Super-sensitive detection of EGFR mutants driving lung cancer recurrence - Super-sensitive detection of EGFR mutants driving lung cancer recurrence Confidential Principal Investigator: Shafer, David A., PhD
PROJECT SUMMARY
Cancer is the leading cause of death worldwide, and lung cancer is the leading cause of cancer-related
death. Several point mutations or deletions in the EGFR gene (L858R exon 21; del codons 746–753 exon 19)
are common in non-small cell lung carcinoma (NSCLC) and exhibit high response rates to first-generation
tyrosine kinase inhibitors (TKIs). Drug-resistance commonly occurs following expansion of a clone(s) harboring
the EGFR T790M mutation, which is often present at low levels before targeted therapy is initiated, but can also
arise during treatment. Several third-generation TKIs have been developed that irreversibly inhibit T790M.
However, third-generation TKIs fail after the emergence of subclones harboring one of >10 additional EGFR
mutations, such as C797S, L718Q, or L844V. Treatment outcomes can be influenced by whether T790M is
present in cis (same allele) or trans (different alleles) with respect to variants conferring resistance to third-
generation TKIs. Recently, a fourth-generation allosteric inhibitor of T790M and C797S was reported.
Collectively, these findings indicate that effective NSCLC therapy depends upon the presence and cis/trans
status of first/third-generation TKI-resistance mutations, both before treatment and during remission. The overall
objective of this Phase I SBIR project is to develop a PCR-based, liquid-biopsy assay for detecting circulating
EGFR mutations in NSCLC patients that confer drug-resistance, and thus lead to recurrence.
GeneTAG Technology develops molecular diagnostic assays for cancer and infectious diseases. Our
primary system, the internal DNA-Detection Switch (iDDS) probe system, comprises two interacting components:
a fluor-labeled probe, and a quencher-labeled antiprobe that is nearly complementary to the probe. In the
absence of the intended target, the paired probes and antiprobes bind together, quenching fluorescence and
preventing off-target detection. This unique probe system shows superior single-base discrimination over a wider
annealing-temperature range (10–30ºC) than common methods. Recently, we merged our iDDS probe
technology with our Wild Terminator (WTx) methods that enable detection of rare mutants (0.1–0.01% frequency)
by blocking amplification of the wild-type sequence. In pilot studies, the combined method showed greater
sensitivity in detecting circulating EGFR variants from lung cancer patients than the FDA-approved cobas®
EGFR Mutation Test, v2. This enhanced qPCR sensitivity meets or exceeds the sensitivity of specialized
platforms without a high instrumentation cost. Our higher sensitivity and specificity will enable development of a
platform-independent, liquid biopsy assay. Our Specific Aims are: 1) to develop XNA-enhanced WTx-iDDS probe
assays for detecting EGFR drug-resistance mutations, and 2) to study drug-resistance EGFR mutations in
plasma from NSCLC patients in remission. Success in Phase I will justify expanded Phase II assay-validation
studies in preparation for filing for FDA approval. More sensitive detection against an expanded set of EGFR
drug-resistance mutations associated with recurrence will guide physicians in selecting first-line therapy.
