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Old 04-29-2016, 01:03 PM
gdpawel gdpawel is offline
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Default Aggressive Prostate Cancer and BRCA Gene Mutations

Robert A. Nagourney, M.D.

A 42 year old gentleman presented in 2003 with an aggressive prostate cancer. He underwent surgery, radiation, hormones, chemotherapy, immunotherapy and both 2nd and 3rd line androgen ablation (male hormone reduction), yet his disease progressed. When we first met, his 2012 PET scan was strongly positive and his PSA was rising. I succeeded in controlling his disease for 3 years until the growth of pelvic lymph nodes led to a biopsy in November of 2015.

We used our EVA-PCD assay to explore his tumor’s drug sensitivity but found his cancer to be drug resistant, that is, with the exception of sensitivity to platinum drugs. With his diagnosis at a young age (42) and this new found platinum sensitivity, known to be characteristic of BRCA gene mutations, I wondered whether he might be a BRCA patient.

The BRCA genes discovered in 1990 found a direct link between BRCA mutations and the early onset of breast and ovarian cancer. BRCA patients have a 30-80% lifetime risk. Widespread BRCA testing has shown that 5-10% of all patients with breast and ovarian cancers carry a “germ line” BRCA mutation that is passed down from generation to generation. And it has now been shown that BRCA mutations can increase the risk of a wide variety of other cancers.

I referred the patient to a genetic counselor who performed a blood-test for the BRCA mutation. The result returned negative. He did not have a “germ-line” BRCA mutation. As a result he would not qualify for treatment with the new and very effective PARP inhibitors that have been FDA-approved exclusively for BRCA positive patients.

I was puzzled: Young age, aggressive disease and a high degree of platinum sensitivity in our laboratory analysis. The results did not add up. No matter what his gene profile found, I had evidence from platinum sensitivity that he was BRCA mutated.

I decided to send the patient's actual tumor tissue (not his blood) for BRCA analysis. Lo and behold, despite the negative “germ line” analysis, his tumor, did carry a BRCA2 mutation, entirely consistent with our laboratory’s platinum-sensitive result.

How could this be? We now recognize that some people with BRCA abnormalities do not have the familial form. For them the terms “BRCA-ness” or “BRCA-like” are applied. Despite negative family histories these patients develop their own unique BRCA mutations within their tumors that are every bit as dangerous. These are known in the scientific literature as “somatic” mutations, from the Greek soma, for body.

Our perseverance and laboratory insights paid off as this patient is now a candidate for the PARP inhibitors that target his BRCA gene and I anticipate a good response. We learn once again that each patient’s cancer is unique and that we must be prepared to use each patient’s tissue to identify active treatments. There is much to learn about human cancer, much more it would seem than can be found through the use of generic gene profiles.
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Old 05-17-2016, 12:15 PM
gdpawel gdpawel is offline
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Default BRCA Prostate Cancer: Lessons Learned from a Successful Outcome

Robert A. Nagourney, M.D.

We reported the story of a 55 year old man diagnosed 13 years earlier with prostate cancer who had failed many prior courses of therapy. When our functional profile performed on a surgical biopsy revealed an unexpected degree of activity for platinum-based chemotherapy, I decided to pursue the possibility that this patient’s tumor might carry a mutation in the BRCA genes.

I referred him to a genetic counselor who conducted a genomic screen to rule out familial (hereditary) BRCA mutation. These results proved to be negative. Based upon our laboratory results however, my suspicions remained high and I submitted a second analysis that specifically examined his cancerous tissue (not his blood) for the BRCA mutation that was not identified by the first test. Our determination paid off as his prostate tumor tissue proved positive for BRCA. Thus the platinum sensitivity identified in our laboratory, characteristic of BRCA patients, led us to the correct diagnosis of BRCA positivity.

I wrote that we had petitioned the insurers to provide the patient Olaparib (Lynparza), the first FDA approved PARP inhibitor that is used for the treatment of BRCA patients. As the request was somewhat unusual, with most BRCA patients being women with breast and ovarian cancer, procuring the drug took some legwork. Nonetheless, we ultimately prevailed and the patient received his first cycle of this simple oral therapy beginning in mid-April.

I am delighted to report that my recent visit with this patient and his wife could not have been a happier occasion. Only three weeks after starting oral Olaparib, the patient’s very firm and enlarged left neck lymph node was demonstrably smaller, his prostatic acid phosphatase (PAP), a measure of prostate cancer, had fallen from 43 to 13.9 (normal 0-4) and his PSA, another prostate cancer test, from 26 to 13 (normal 0-4).

Although he complained of some fatigue, he has been spared the hair loss, nausea, vomiting and bone marrow suppression that is so commonly associated with classic chemotherapy treatments.

This gratifying response to a simple oral medication reflects the growing recognition that each patient must be treated with those drugs and/or combinations that are found effective for their unique disease. Without the insights gained from our laboratory analyses, this patient would be now confronting the grim prospect of ineffective and toxic chemotherapy or worse still, disease progression. We are delighted with his good results.
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Old 05-17-2016, 12:23 PM
gdpawel gdpawel is offline
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Default Caveat about blood assays

Getting tumor cells from blood maybe feasible for solid tumors, though ususally only when the tumor is very advanced, and then only in small numbers. It seems plausible that you can get enough specimen from circulating tumor cells for solid tumors. It may be possible using PCR (Polymerase Chain Reaction) or similar technology for specific agents.

Only minute quantities of DNA are necessary for PCR. DNA can be amplified from a single cell. PCR amplification techniques raise considerable concerns regarding contamination from one specimen to another, creating the potential for false positive results. Clinical interpretation of PCR results may also be challenging.

But, PCR may be useful when culture is difficult due to the low numbers of the organisms, for lengthly culture requirements, or when there is difficulty in collecting an appropriate sample. Don't know if the results would be indicative of what would happen inside the human body.

They usually proliferate (grow) cancer cells from a small sample and subject those cells to chemo. Cells 'grown' in the lab will not behave the same way as the actual cancer cells do in your body's own environment. Because they test on subcultured cells (as opposed to fresh tumor cultures) and test the cells in monolayers (as opposed to three dimensional cell clusters), the cell grown in the lab will not behave the same way as the actual cancer cells do in your body's own environment.

Older technology assay tests failed because scientists looked to see which drugs inhibited the cancer cells' growth (cell-growth endpoint), not which chemotherapies actively killed the tumor cells (cell-death endpoint). Cancer wasn't growing faster than other cells, it's just dying slower. The newer assay testing technology connects drugs to patients by what 'kills' their cells, not by what 'slows' them down.

All of the work in the past twenty years in the cell culture field has been carried out largely on three dimensional (3D) clusters of cells (not monolayers). Work is done exclusively with three dimensional, floating, tumor spheroids. When you test the cells as three dimensional spheroids, they are many-fold resistant in vitro, just as they are in vivo (multicellular resistance). Even Johns Hopkins and the Washington University at St. Louis has discovered that 3D analysis is more accurate.

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.

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.

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.

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.

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