Scientists from the Icahn School of Medicine at Mount Sinai have developed a new technique to more precisely analyse bacterial populations, to reveal epigenetic mechanisms that can drive virulence.  The new methods hold the promise of a potent new tool to offset the growing challenge of antibiotic resistance by bacterial pathogens.

The information content of the genetic code in DNA is not limited to the primary nucleotide sequence of A’s, G’s, C’s and T’s. Individual DNA bases can be chemically modified, with significant functional consequences.  In the bacterial kingdom, the most prevalent base modifications are in the form of DNA methylations, specifically to adenine and cytosine residuals.  Beyond their participation in host defence, increasing evidence suggests that these modifications also play important roles in the regulation of gene expression, virulence and antibiotic resistance.

The research team employed the PacBio RS II system which can collect data on base modifications simultaneously as it collects DNA sequence data. PacBio’s single molecule, real-time sequencing enables the detection of N6-methyladenine and 4-methylcytosine, two major types of DNA modifications comprising the bacterial methylome. However, existing methods for studying bacterial methylomes rely on a population-level consensus that lack the single-cell resolution required to observe epigenetic heterogeneity.

“We created a technique for the detection and phasing of DNA methylation at the single molecule level.  We found that a typical clonal bacterial population that would otherwise be considered homogeneous using conventional techniques has epigenetically distinct subpopulations with different gene expression patterns’ said Gang Fang, PhD, Assistant Professor of Genetics and Genomics at the Icahn School of Medicine at Mount Sinai and senior author of the study.  “Given that phenotypic heterogeneity within a bacterial population can increase its advantage of survival under stress conditions such as antibiotic treatment, this new technique is quite promising for future treatment of bacterial pathogens, as it enables de novo detection and characterization of epigenetic heterogeneity in a bacterial population.”

The researchers studied seven bacterial strains, demonstrating the new technique reveals distinct types of epigenetic heterogeneity. For Helicobacter pylori, a pathogenic bacterium that colonizes over 40% of the world population and is associated with gastric cancer, the team discovered that epigenetic heterogeneity can quickly emerge as a single cell divides, and different subpopulations with distinct methylation patterns have distinct gene expressions patterns. This may have contributed to the increasing rate of antibiotic resistance of Helicobacter pylori.

“The application of this new technique will enable a more comprehensive characterization of the functions of DNA methylation and their impact on bacterial physiology.  Resolving nucleotide modifications at the single molecule, single nucleotide level, especially when integrated with other single molecule- or single cell-level data, such as RNA and protein expression, will help resolve regulatory relationships that govern higher order phenotypes such as drug resistance” said Eric Schadt, PhD, Founding Director of the Icahn Institute and Professor of Genomics at the Icahn School of Medicine at Mount Sinai.  “The approach we developed can also be used to analyze DNA viruses and human mitochondrial DNA, both of which present significant epigenetic heterogeneity.” Mount Sinai Health System

A study shows that genetic test results, as revealed by non-invasive prenatal testing for foetal chromosome abnormalities, may also detect underlying conditions in the mother, including cancer. The study reports on a case series of eight women who had abnormal non-invasive prenatal testing results. While their foetuses had normal chromosomes, retrospective genomic analysis showed that the abnormal findings were due to a variety of undiagnosed cancers in the mothers.

A team of scientists and clinicians, led by Diana W. Bianchi, MD, Executive Director of the Mother Infant Research Institute at Tufts Medical Center, reports the results of their DNA sequencing analysis. Their findings demonstrate that previously undetected maternal cancers may provide a biological explanation for some prenatal screening results that differ from results of prenatal diagnostic tests.

Non-invasive prenatal screening is a recent clinical advance that provides pregnant women with information about possible chromosomal abnormalities, such as Down syndrome, in their foetuses. The screening test, which can be offered as early as the tenth week of pregnancy, analyses fragments of placental and maternal DNA that circulate in the maternal plasma. In women with cancer, the plasma sample also contains cancer DNA.

