Background: Colorectal cancer is the second most common cause of cancer related deaths
in the United States, yet it is potentially the most treatable and preventable cancer with
effective screening. Stool DNA testing offers a noninvasive, accurate, affordable, and userfriendly
approach to achieve effective detection of both colorectal cancer and precancerous
adenomas. Exact Sciences is working on a next generation stool DNA screening test and
has developed a multiplexed quantitative Invader® assay for the simultaneous detection of
methylated and unmethylated sequences in the promoter region of the vimentin gene, a
known colorectal cancer marker. We herein report the performance of this DNA assay on
colorectal tissues from cancers, pre-malignant adenomas, and normal epithelia.
Objective: To develop a qInvader approach for sensitive detection of methylated markers
based on real-time fluorescence measurement, and to evaluate its performance in detection
of methylated vimentin in colorectal neoplasms.
Methodology: qInvader combines PCR with Invader chemistry and detects Invader
signal during each amplification cycle in a similar fashion to real-time PCR. A two dye
multiplexed qInvader assay was designed to detect both methylated and unmethylated
vimentin. The dynamic range of this assay for quantifying methylated and unmethylated
vimentin was determined using 10-fold serially diluted plasmids. Analytical sensitivity
was measured by detecting methylated vimentin spiked in unmethylated vimentin and
vice versa at 10, 1.0, 0.1, and 0.01% ratios. In addition, detection of vimentin methylation
in tissues from 22 colorectal cancers, 10 pre-malignant advanced adenomas (i.e. size >/=
1cm), and 12 normal colorectal epithelia was also performed, with all patients and normal
controls age and gender matched. Methylation level was defined as the ratio of methylated
vimentin copies to unmethylated copies multiplied by 10,000.
Results: qInvader linearly detected methylated or unmethylated vimentin down to <10
copies. No cross reactivity was observed when methylated and unmethylated assays
were used to amplify 105 copies of unmethylated and methylated genes, respectively.
The multiplex assay detected methylated vimentin spiked in unmethylated vimentin at
0.01% ratio and vice versa. Median methylation level was 76 (range; 0~1,089,202) in
cancers, 4766 (range; 0~7,329,650) in adenomas, and 0 (range; 0~0.1) in normal epithelia.
Vimentin methylation level was significantly higher in cancer and adenoma than in normal
epithelia (p<0.01). At a specificity cutoff of 100%, methylated vimentin was detected in
68% (15/22) of cancers and in 90% (9/10) adenomas. Notably, methylated vimentin was
not detected in 11 of 12 normals.
Conclusions: Methylation specific qInvader is a promising approach for quantifying
methylated DNA markers. The vimentin qInvader assay discriminated between colorectal
neoplasia and normal samples at high sensitivity and specificity.
From the AACC site:
Annual Meeting Abstracts
'We generally begin soliciting abstracts for poster presentations at the Annual Meeting in November of the prior year with an early January deadline. Presenters are notified in April whether their abstracts have been accepted for the meeting.'
FDA stopped sales of EXAS SDNA tests (which used multiple markers), requiring full FDA trials. (They never suggested it wasn't safe and effective.) For technical reasons, Labcorp was able to get around the FDA objection by going to a single marker test, which they did (Colosure, vimentin only). So, the FDA forced us to replace a previous generation test with one that was less effective. What really happened IMO is Medicare asked FDA's opinion about whether to reimburse, FDA didn't want to assume that respnsibility without full trials (which would take a year or two and more money than EXAS had). So, they more rigidly enforced the rules re lab developed tests ("home brews"). Net result, EXAS nearly ceased to exist, and tens of thousands more people died for lack of a patient friendly CRC screening test. FDA could have handles it a lot better IMO, certainly prior EXAS management also contributed greatly to what has become an insane amount of delay deploying desparately needed life-saving technology. Between the two of them, and imperfect laws, probably resulted in as many American deaths as Vietnam (or ten Iraq Wars), IMO.
I think laws re FDA approval need revision. For technology that will save lives that wouild otherwise be lost, when there is substantial evidence it is safe and effective, there should be a way to make it available (with full disclosure) during the year or two it takes to do full FDA trials. The lab developed test option was supposed to help provide that, but it doesn't seem to work lately. (DNDN story is another example.)
