"While mutations of the [EGFR] predict sensitivity to tyrosine kinase inhibitors [TKIs] in patients with lung adenocarcinoma, these patients often go on to develop resistance to these drugs, to EGFR [TKIs], within 6 to 12 months," explained lead investigator Maria Arcila, MD, from the Department of Molecular Pathology at Memorial Sloan-Kettering Cancer Center in New York City, during a poster presentation.
She reported that although up to 50% of this patient population has shown a secondary EGFR mutation (T790M) in their tumor tissue, previous studies suggest that this number might actually be much higher.
"Standard sequencing techniques may lead to many T790M mutations going undetected," said Dr. Arcila. "We wanted to know, if we used a more sensitive method, whether those patients did have the mutation and we just weren't seeing it — that it could be that the cells that have the mutations are just very diluted within the other tumor cells."
T790M Resistance: As High as 72%?
For this study, Dr. Arcila and her team retrospectively reviewed 39 tumor samples that were tested by standard sequencing from August 2008 to May 2009. DNA from these samples was gathered from biopsies of patients with metastatic or recurrent lung adenocarcinomas.
A total of 18 of the 24 stored mutation-negative samples were retested using a "locked nucleic acid to develop a higher-sensitivity PCR sequencing assay to allow the detection of EGFR T790M in patients with a low mutant allele burden," according to the presentation. "This step was designed to suppress the amplification of nonmutant DNA," explained Dr. Arcila.
Results showed that 15 of the standard sequencing samples (38%) were found to be positive for the T790M mutation on initial testing.
The locked nucleic acid test detected the mutation in another 10 cases (56%). In addition, "the detection sensitivity of similar locked nucleic acid assays for specific point mutations, based on serial dilutions of mutated heterozygous cell lines mixed with a normal control sample, was at least 0.1%," reported Dr. Arcila.
She added that "since 56% of the cases that were negative by conventional sequencing were positive with a more sensitive assay, we estimate that the prevalence of the T790M resistance mutation in patients with established EGFR TKI resistance may be as high as 72%, which is significantly higher than the 50% usually reported in the literature."
Dr. Arcila said that her team was a little surprised that the assay worked so well. "We did think that these mutations were not being detected easily because standard PCR is known to have a very low sensitivity. But that the assay is able to pick up mutations at such a low, low dilution level was nice to see."
"I'd say the number 1 take-away message is the fact that these mutations are more common than people actually think and the improved detection by higher-sensitivity methods, such as nucleic acid, may have significant clinical implications," said Dr. Arcila. "After detection, these patients could then be treated with something else or be assigned to other clinical trials that they could benefit from."
Early but Promising Results
"This study really represents many of the studies that were presented here [at the meeting], in that it addresses an important problem in medicine — one that's related to understanding better treatment response in developing the best possible diagnostics," said AMP program chair Timothy O'Leary, MD, PhD, new editor (beginning in January) of The Journal of Molecular Diagnostics, which is published by the AMP and the American Society for Investigative Pathology. Dr. O'Leary was not involved with this study.
He noted that the investigators "have done a very careful study of several techniques and it suggests that there are, which is always true in the field, improvements that can be made in the diagnostic methods that will eventually, presumably, devise better selections of patients for treatment."
"Overall, I think the results they found are very promising. They are early and it is a relatively small number of patients. It's not something that folks should necessarily be running out to do. Nonetheless, it looks like an approach that offers promise and is absolutely worth investigating further in larger numbers of patients," concluded Dr. O'Leary.
Dr. Arcila and Dr. O'Leary have disclosed no relevant financial relationships.
Association for Molecular Pathology (AMP) 2009 Annual Meeting: Abstract ST21. Presented November 20, 2009.
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