Diagnosis of cancer during pregnancy is relatively uncommon, with an incidence of about 1 in 1,000 women. Cancer detected during pregnancy most often occurs in the breast, cervix, ovary and colon, as well as melanoma, lymphoma and leukaemia. “This study provides one explanation for when non-invasive prenatal testing results are different from the foetal karyotype. It highlights the need to perform a diagnostic procedure to determine true foetal karyotype whenever non-invasive prenatal testing suggests chromosomal abnormalities,” said Dr. Bianchi, an international expert on non-invasive prenatal testing.

The cases in this study came from a larger group of 125,426 samples submitted from asymptomatic pregnant women who underwent non-invasive prenatal testing for foetal chromosomal abnormalities between 2012 and 2014. Of these, 3757 cases were positive for one or more abnormalities in the number of chromosomes 13, 18, 21, X or Y.  The women’s physicians later reported ten cases of cancer to the laboratory that originally conducted the non-invasive prenatal testing. The study analysed eight of the ten cases in depth. All of the women had abnormal non-invasive prenatal test results, and most frequently, more than one chromosomal abnormality was detected, which is a very unusual result. Cancer was diagnosed during pregnancy or postpartum in these women at an average of 16 weeks following the initial non-invasive prenatal testing.

Some women were tested more than once, and some were tested both during pregnancy and after. One patient had testing after treatment for colorectal cancer, and the abnormal pattern was no longer evident, suggesting a response to treatment. “Non-invasive prenatal testing results may lead to findings of an underlying maternal condition, which, in these cases, was due to cancer,” said Dr. Bianchi. “The take-home message is that women should be aware of this possibility when they seek testing. More research needs to be done to further study this occurrence to help guide physicians on how to counsel women and manage their follow-up care.” Tufts Medical Center

A genetic analysis led by UT Southwestern Medical Center researchers suggests that most pancreatic cancers harbour genetic alterations that could be targeted by existing drugs, using their genetic features as a roadmap for treatment. The findings support a precision approach to treating pancreatic cancer, the fourth most deadly cancer for both men and women.

A comprehensive DNA sequencing of pancreatic cancer cases revealed not only a plethora of damaged genes, but potential diagnostic biomarkers that could help identify those with longer or shorter survival, and provide opportunity for new therapeutic interventions.

“We identified a wealth of genetic diversity, including multiple mutated genes that were previously unknown to pancreatic cancer − an important step in gaining a better understanding of this difficult and particularly deadly disease,” said lead author Dr. Agnieszka Witkiewicz, Associate Professor of Pathology and a member of the Harold C. Simmons Comprehensive Cancer Center at UT Southwestern. “Importantly, the team was able to identify several genes that may be able to help us to predict outcomes in certain circumstances or serve as good candidates for therapeutic efforts.”

Researchers have long hoped that genetic analysis would provide insight into the biology of pancreatic cancer and define new targets for more effective treatment. Achieving this goal has been hampered by the technical difficulty of isolating pure cancer cells out of the tumour tissue that contains both tumour cells as well as normal cells. The new study overcame this limitation by selectively dissecting cancer cells from pieces of tumour tissue. This method was applied to specifically determine the genetic features of 109 different tumours. 

The data showed that the genetic architecture of pancreatic cancer is complex, and each patient’s tumour was found to be unique. The genetic features illuminated ways in which the disease arises, defined events associated with survival, and yielded potential targets for therapeutic intervention.

“While we suspect that genetics can be used as the basis of targeted treatments, this point will only be proven through extensive research and clinical studies, hopefully leading to improved outcomes for patients,” said senior author Dr. Erik Knudsen,  Professor of Pathology, and member of the Simmons Cancer Center who holds the Dr. Charles T. Ashworth Professorship in Pathology. “I am considerably more optimistic of the utility of a genetically targeted therapy for pancreatic cancer today than when we began this work.”

Pancreatic cancer is particularly difficult to treat, and is often diagnosed at a late stage when it is no longer amenable to surgical removal. Chemotherapy has a modest effect, and unfortunately the disease progresses in the vast majority of cases. Therefore, new therapeutic regimens are urgently needed.  UT Southwestern Medical Center

Indiana University cancer researchers found that a particular microRNA may be a potent therapeutic agent against pancreatic cancer.