Markowitz/Case Western discovered and patented Vimentin for CRC screening and EXAS purchased the exclusive global license for it (for SDNA). EXAS for years has had an ongoing research relationship with Case Western and others (including Vogelstein at Johns Hopkins).
EXAS funded the research published by Itzkowitz et al 2007 (Markowitz was a co-author), which helped to both externally validate and improve EXAS SDNA technology (Itzkowitz was at Mount Sinai Medical Center).
Naturally, when the SDNA R&D was essentially stopped for a year and the company rebuilt with an entirely new team (retaining only medical officer Dr. Berger), the new team made some course changes (thank goodness, they've made some wonderful improvements). Naturally, with all the improvements, the validation process is being started fresh. (More markers, automated, smaller sample, improved DNA extraction, improved methylation detection, FIT.)
Labcorp put together Colosure with just one marker (vimentin), which got around the technical objection FDA made. It is not automated, they don't make any money doing it, their license from EXAS is short term without expectation of renewal, and without full FDA approval no CRC screening test is very marketable, so Labcorp has never attempted to promote it, even a little. (It's hard to find info about it from them even if you go digging for it.) It's no surprise it doesn't sell much, even though it is by far the most effective CRC screening test that doesn't require bowel prep. Most doctors have no idea it exists. It also requires collection of a whole stool, which is very inconvenient.
"The FDA issues never did shut down the sales by Lab Corp or did the sales start after the fact?"
This is all "water under the bridge" but EXAS received an FDA "Warning Letter" back in the Fall of 2008 because the previous CEO's at Exact ( Hardison and Luber ) were such naive knuckle-heads that believed that they didn't have to achieve FDA approval for PreGen+. They felt that they only needed to go the simplistic "Homebrew" route with LabCorp and thus not have to jump through the hoops of the FDA.
It appears as though the FDA had been trying to shepard Hardison and Luber in the right direction all along, but they were simply "tone-deaf". It takes a lot to receive a "Warning Letter" from the FDA, and these morons were clearly deaf, dumb, and blind.
They did not figure out that in order for a cancer screening test to qualify for "Centers for Medicare & Medicaide Services" (CMS) reimbursement, they needed to get the screening test ( PreGen+ ) FDA approved.
What little sales there are of the one-marker "Colosure" by LabCorp are trivial given that they are not allowed to commercially MARKET the screening test per FDA rules. Thus, "Colosure" never got off the ground because no one even really knows about it.
The difference between the current management team of Conroy, Arora, and Lidgard vs the previous management team of Luber and Hardison is "night and day".
As Conroy and Arora have eluded to on many occasions via conference calls and presentations, EXAS is currently working with the FDA and CMS in order to not be "surprised" by any further requirements once the clinical trial has been completed.
The FDA issues never did shut down the sales by Lab Corp or did the sales start after the fact?
What exactly was the settelement between the FDA and Exact?
Sorry if I am rehashing old things but there may be others here like me that don't know all the details.
Yes I had read about the peoblems with the FDA.
So the research by Dr. Markowitz was being conducted specifically for Exact Sciences?
The prospective clinical trial that was being run was by or for Exact Sciences and it was shut down when the FDA problems started and has to do with the validation study now being run?
And of course if it is one and the same study the one being conducted now is a little different? More markers etc.?
Looks like a pretty good article (I hadn't seen it), Itzkowitz published key SDNA paper in 2007, he is excellent, although not sure how in the loop he is re the newest stuff.
Keep looking into this, it's worth it. Looks like another DNDN or better, IMO. Check the Mayo Clinic site re SDNA. Ahlquist is a key researcher now, also Zou.
In 2007 the FDA took steps that shut down commercializtion of SDNA by EXAS, which eventually resulted in the complete cessation of R&D and the near destruction of the company (based on legal technicalities, not based on any problem with the test). So, planned research did not happen. It will be released later this year (val study).
We've been back on track with since new management since 2009. There has been amazing progress the past year, vastly improved DNA extraction and ability to discriminate methylated vs unmethyated vimentin molecules (100 fold improvement of a crucial step, reported in recent abstract). Also, new results include pre-cancer not in previous Markowitz research.