A new technology developed by Harvard Medical School researchers at the Massachusetts General Hospital Center for Systems Biology allows the simultaneous analysis of hundreds of cancer-related protein markers from minuscule patient samples gathered through minimally invasive methods. This powerful and sensitive technology uses antibodies linked to unique DNA ‘barcodes’ to detect a wide range of target proteins.

It could serve as a tool to help clinicians gain insights into the biology of cancer progression as well as determine why certain cancer therapies stop working or are ineffective to begin with.

Minimally invasive techniques—such as fine-needle aspiration or circulating tumour cell analysis—are increasingly employed to track treatment response over time in clinical trials, as the tests can be simple and cheap to perform. Fine-needle aspirates are also much less invasive than core biopsies or surgical biopsies, since very small needles are used. The challenge has been to comprehensively analyse the very few cells that are obtained via this method.

‘What this study sought to achieve was to vastly expand the information that we can obtain from just a few cells,’ explained Cesar Castro, HMS instructor in medicine at Mass General and a co-author of the paper. ‘Instead of trying to procure more tissue to study, we shrank the analysis process so that it could now be performed on a few cells.’

Until now, pathologists have been able to examine only a handful of protein markers at a time for tumour analyses. With this new technology, the researchers have demonstrated the ability to look at hundreds of markers simultaneously, down to the single-cell level.

‘We are no longer limited by the scant cell quantities procured through minimally invasive procedures,’ Castro said. ‘Rather, the bottleneck will now be our own understanding of the various pathways involved in disease progression and drug target modulation.’

The new method uses an approach known as DNA-barcoded antibody sensing, in which unique DNA sequences are attached to antibodies against known cancer marker proteins. The DNA ‘barcodes’ are linked to the antibodies with a special type of glue that breaks apart when exposed to light. When mixed with a tumour sample, the antibodies seek out and bind to their targets; then a light pulse releases the unique DNA barcodes of these bound antibodies that are subsequently tagged with fluorescently labelled complementary barcodes. The tagged barcodes can be detected and quantified via imaging, revealing which markers are present in the sample.

After initially demonstrating and validating the technique’s feasibility in cell lines and single cells, the team tested it on samples from patients with lung cancer. The technology was able to reflect the great heterogeneity—differences in features such as cell-surface protein expression—of cells within a single tumour and to reveal significant differences in protein expression between tumours that appeared identical under the microscope. Examination of cells taken at various time points from participants in a clinical trial of a targeted therapy drug revealed patterns that distinguished those who did and did not respond to treatment.

‘We showed that this technology works well beyond the highly regulated laboratory environment, extending into early-phase clinical trials,’ said Castro, who is also a medical oncologist in the Mass General Cancer Center and director of the Cancer Program within the hospital’s Center for Systems Biology. ‘In this era of personalised medicine, we could leverage such technology not only to monitor but actually to predict treatment response. By obtaining samples from patients before initiating therapy and then exposing them to different chemo-therapeutics or targeted therapies, we could select the most appropriate therapy for individual patients.’ Harvard Medical School

In a new study, researchers from North Carolina State University, UNC-Chapel Hill and other institutions have taken the first steps toward creating a roadmap that may help scientists narrow down the genetic cause of numerous diseases. Their work also sheds new light on how heredity and environment can affect gene expression.
Pinpointing the genetic causes of common diseases is not easy, as multiple genes may be involved with a disease. Moreover, disease-causing variants in DNA often do not act directly, but by activating nearby genes. To add to the complexity, genetic activation is not like a simple on/off switch on a light, but behaves more like a ‘dimmer switch’ – some people may have a particular gene turned all the way up, while others have it only turned halfway on, completely off, or somewhere in between. And different factors, like DNA or the environment, play a role in the dimmer switch’s setting.
According to Fred Wright, NC State professor of statistics and biological sciences, director of NC State’s Bioinformatics Center and co-first author of the study, ‘Everyone has the same set of genes. It’s difficult to determine which genes are heritable, or controlled by your DNA, versus those that may be affected by the environment. Teasing out the difference between heredity and environment is key to narrowing the field when you’re looking for a genetic relationship to a particular disease.’
Wright, with co-first author Patrick Sullivan, Distinguished Professor of Genetics and Psychiatry at UNC-Chapel Hill and director of the Center for Psychiatric Genomics, and national and international colleagues, analyzed blood sample data from 2,752 adult twins (both identical and fraternal) from the Netherlands Twin Register and an additional 1,895 participants from the Netherlands Study of Depression and Anxiety. For all 20,000 individual genes, they determined whether those genes were heritable – controlled by the DNA ‘dimmer switch’ – or largely affected by environment.
‘Identical twins have identical DNA,’ Wright explains, ‘so if a gene is heritable, its expression will be more similar in identical twins than in fraternal twins. This process allowed us to create a database of heritable genes, which we could then compare with genes that have been implicated in disease risk. We saw that heritable genes are more likely to be associated with disease – something that can help other researchers determine which genes to focus on in future studies.’
‘This is by far the largest twin study of gene expression ever published, enabling us to make a roadmap of genes versus environment,’ Sullivan says, adding that the study measured relationships with disease more precisely than had been previously possible, and uncovered important connections to recent human evolution and genetic influence in disease.
The Netherlands Twin Register has followed twin pairs for over 25 years and in collaboration with the longitudinal Netherlands Study of Depression and Anxiety established a resource for genetic and expression studies. Professor Dorret Boomsma, who started the twin register, says, ‘in addition to the fundamental insights into genetic regulation and disease, the results provide valuable information on causal pathways. The study shows that the twin design remains a key tool for genetic discovery.’ EurekAlert

