During breast-tissue development, a transcription factor called SLUG plays a role in regulating stem cell function and determines whether breast cells will mature into luminal or basal cells.

Studying factors, such as SLUG, that regulate stem-cell activity and breast-cell identity are important for understanding how breast tumours arise and develop into different subtypes. Ultimately, this knowledge may help the development of novel therapies targeted to specific breast-tumour subtypes.

Background: Stem cells are immature cells that can differentiate, or develop, into different cell types. Stem cells are important for replenishing cells in many tissues throughout the body. Defects that affect stem-cell activity can lead to cancer because mutations in these cells can cause uncontrollable growth. Some transcription factors regulate the differentiation or ‘programming’ of breast stem cells into the more mature cells of the breast tissue. Abnormal expression of these transcription factors can change the normal programming of cells, which can lead to imbalances in cell types and the over-production of cells with enhanced properties of stem cells.

Breast tissue has two main types of cells: luminal cells and basal cells. Transcription factors, like SLUG, help control whether cells are programmed to become luminal cells or basal cells during normal breast development. In cancer, transcription factors can become deregulated, influencing what type of breast tumour will form. In aggressive basal-type breast tumours, SLUG is often over-expressed.

Previous work led by Charlotte Kuperwasser, principal investigator and senior author, determined that some common forms of breast cancer originate from luminal cells, whereas rare forms of breast cancer originate from basal cells. This difference in origins suggests that genes that affect the ability of a cell to become luminal or basal may also affect the formation of breast tumours. Because SLUG can regulate breast-cell differentiation, Kuperwasser’s team investigated SLUG’s role in breast-cell differentiation and tumor growth.

The research team reduced the expression of the SLUG gene in human-derived breast cells and then used cell-sorting techniques to separate the cells into groups of luminal, basal, and stem cells. Next, they used mathematical modelling to measure the rate and frequency that each of the three cell types changed into another cell type. By comparing the rates between control cells and cells in which SLUG was reduced, the team was able to determine the role of SLUG in luminal-, basal-, and stem-cell transitions.

To test the result of their mathematical model, the research team examined and compared breast-tissue samples from mice in two groups: a control group with normal SLUG and an experimental group that did not express SLUG. Mammary glands from the experimental and control groups were analyzed for changes in structure, the amount and distribution of luminal and basal cells in the gland, and whether these cells had stem-cell activity.

The SLUG-deficient mice exhibited defects in breast-cell differentiation. The mammary glands of these mice had too many luminal cells and defective basal cells that had luminal-cell characteristics. The control group of normal mice had a normal ratio of luminal to basal cells.

The SLUG-deficient mice showed defects in stem-cell function: Specifically, tumour formation and tissue regeneration was inhibited, an indication of defective stem cells, suggesting that SLUG was necessary to maintain normal luminal and basal cells within the mammary gland.

Additionally, SLUG-deficient cells when transplanted could not regenerate the mammary gland of the mouse, suggesting that SLUG is necessary for mammary stem-cell function. Tumour formation was also inhibited in SLUG-deficient mice, suggesting that SLUG may affect stem-cell activity necessary for tumour formation. Tufts University

Dartmouth Institute for Quantitative Biomedical Sciences (iQBS) researchers developed a new gene expression analysis approach for identifying cancer genes. The study results challenge the current paradigm of microarray data analysis and suggest that the new method may improve identification of cancer-associated genes.

Typical microarray-based gene expression analyses compare gene expression in adjacent normal and cancerous tissues. In these analyses, genes with strong statistical differences in expression are identified. However, many genes are aberrantly expressed in tumours as a byproduct of tumorigenesis. These ‘passenger’ genes are differentially expressed between normal and tumour tissues, but they are not ‘drivers’ of tumorigenesis. Therefore, better analytical approaches that enrich the list of candidate genes with authentic cancer-associated ‘driver’ genes are needed.

