this group, 80 percent experienced cancer relapse within about two years. Of the 164 patients whose blood tested negative for ctDNA, only 10 percent relapsed.

‘A positive ctDNA test is an indicator that cancer cells from the original tumor are hiding somewhere in the body,’ said Peter Gibbs, a Ludwig investigator at WEHI who co-led the study with Tie and Vogelstein.

The team also looked at whether ctDNA could be used to gauge the impact of chemotherapy treatments. Six of the patients who tested positive for ctDNA following surgery also underwent adjuvant chemotherapy. The scientists continued to collect blood samples from these patients and found that in two patients, ctDNA readings changed from positive after surgery to negative following chemotherapy.

‘To an oncologist, that’s probably the most exciting aspect of a ctDNA screening test—that it can be used not only to determine the risk of recurrence, but also as a real-time marker of chemotherapy benefits,’ said Gibbs.

In the current study, the ctDNA assays were custom-tailored to each patient’s unique cancer mutations, but the scientists are also developing a ctDNA screening test that covers frequently occurring colorectal cancer mutations.

‘When such a generic test is developed, it could still catch more than 90 percent of colorectal cancers, and it would eliminate the need to retrieve and test individual tumour samples, thus saving time, effort and money,’ Gibbs said.

www.ludwigcancerresearch.org/news/new-screening-test-using-blood-biomarkers-may-identify-risk-colon-cancer-recurrence-early

In a landmark multi-country study, Australian researchers have transformed our understanding of the genes that affect our risk of cancer. The researchers uncovered numerous new genetic risk factors for the bone and soft-tissue cancer, sarcoma – and, in a world first for any cancer, they showed that carrying several of these genetic mutations markedly increases an individual’s cancer risk. The findings have immediate implications for how sarcomas and other cancers are treated.

In a landmark study of over 1000 sarcoma patients, the researchers uncovered numerous new genetic risk factors for the cancer – and, in a world first for any cancer type, they showed that carrying two or more of these rare mutations increases an individual’s cancer risk.

Sarcomas are cancers of connective tissues that disproportionately affect the young. They are one of the three leading causes of disease-related death among children and young adults in Australia, and sarcoma survivors are at higher risk of developing a second cancer.

The new findings relating to cancer risk were uncovered through the International Sarcoma Kindred Study (ISKS), an Australian-led international consortium that is exploring the genetic basis of sarcoma in over 1000 individuals – the largest study ever conducted in this disease.

The ISKS team used a ‘gene panel’ of 72 genes to detect mutations in each study participant. They identified mutations in a number of new genes that significantly increase the risk of developing sarcoma, including in the genes ERCC2, ATR, BRCA2 and ATM.

Importantly, in individuals carrying mutations in two genes, the risk of developing sarcoma was measurably higher than in those with a mutation in only one gene. And in carriers of three or more mutations, the risk was greater still.

“This is the first time – in any cancer – that anyone has quantified the effect of multiple rare genetic mutations on cancer risk,” says Professor David Thomas (Head of The Kinghorn Cancer Centre and the Cancer Division of the Garvan Institute of Medical Research), who led the study.

“Until now, we’ve been limited to single-gene thinking, so we tell patients, for instance, that carrying a BRCA1 mutation means their breast cancer risk is higher, or that their risk of sarcoma and other cancers is higher if they’ve got a particular mutation in the p53 gene.

“The study shows us that the landscape of cancer risk is far more complex than that. We can now see that the risk for developing sarcoma is increased through the combined effect of multiple genes, and that the more mutations someone carries, the earlier the onset of cancer.

“These previously invisible effects are at least as large as the impact of mutations in the p53 gene itself, which is currently the strongest known genetic cause of sarcoma.”

Dr Mandy Ballinger (Garvan), who co-ordinates the ISKS globally, says the study will radically change how sarcoma risk is understood.

“It’s well accepted for a few cancers – like breast cancer and bowel cancer – that cancer risk is substantially determined by the genes we inherit from our parents. Our study brings sarcoma into that select group.

“About half the study participants carried at least one of these apparently cancer-promoting mutations, and almost a quarter carried more than one, which really underscores that sarcoma risk is inherited to a large extent from one’s parents.”

“We’ve never been able to identify these at-risk individuals, and their families, before. Now we can,” adds Prof Thomas. “That means we can manage risk better, and help those people to get the care they need, when they need it.”

Garvan Institute www.garvan.org.au/news/news/beyond-single-gene-thinking-garvan-researchers-uncover-complex-genetic-secrets-of-cancer-risk