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Old 12-28-2016, 05:50 PM
gdpawel gdpawel is offline
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Default Liquid Biopsy Monitors Response in Ovarian Cancer

Medscape Medical News

Another study shows that a "liquid biopsy" that measures levels of circulating tumor DNA (ctDNA) can be useful for monitoring cancer patients – this time patients with ovarian cancer.

At a recent cancer research meeting, experts predicted that liquid biopsies are "the future." Jean-Charles Soria, MD, chair of the scientific committee for the EORTC-NCI-AACR symposium and director of the Site de Recherche Intégrée sur le Cancer (SIRIC) Socrate project at Gustave Roussy Cancer Campus, Paris, said that liquid biopsies are going to "completely change the rules of engagement" for patient management and clinical practice.

Dr Soria continued: "I really think ― and I'm ready to bet ― that this is the most transformational thing that's going to happen in oncology in terms of how it's going to impact cancer clinical trials and cancer daily management for the next 5 years."

The latest study, conducted by researchers from Cancer Research UK Cambridge Institute, was published online December 20 in PLOS Medicine. The researchers found that ctDNA test results correlated with the size of ovarian cancers and was predictive of response to treatment or time to disease progression.

"These findings have strong potential for clinical utility owing to the ease of assaying DNA in plasma and the low cost and speed of ctDNA testing," write the authors, led by Nitzan Rosenfeld, PhD, and James Brenton, MD, PhD, both of Cancer Research UK. "Having very early information on response would empower patients and physicians to test alternative treatment options and have high utility in trials that link biomarkers to targeted therapy."

The standard clinical blood test used in ovarian cancer is the serum protein cancer antigen 125 (CA-125), which is sensitive but lacks specificity for detecting ovarian cancer, note the authors. In addition, CA-125 levels do not change quickly enough following treatment to inform decisions regarding changes in chemotherapy after one or two cycles in cases in which the patient is not responding.

Therefore, there is a need for better tumor markers, and ctDNA is a promising candidate, the authors suggest.

Sensitive to Treatment Response

Somatic TP53 mutations are a defining feature of high-grade serous ovarian carcinoma (HGSOC). Dr Rosenfeld, Dr Brenton, and colleagues evaluated the use of these mutations as personalized markers to monitor tumor burden, early response to treatment, and time to progression.

They conducted a retrospective analysis using 318 blood samples that had been obtained from 40 HGSOC patients. Patient-specific TP53 assays were designed and used to quantify the amount of ctDNA in the samples that were collected before, during, and after chemotherapy.

Of these samples, 261 were collected during treatment of relapsed disease. Treatment included 54 courses of chemotherapy (n = 32 patients). An additional 57 specimens were collected from 12 patients during first-line treatment with chemotherapy.

The findings showed that the fraction of mutated TP53 in ctDNA (TP53MAF) had a significant correlation with disease volume, as measured by CT scan (Pearson r = 0.59; P < .001). Pretreatment TP53MAF levels correlated with the time to progression.

These findings are consistent with previous studies across a range of tumor types, note the authors, which found that ctDNA levels increase as stage increases.

Patient response to chemotherapy was seen much earlier with ctDNA, with a median time of 37 days, as compared to 84 days with CA-125.

The ratio of TP53MAF to volume of disease was higher in relapsed patients (0.04%/cm3) as compared to patients who were untreated (0.0008%/cm3; P = .004).

In 49 treatment courses for relapsed disease, pretreatment TP53MAF concentration, but not CA-125, was associated with time to progression.

Among patients being treated for relapsed disease, a decrease in TP53MAF of >60% was an independent predictor of longer time to progression (hazard ratio, 0.22; P = .008), whereas a decrease in TP53MAF of ≤60% was associated with poor response and could identify patients for whom time to progression was less than 6 months (sensitivity, 71%; specificity, 88%).

"TP53 ctDNA has the potential to be a clinically useful blood test to assess prognosis and response to treatment in women with HGSOC," they conclude.

These findings need to be confirmed in larger, prospective studies with patients receiving uniform treatment, the authors comment. If the findings do hold up, then "TP53 ctDNA could be used in HGSOC clinical trials and routine practice to identify earlier whether treatment is effective," the authors add.

The study was supported by Cancer Research UK. Dr Rosenfeld, Dr Brenton, and coauthor Davina Gale are cofounders, shareholders, and officers/consultants of Inivata Ltd, a cancer genomics company that commercializes ctDNA analysis.

PLoS Med. Published online December 20, 2016.