Lead authors of the study, Ivan P. Gorlov, PhD, Associate Professor of Community and Family Medicine and Christopher Amos, PhD, Professor of Community and Family Medicine and Director of the Center for Genomic Medicine described a new method to analyse microarray data. The research team demonstrated that ranking genes based on inter-tumour variation in gene expression outperforms traditional analytical approaches. The results were consistent across four major cancer types: breast, colorectal, lung, and prostate cancer.

The team used text-mining to identify genes known to be associated with breast, colorectal, lung, and prostate cancers. Then, they estimated enrichment factors by determining how frequently those known cancer-associated genes occurred among the top gene candidates identified by different analysis methods. The enrichment factor described how frequently cancer associated genes were identified compared to the frequency of identification that one could expect by pure chance. Across all four cancer types, the new method of selecting candidate genes based on inter-tumour variation in gene expression outperformed the other methods, including the standard method of comparing mean expression in adjacent normal and tumour tissues. Dr. Gorlov and colleagues also used this approach to identify novel cancer-associated genes.

The authors cite tumour heterogeneity as the most likely reason for the success of their variance-based approach. The method is based on the knowledge that different tumours can be driven by different subsets of cancer genes. By identifying genes with high variation in expression between tumours, the method preferentially identifies genes specifically associated with cancer. This same feature, tumour heterogeneity, may reduce the ability to identify critical gene expression changes when comparing mean gene expression in adjacent tumor and normal tissues, as tumors of the same type may have different sets of genes differentially expressed.

The results of the study challenge the model that comparing mean gene expression in adjacent normal and cancer tissues is the best approach to identifying cancer-associated genes. Indeed, the team identified high variation in adjacent ‘normal’ tissue samples, which are typically used as control samples for comparison in analyses based on mean gene expression. The study suggests that methods based on variance may help get the most from existing and future global gene expression studies. Dartmouth Institute for Quantitative Biomedical Sciences

EKF Diagnostics has acquired Separation Technology, Inc. (STI), the Florida based manufacturer of in vitro diagnostics devices for hematology testing from Thermo Fisher Scientific, Inc. This acquisition complements EKF’s existing offering in the hemoglobin testing market place, which includes Hemo Control (also sold as HemoPoint H2 in USA and Asia). Notably, STI’s primary instrument is the UltraCrit hematocrit measurement device which is FDA-cleared for blood donor screening. STI develops, manufactures and markets specialty IVD devices including ultrasound instruments and tabletop centrifuges for the hematology testing market. STI also has an in-house engineering capability, including product design, production support and new product development. STI’s UltraCrit is the first and only hematocrit/hemoglobin device to use ultrasound technology. The hematocrit reading is displayed automatically in about 30 seconds and provides a hematocrit value that allows for standardization for all collections, including whole blood, apheresis and double red cell collections. UltraCrit uses reagentless cuvettes, a major point of differentiation between different analysers.