Women who are members of families with BRCA2 mutations but who test negative for the family-specific BRCA2 mutations are still at greater risk for developing breast cancer compared with women in the general population.
Women with certain mutations in their BRCA1 or BRCA2 genes are at increased risk for breast cancer. However, if a woman who comes from a BRCA family tests negative for her family-specific BRCA mutation, her risk for breast cancer is considered to be the same as someone in the general population, according to the National Cancer Institute. This study, however, suggests that it may not always be true.
‘We found that women who test negative for family-specific BRCA2 mutations have more than four times the risk for developing breast cancer than the general population,’ said Gareth R. Evans, M.B.B.S., M.D., M.R.C.P., F.R.C.P., honorary professor of medical genetics and cancer epidemiology at the Manchester Academic Health Science Center at the University of Manchester in the United Kingdom. ‘We also found that any increased risk for breast cancer is largely limited to BRCA2 families with strong family history and other genetic factors.
‘It is likely that these women inherit genetic factors other than BRCA-related genes that increase their breast cancer risk,’ he explained. ‘About 77 single nucleotide polymorphisms [SNPs—genetic variations that can help track the inheritance of disease genes within families] are linked to breast cancer risk. Identification of additional SNPs is necessary to understand why some of the BRCA-negative women from BRCA families are at higher risk.’
The authors note that specialists should use caution when stating that a woman’s breast cancer risk is the same as that of the general population following a negative test, because it may not be true for some women who come from BRCA2 families with a strong family history.
Evans and colleagues used data from the M6-Inherited Cancer in England study, which has screened families of individuals with breast and/or ovarian cancer for mutations in BRCA1 and 2 since 1996. Details on affected individuals, and all tested and untested relatives, were entered into a Filemaker Pro-7 database. From 807 BRCA families, the researchers identified 49 women who tested negative for the family-specific BRCA mutation, but subsequently developed breast cancer. The researchers called these women ‘phenocopies.’
Of the 49 phenocopies identified, 22 were among 279 women who tested negative from BRCA1 families, and 27 were among 251 women who tested negative from BRCA2 families. When the researchers stratified the phenocopies based on their age (30-39, 40-49, 50-59, and 69-80), they found that in each age range there were about twice as many cases of breast cancer as would have been expected from the general population.
Next, to conduct risk analyses, Evans and colleagues calculated the ‘observed versus expected ratio’ (O/E), a ratio of observed risk for breast cancer in BRCA-negative women from BRCA families, versus the risk expected for any woman in the general population.
They found the O/E for phenocopies from BRCA1 families was not substantially higher than that of the general population; however, the O/E for phenocopies from BRCA2 families was 4.57, leading them to conclude that the more than fourfold increased risk for breast cancer among BRCA-negative women is largely limited to BRCA-negative women from BRCA2 families.
When the researchers considered the date of predictive testing (the date on which BRCA testing was done for an individual) instead of the date of family ascertainment (the date the first family member of the individual was referred to genetic service), O/E dropped from 4.57 to 2.01, ‘because there is less follow-up in the predictive test group from time of testing and we may be unaware of breast cancers that have occurred in the near past,’ explained Evans. American Association for Cancer Research

Researchers have developed a novel technique for detecting ALK rearrangements in non-small cell lung cancers (NSCLCs) that is more sensitive and easier to perform than currently available techniques. The technique can help enhance the routine practice of diagnostic ALK testing on NSCLCs, which is crucial for identifying patients with advanced NSCLC who are most likely to benefit from targeted therapy with an ALK inhibitor.

None of the current three routine methods used to detect ALK rearrangements in NSCLC is without drawbacks, especially for tissue specimens that are fixed in formalin and embedded in paraffin. Fluorescent in situ hybridisation (FISH) is the only approved technique for ALK testing, but it is not always feasible because of high cost, the time required for testing, and the need for specialized equipment and expertise. Interpretation of immunohistochemistry (IHC) results can be challenging because of weak and variable immunoreactivity. Reverse transcription-polymerase chain reaction (RT-PCR) is highly sensitive, but requires high-quality RNA, which is often difficult to obtain, and cannot detect rearrangements with unknown partners.

The novel technique, based on quantitative (q)RT-PCR, overcomes these issues by capitalising on the sensitivity of RT-PCR and including two features: an RNA isolation method that was optimised to reverse formaldehyde modification and small RT-PCR amplicons to allow for the use of fragmented nucleic acids for efficient amplification of ALK cDNA. The novel qRT-PCR test measures the expression of the 5’ and the 3’ portions of the ALK transcript separately; it detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) of 523 interpretable NSCLC specimens and full-length ALK transcript expression in six tumors (1.1%). Both FISH and qRT-PCR testing were done on 182 tumors; qRT-PCR accurately typed 97% of 19 tumours with ALK rearrangements and 158 with no rearrangements.

‘The qRT-PCR technique reliably detects ALK-rearranged tumours independently of the fusion partner and also identifies tumours with full-length transcript expression of the gene that is not detectable by FISH but may be relevant for ALK inhibitor therapy as well,’ says lead author Claudia Kalla, PhD, of the Department of Clinical Pathology, Robert-Bosch-Krankenhaus and theDr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany. ‘The technique seems to be a sensitive, easy-to-perform, and high-throughput method suitable for the routine diagnosis of ALK activation not only in lung cancer, but also in other tumour entities where rearrangements with alternative fusion partners or transcriptional upregulation are prevalent.’ The International Association for the Study of Lung Cancer (IASLC)