[url]http://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1002198

Citation: Liquid Biopsy Monitors Response in Ovarian Cancer Medscape Medical News Roxanne Nelson December 27, 2016.
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  #2  
Old 12-28-2016, 05:53 PM
gdpawel gdpawel is offline
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Default Heterogeneous populations of circulating tumor cells

The cells that slough off from a cancerous tumor into the bloodstream are a genetically diverse bunch, Stanford University School of Medicine researchers have found. Some have genes turned on that give them the potential to lodge themselves in new places, helping a cancer spread between organs. Others have completely different patterns of gene expression and might be more benign, or less likely to survive in a new tissue. Some cells may even express genes that could predict their response to a specific therapy. Even within one patient, the tumor cells that make it into circulating blood vary drastically.

The finding underscores how multiple types of treatment may be required to cure what appears outwardly as a single type of cancer, the researchers say. And it hints that the current cell-line models of human cancers, which showed patterns that differed from the tumor cells shed from human patients, need to be improved upon.

The new study, which was published online in PLoS ONE, is the first to look at so-called circulating tumor cells one by one, rather than taking the average of many of the cells. And it's the first to show the extent of the genetic differences between such cells.

"Within a single blood draw from a single patient, we're seeing heterogeneous populations of circulating tumor cells," said senior study author Stefanie Jeffrey, MD, professor of surgery and chief of surgical oncology research.

For over a century, scientists have known that circulating tumor cells, or CTCs, are shed from tumors and move through the bloodstreams of cancer patients. And over the past five years, there's been a growing sense among many cancer researchers that these cells - accessible by a quick blood draw - could be the key to tracking tumors non-invasively. But separating CTCs from blood cells is hard; there can be as few as one or two CTCs in every milliliter of a person's blood, mixed among billions of other blood cells.

To make their latest discovery, Jeffrey, along with an interdisciplinary team of engineers, quantitative biologists, genome scientists and clinicians, relied on a technology they developed in 2008. Called the MagSweeper, it's a device that lets them isolate live CTCs with very high purity from patient blood samples, based on the presence of a particular protein - EpCAM - that's on the surface of cancer cells but not healthy blood cells.

With the goal of studying CTCs from breast cancer patients, the team first tested whether they could accurately detect the expression levels of 95 different genes in single cells from seven different cell-line models of breast cancer - a proof of principle since they already knew the genetics of these tumors. These included four cell lines generally used by breast cancer researchers and pharmaceutical scientists worldwide and three cell lines specially generated from patients' primary tumors.

"Most researchers look at just a few genes or proteins at a time in CTCs, usually by adding fluorescent antibodies to their samples consisting of many cells," said Jeffrey. "We wanted to measure the expression of 95 genes at once and didn't want to pool our cells together, so that we could detect differences between individual tumor cells."

So once Jeffrey and her collaborators isolated CTCs using the MagSweeper, they turned to a different kind of technology: real-time PCR microfluidic chips, invented by a Stanford collaborator, Stephen Quake, PhD, professor of bioengineering. They purified genetic material from each CTC and used the high-throughput technology to measure the levels of all 95 genes at once. The results on the cell-line-derived cells were a success; the genes in the CTCs reflected the known properties of the mouse cell-line models. So the team moved on to testing the 95 genes in CTCs from 50 human breast cancer patients - 30 with cancer that had spread to other organs, 20 with only primary breast tumors.

"In the patients, we ended up with 32 of the genes that were most dominantly expressed," said Jeffrey. "And by looking at levels of those genes, we could see at least two distinct groups of circulating tumors cells." Depending on which genes they used to divide the CTCs into groups, there were as many as five groups, she said, each with different combinations of genes turned on and off. And if they'd chosen genes other than the 95 they'd picked, they likely would have seen different patterns of grouping. However, because the same individual CTCs tended to group together in multiple different analyses, these cells likely represent different types of spreading cancer cells.

The diversity, Jeffrey said, means that tumors may contain multiple types of cancer cells that may get into the bloodstream, and a single biopsy from a patient's tumor doesn't necessarily reflect all the molecular changes that are driving a cancer forward and helping it spread. Moreover, different cells may require different therapies. One breast cancer patient studied, for example, had some CTCs positive for the marker HER2 and others lacked the marker. When the patient was treated with a drug designed to target HER2-positive cancers, the CTCs lacking the molecule remained in her bloodstream.

When the team went on to compare the diverse genetic profiles of the breast cancer patients' CTCs with the cells they'd studied from the cell lines, they were in for another surprise: None of the human CTCs had the same gene patterns as any of the cell-line models.

"These models are what people are using for drug discovery and initial drug testing," said Jeffrey, "but our finding suggests that perhaps they're not that helpful as models of spreading cancers." While the human cell-line cells did show diversity between each of the seven cell lines, they didn't fall into any of the same genetic profiles as the CTCs from human blood samples.