Led by Janaiah Kota, Ph.D., assistant professor of medical and molecular genetics at the IU School of Medicine and a researcher at the Indiana University Melvin and Bren Simon Cancer Center, the researchers found that restoring missing microRNA-29 (miR-29) in pancreatic cancer stromal cells reduced the viability and growth of the cancerous cells.

A thick fibrotic shell around the cancer cells is known as ‘stroma,’ which protects the pancreatic cancer cells from anticancer drugs such as chemotherapy.

‘We found that the loss of miR-29 is a common phenomenon of pancreatic cancer stromal cells, and that by restoring it, the stromal accumulation and cancer growth was reduced,’ Kota said. ‘The use of miR-29 as a therapeutic agent may be more effective in targeting reactive stroma, as a single miRNA regulates the expression of several genes associated with disease mechanisms.’

‘In healthy cells and tissues, a single miRNA controls the expression of hundreds of genes, and any alterations in their normal expression leads to abnormal overexpression of bad genes that are favourable for the growth of cancer cells and are harmful to normal cells,’ Kota explained.

Kota and his colleagues were studying the role of small non-coding RNAs called miRNAs in molecular mechanisms associated with pancreatic cancer stroma to evaluate their use for therapeutic intervention in pancreatic cancer. They found that there is loss of miR-29 in stroma of the pancreatic tumours compared to the healthy pancreas. The researchers expected its expression in stromal cells would restore normal function of stromal cells and reduce the abundance of fibrotic stromal proteins. However, they were surprised that when they co-cultured miR-29 overexpressing stromal cells with cancer cells, it also reduced the viability and growth of cancer cells for unknown factors.

They are currently performing additional studies to understand the molecular mechanisms associated with the effect of miR-29 overexpression in stromal cells on cancer cells as well as in preclinical animal models.

‘This is a novel approach that has the potential to overcome the problems associated with current anti-stromal drugs and that could lead to improved therapeutic strategies, enhanced drug delivery to the tumour bed, and, in the future, improved patient survival,’ said Murray Korc, M.D., the Myles Brand professor of cancer research at the IU School of Medicine and a researcher at the IU Simon Cancer Center. Korc is also director of the Pancreatic Cancer Signature Center.

The need for new therapies for pancreatic cancer patients is great as only 7 percent of people with the disease survive more than five years after diagnosis. According to the National Cancer Institute, there will be an estimated 48,960 new cases of pancreatic cancer and 40,560 deaths from the disease in 2015. Indiana University

Australian researchers have found that so-called ‘triple-negative breast cancers’ are two distinct diseases that likely originate from different cell types. This helps explain why survival prospects for women with the diagnosis tend to be either very good or very bad.

The Sydney-based research team has found a gene that drives the aggressive disease, and hopes to find a way to ‘switch it off’. The aggressive form of triple-negative breast cancer appears to arise from stem cells, while the more benign form appears to arise from specialised cells.

Stem cells have many of the same features as cancers. They are plastic and flexible, and have the ability to proliferate and spread into other tissues – deadly traits in cancers.

Previous studies have shown that breast stem cells are needed for breast growth and development during puberty and pregnancy, although how they evolve from stem cells into specialist cells has been unclear.

The new study has shown that a gene known as ‘inhibitor of differentiation’ (ID4) determines whether a stem cell remains a stem cell, or whether it differentiates into a specialist cell.

Notably, when the high levels of ID4 in a stem cell are ‘switched off’, other genes that drive cell specialisation are ‘switched on’.

Drs Alex Swarbrick and Simon Junankar from Sydney’s Garvan Institute of Medical Research spearheaded this large interdisciplinary study, which links the development of the mammary gland in mice with human breast cancer. Its main finding, that ID4 not only ‘marks’, but appears to control, the highly aggressive form of triple negative breast cancer.

“We found that ID4 is produced at high levels in roughly half of all triple negative breast cancers, and that these cancers have a particularly poor prognosis,” said project leader Dr Alex Swarbrick.

“We also showed that if you block the ID4 gene in experimental models of triple negative breast cancer, the tumour cells stop dividing.” Garvan Institute of Medical Research