EKF Diagnosticswww.ekfdiagnostics.com

A newly identified difference between the brains of women and men with multiple sclerosis (MS) may help explain why so many more women than men get the disease, researchers at Washington University School of Medicine in St. Louis report.
In recent years, the diagnosis of MS has increased more rapidly among women, who get the disorder nearly four times more than men. The reasons are unclear, but the new study is the first to associate a sex difference in the brain with MS.
Studying mice and people, the researchers found that females susceptible to MS produce higher levels of a blood vessel receptor protein, S1PR2, than males and that the protein is present at even higher levels in the brain areas that MS typically damages.
‘It was a ‘Bingo!’ moment – our genetic studies led us right to this receptor,’ said senior author Robyn Klein, MD, PhD. ‘When we looked at its function in mice, we found that it can determine whether immune cells cross blood vessels into the brain. These cells cause the inflammation that leads to MS.’
An investigational MS drug currently in clinical trials blocks other receptors in the same protein family but does not affect S1PR2. Klein recommended that researchers work to develop a drug that disables S1PR2.
MS is highly unpredictable, flaring and fading at irregular intervals and producing a hodgepodge of symptoms that includes problems with mobility, vision, strength and balance. More than 2 million people worldwide have the condition.
In MS, inflammation caused by misdirected immune cells damages a protective coating that surrounds the branches of nerve cells in the brain and spinal column. This leads the branches to malfunction and sometimes causes them to wither away, disrupting nerve cell communication necessary for normal brain functions such as movement and co-ordination.
For the new research, Klein studied a mouse model of MS in which the females get the disease more often than the males. The scientists compared levels of gene activity in male and female brains. They also looked at gene activity in the regions of the female brain that MS damages and in other regions the disorder typically does not harm.
They identified 20 genes that were active at different levels in vulnerable female brain regions. Scientists don’t know what 16 of these genes do. Among the remaining genes, the increased activity of S1PR2 stood out because researchers knew from previous studies that the protein regulates how easy it is for cells and molecules to pass through the walls of blood vessels.
Additional experiments showed that S1PR2 opens up the blood-brain barrier, a structure in the brain’s blood vessels that tightly regulates the materials that cross into the brain and spinal fluid. This barrier normally blocks potentially harmful substances from entering the brain. Opening it up likely allows the inflammatory cells that cause MS to get into the central nervous system.
When the researchers tested brain tissue samples obtained from 20 patients after death, they found more S1PR2 in MS patients’ brains than in people without the disorder. Brain tissue from females also had higher levels of S1PR2 than male brain tissue. The highest levels of S1PR2 were found in the brains of two female patients whose symptoms flared and faded irregularly, a pattern scientists call relapsing and remitting MS.
Klein is collaborating with chemists to design a tracer that will allow scientists to monitor S1PR2 levels in the brains of people while they are living. She hopes this will lead to a fuller understanding of how S1PR2 contributes to MS. Washington University School of Medicine

Using next generation DNA sequencing, Dartmouth scientists have identified potentially actionable mutations in cancers of the appendix. When specific mutations for a cancer type are identified, patients can be treated with chemotherapy or other targeted agents that work on those mutations.
Little is known about the molecular biology of two types of appendix tumours, low-grade appendiceal mucinous neoplasm (LAMN) and adenocarcinoma, but both can lead to pseudomyxoma peritonea (PMP), a critical condition in which cancerous cells grow uncontrollably along the wall of the abdomen and can crush digestive organs.

Dartmouth pathologists studied 38 specimens of LAMN and adenocarcinoma tumors (some of which had progressed to PMP) from their archives to look for shared genetic errors that might be responsible for the abnormal cell growth. Tissue samples were sequenced using the AmpiSeq Hotspot Cancer Panel v2, which pathologists had verified for the clinical screening of mutations in 50 common cancer-related genes for which treatments exist. This was the first study making use of a multigene panel in appendiceal cancers to support the use of potential targeted therapies.

‘We routinely use this molecular profiling approach on all of our lung adenocarcinomas, melanomas, colon cancers, and gliomas,’ said Gregory Tsongalis, PhD, principal investigator for the study and director of Molecular Pathology at Dartmouth-Hitchcock Norris Cotton Cancer Center. He says examining an individual tumour profile has the potential to significantly alter patient outcome in a positive way.

KRAS and GNAS mutations were the most common alterations identified in the study. Twelve distinct abnormalities were mapped to the KRAS gene. Additional mutations were identified (i.e., AKT1, APC, JAK3, MET, PIK3CA, RB1, and STK11 for LAMN and TP53, GNAS, and RB1 for adenocarcinoma) in the four sample types studied. Seven of these mutations were shared by more than one group, which suggests there is some molecular similarity.

‘These findings suggest that tumours of the appendix, although rare and very aggressive, are distinct entities and have subclasses of disease within each category that are different from each other based on their mutation profile,’ said Tsongalis. ‘New therapeutic approaches may be able to target those pathways that are mutated in these tumour types.’

This laboratory research has the potential to change clinical practice if physicians now develop treatment plans to target the identified genetic mutations. ‘Our success in the Dartmouth-Hitchcock Medical Center Department of Pathology at the Norris Cotton Cancer Center is attributed to our multidisciplinary approach to these discoveries, which truly allow us to bring scientific findings from the bench to the bedside,’ said Tsongalis. Norris Cotton Cancer Center at Dartmouth-Hitchcock