These results don't have immediate impacts for cancer patients in the clinic because more work is needed to discover whether different types of CTCs respond to different therapies and whether that will be clinically useful for guiding treatment decisions. But the finding is a step forward in understanding the basic science behind the bits of tumors that circulate in the blood. It's the first time that scientists have used high-throughput gene analysis to study individual CTCs, and opens the door for future experiments that delve even more into the cell diversity. The Stanford team is now working on different methods of using CTCs for drug testing as well as studying the relationship between CTC genetic profiles and cancer treatment outcomes. They've also expanded their work to include primary lung and pancreatic cancers as well as breast tumors.

Source: Stanford University Medical Center

[url]http://www.medscape.com/viewarticle/782543

Note: There is not a single validation of a molecular marker in CTCs (Liquid Biopsy) that provides prognostic information or predicts response to cancer therapies.

According to laboratory oncologist Dr. Robert A. Nagourney, liquid biopsy can mean several different things. On the one hand it can be a multiplexed biochemical, proteomic, circulating DNA types of analyses on serum. On the other, it can be circulating tumor cell (CTC) extraction mostly for genomics. The CTC approach is offered commercially and has use in target identification, when distinct driver mutations are found, but it does not capture the cellular microenvironment (e.g. stroma, vasculature, cytokines) critical to accurate response prediction of many classes of drugs. This is why cell function analysis exclusively uses fresh tissue explants.
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  #3  
Old 12-28-2016, 05:54 PM
gdpawel gdpawel is offline
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Default It's not that unusual for healthy people to have an occasional cancer cell in blood

In an Opinion column on CNN, Dr. H. Gilbert Welch, M.P.H., a professor of medicine at the Dartmouth Institute of Health Policy & Clinical Practice and the author of Overdiagnosed: Making People Sick in the Pursuit of Health" (Beacon Press 2011), raises questions about this simple new blood test that is able to detect minute quantities of cancer cells that might be circulating in your bloodstream.

“The conventional wisdom is people either have a disease or they do not. But, in fact, there are a lot of people somewhere in between. . . I don't know whether this test will help some patients. It might, but it will take years to figure that out... Ironically, what this test might actually teach us is that it's not that unusual for healthy people to have an occasional cancer cell in their blood.”

[url]http://www.cnn.com/2011/OPINION/01/11/welch.overdiagnosed.cancer/index.html?npt=NP1

Dr. Elaine Schattner pointed out that Dr. Welch may have mised the point of this technology. It was developed primarily to help oncologists monitor tumors in patients who already are known to have disease. For example, doctors could check for new, resistance-conferring mutations in patients who are already on a cocktail of meds for lung cancer.

The blood test could obiate the need for repeatedly doing CT scans and biopsies to measure disease, the extent of disease and new mutations in people undergoing cancer treatment.

The June issue of Oncology News International (June 2010, V 19, No 6) quotes a Duke University study of the use of high-tech cancer imaging, with one representative finding being that the average Medicare lung cancer patient receives 11 radiographs, 6 CT scans, a PET scan, and MRI, two echocardiograms, and an ultrasound, all within two years of diagnosis. A study co-author (Dr. Kevan Schulman) asks: "Are all these imaging studies essential? Are they all of value? Is the information really meaningful? What is changing as a result of all this imaging?"

So the investment by Johnson and Johnson, which was what the news was about, makes it more likely this will actually happen in non-research clinics. The technology has the potential to make cancer patients' lives easier and less costly and for doctors to stop giving them meds to which they've acquired resistance.

[url]http://www.scientificamerican.com/article.cfm?id=a-chip-against-cancer

My comment is not really about early cancer diagnosis. It is about prognostication and drug selection with the CTC (circulating tumor cells) technique. The number of cells discovered in the CTC technique has turned out to be a good prognosticator of how well treatments are working. Monitoring CTCs could be utilized for confirmation after the patient is administered either empiric or assay-directed most beneficial therapeutic agents.

But CTCs really aren't useful with respect to drug selection. The problem is with isolating (even by size) and analying single cancer cells. The supposition is that common cancers can be detected and cured through analysis at a genetic level of a small number of cells or even a single wayward cell. CTCs are free-floating cancer cells that can remain in isolation from a tumor for over twenty years.

And what is the relationship of such long-lasting cells to the tumor cells that needed to be attacked through tested substances? And in regards to some molecular tests utilizing living cells, generally of individual cancer cells in suspension, sometimes derived from tumors and sometimes derived from CTCs, this was tried with the old human clonogenic assay, which had been discredited long ago.

One testing approach to find CTCs actually can miscount non-tumor epithelial cells as tumor cells. And also highly invasive cells may not be detected if you are looking for epithelial antigens because the CTC also goes through a phase called "epithelial to mesenchymal transition", where you will miss locating that tumor cell if you are targeting the antigen.

The key is to look for the tumor cell and not something else that "hangs with the tumor cell."

Basically, CTC labs use "negative selection" to isolate alleged circulating tumor cells. What that means is methods to "selectively" remove circulating normal cells, such as monocytes, lymphocytes, neutrophils, circulating endothelial cells, etc. The problem is that these normal cells outnumber circulating tumor cells by a factor of a million to one, and no "negative selection" procedure (or combination of procedures) can possibly strip away all the normal cells, leaving behind a relatively pure population of tumor cells.

What you have to do is to use a "positive selection" procedure, meaning selectively extracting the tumor cells out of the vastly larger milieu of normal cells. The problem is, when you do this, there is only a teeny tiny yield of tumor cells:

Here's from Wikipedia:

Circulating tumor cells are found in frequencies on the order of 1-10 CTC per mL of whole blood in patients with metastatic disease. For comparison, a mL of blood contains a few million white blood cells and a billion red blood cells.

So, from a typical 7 ml blood draw into a purple top tube, you are going to get, on average, 7 to 70 tumor cells -- total. This may be sufficient for certain molecular type tests (although the degree to which this tiny sample of cells is representative may be questioned), but it isn't nearly sufficient to test even a single drug in a cell culture assay, where one requires millions of cells for quality testing, including requirements for negative and positive controls.
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Old 12-28-2016, 05:56 PM
gdpawel gdpawel is offline
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Default Can circulating tumor cells be effective in the determination of treatment?

Many people are interested to learn about a new blood test (which is still in early stages) said to capture migrating cancer cells so that cancer can be detected earlier; determine whether it is spreading; or if the current treatment is even working. These circulating cells are at such a low level in the bloodstream, they are normally hard (or impossible) to detect. The only test currently on the market to find tumor cells in the blood, CellSearch by Johnson & Johnson, can give a cell count, however it doesn't capture the whole cells that doctors need to analyze.

Although the test offers some amazing advances for the diagnosis of early-stage disease, Robert Nagourney, MD, medical director of Rational Therapeutics, cautions that it may not be as effective in the determination of treatment. "The circulating tumor cells may be evaluable for very specific gene profiles, but the biological behavior of isolated individual cells in response to drugs and combinations because these few cells may not reflect the same behavior as those in the micro spheroid (cancer cells in clusters, as they exist in the body) environment.

The test uses a microchip, similar to a lab slide. When blood is placed across the slide, the cancer cells stick and can be collected. J&J in conjunction with Veridex, will be working on improving the microchip. They will be trying different designs and a cheaper plastic to make it more practical for mass production.

The American Cancer Society's Dr. J. Leonard Lichtenfeld reminded us on his blog that this is not a new breakthrough. It is not something that has been proven effective in improving cancer detection and treatment. The researchers have signed a contract with a company to further develop this research and determine whether in fact it can be applied successfully to large numbers of patients in a more efficient and less expensive manner.

Oncologist Dr. Elaine Schattner has reminded us on her blog that this technology was developed, primarily, to help oncologists monitor tumors in patients who already are known to have disease. For example, doctors could check for new, resistance-conferring mutations in patients who are already on a cocktail of meds for lung cancer. The blood test could obviate the need for repeatedly doing CT scans and biopsies to measure disease the entent of disease and new mutations in people undergoing cancer treatment.

The June 2010 issue of Oncology News International (V 19, No 6) quotes a Duke University study of the use of high-tech cancer imaging, with one representative finding being that the average Medicare lung cancer patient receives 11 radiographs, 6 CT scans, a PET scan, and MRI, two echocardiograms, and an ultrasound, all within two years of diagnosis. A study co-author (Dr. Kevan Schulman) asks: "Are all these imaging studies essential? Are they all of value? Is the information really meaningful? What is changing as a result of all this imaging?"

The number of cells discovered in the circulating tumor cell (CTC) technique has turned out to be a good prognosticator of how well treatments are working. Monitoring CTCs could be utilized for confirmation after the patient is administered either physician-directed or assay-directed most beneficial therapeutic agents.

But CTCs really aren't useful with respect to drug selection. The problem is with isolating and analying single cancer cells. The supposition is that common cancers can be detected and cured through analysis at a genetic level of a small number of cells or even a single wayward cell. CTCs are free-floating cancer cells that can remain in isolation from a tumor for over twenty years.

Regardless of all of this, most of the cells that leave home don't survive the journey in the blood or lymph systems and many cancerous cells that eventually do lodge in a distant organ simply remain dormant, leaving it up to the immune system to take care of them.

Full-blown metastasis is an extremely challenging trade and the great majority of cancer cells are not up to the task. Even those malignant characters that manage to slither their way into the blood or lymph system, usually fail to do anything further.

Most tumor cells lack the streamlined form of the blood and immune cells that are designed for cross-body trafficking, shear forces in the smaller vessels may rip the intruders apart. These free-floating cancer cells can remain in isolation from a tumor for over twenty years (Gupta, G.P., and J. Massague. 2006. Cancer metastasis: building a framework. Cell. 127:679-95).
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