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Jun 1, 2013 (Vol. 33, No. 11)

Acquired Drug Resistance in Cancer

New Methods Allow Non-invasive Analysis in Blood Plasma

  • Oncologists continually face the challenge of matching the right therapeutic regimen with the right patient, balancing relative benefit with risk to achieve the most successful outcome. But they say, in current clinical practice, physicians and patients are “often disappointed” by suboptimal treatment outcomes.

    As an example, 70–80% of patients receiving cytotoxic therapy for lung cancer obtain little-to-no clinical benefit from their treatment. The marginal success rate achieved in many types of cancer, clinicians say, reflects the complexity of the disease process coupled with an inability to properly guide the use of available therapeutics, and the development of tumor drug resistance.

    The ability to monitor whether a cancer patient’s tumor has acquired a resistance mutation as a result of targeted therapy would allow patients to switch therapies, potentially slowing or halting tumor growth. But sampling tumors repeatedly remains problematic as these procedures are invasive and some tumors are inaccessible. Further, tumors are highly heterogeneous, evolve constantly, and reflect many different cell types, clinicians say. And since biopsies take time in the clinic and only sample a small part of a tumor, they may also not be representative of is occurring with the biology of the entire tumor mass.

    But new, noninvasive blood-plasma based methods to follow the genetic and molecular evolution of solid tumors in patients are emerging. These technologies detect mutations in circulating tumor DNA, allowing monitoring of tumor changes over time.

    In 2012 a team of investigators from Cambridge Research Institute reporting in Science Translational Medicine said it had developed a method for detecting tumor mutations in blood. Using tagged-amplicon deep sequencing (TAm-Seq), the investigators screened 5,995 genomic bases for low-frequency mutations, identifying mutations present in circulating DNA at allele frequencies as low as 2% with sensitivity and specificity of >97%.

    The group identified mutations throughout the tumor suppressor gene TP53 in circulating DNA from 46 plasma samples of advanced ovarian cancer patients. Scientists also used TAm-Seq to noninvasively identify the origin of metastatic relapse in a patient with multiple primary tumors and in another individual, identifying an EGFR mutation in plasma, a mutation not found in an initial ovarian biopsy.

    TAm technology also allowed the scientists to monitor tumor dynamics, and track 10 concomitant mutations in plasma of a metastatic breast cancer patient over 16 months. This low-cost, high-throughput method could facilitate analysis of circulating DNA as a noninvasive “liquid biopsy” they say could be used for personalized cancer genomics.

    Dana-Farber Cancer Institute recently reported that a new blood test revealed more of the gene mutations that sustain certain digestive-tract tumors than did a DNA analysis of a traditional tumor biopsy. Dana-Farber investigators reported their results at a special symposium of the American Association for Cancer Research annual meeting in Washington.

    The blood analysis was performed using Inostics’ BEAMing digital PCR technology.

    Originally developed in the laboratory of Bert Vogelstein, M.D., at Johns Hopkins, BEAMing technology involves conversion of single DNA molecules to single magnetic beads, each containing thousands of copies of the sequence of the original DNA molecule. The number of variant DNA molecules in the population can then be assessed by staining the beads with fluorescent probes and counting them by using flow cytometry. Beads representing specific variants can be recovered through flow sorting and used for subsequent confirmation and experimentation.

    George D. Demetri, M.D., director of the Ludwig Center at Dana-Farber Cancer Institute and Harvard Medical School used BEAMing blood biopsy to follow patients with metastatic gastrointestinal stromal tumors (GIST) in whom disease had progressed despite treatment with imatinib (Gleevec) or sunitinib (Sutent), both multitargeted receptor tyrosine kinase (RTK) inhibitors.

    The research involved patients with metastatic GIST who were participating in a Phase III clinical trial of oral regorafenib, which inhibits vascular endothelial growth factor receptors (VEGFRs) 2 and 3, and Ret, Kit, PDGFR, and Raf kinases.

    The investigators collected DNA from tumor samples from as many patients as possible and analyzed them for mutations in the genes for KIT and PDGR. “We expected this traditional method would enable us to detect the original mutations within these genes, but not the mutations that cropped up after treatment with imatinib and the second-line drug sunitinib,” Dr. Demetri said. The researchers then analyzed blood samples drawn from these patients after the disease had become resistant to both imatinib and sunitinib.

    Dr. Demetri and his colleagues compared whether BEAMing technology or traditional tissue analysis was better at picking up “secondary” resistance mutations in the gene for KIT—abnormalities that emerged after the disease had become resistant to imatinib and sunitinib. The BEAMing technology proved superior, finding such mutations in 48% of the blood samples, compared to only 12% found in tissue samples using traditional methods.

  • Study Results

    According to Dr. Demetri, the results show a clear association between the presence of different cancer-driving gene mutations in patients’ blood samples and clinical outcomes. “By using this technology, we hope to develop the most rational drug combinations and better tests to match patients with the most effective therapies going forward,” he said.

    In another report using the BEAMing approach, Giulia Siravegna, and colleagues at IRCC, Candiolo, Italy; Falck Division of Medical Oncology, Ospedale Niguarda Ca’ Granda, Milan; and the Ludwig Center for Cancer Genetics and Therapeutics, Johns Hopkins Kimmel Cancer Center reported blood-based molecular detection of acquired resistance to anti-EGFR therapies in colorectal cancer patients. These investigators had studied the molecular basis of the resistance that colorectal cancers develop to the anti-EGFR monoclonal antibodies cetuximab and panitumumab, usually within 3–12 months after therapy initiation. They had previously reported that KRAS mutant alleles emerged in the circulation of approximately 50% of patients treated with anti-EGFR antibodies months before radiographic documentation of disease progression.

    In a new approach, Siravegna and colleagues had a look at the 50% of patients in which KRAS mutations had not emerged during anti-EGFR therapy. Exome sequencing identified amplification of the MET proto-oncogene in biopsies from patients who did not develop KRAS mutations. The authors found, they said, that MET amplification could be identified in a noninvasive manner through the examination of ctDNA months before relapse becomes clinically manifest. And they concluded, as multiple anti-MET therapeutic strategies are available, these findings offer immediate opportunities to design clinical studies.

    Philipp Angenendt, Ph.D., CTO at Inostics, a company focused on the blood-based detection of genetic alterations, said in an e-mail to GEN that he is pleased to see that more scientific evidence is gathered to underline the importance of blood-based detection of acquired resistance. He is convinced that the early detection of resistance together with the advent of second generation drugs that target the resistance will make a remarkable impact on patient management and overall survival.

    Dr. Angenendt told GEN that Inostics had recently received CLIA approval for its lab in Baltimore.“Through our CLIA-certified lab in Baltimore we are committed to bringing blood-based testing to patients,” he said. “Our current challenge is to increase awareness among clinicians that this method is useful for the therapy of a patient.”

    Advances in assays to analyze tumor DNA circulating in the blood of cancer patients may eventually allow routine cancer treatment based on tumor genomic profiles and enable physicians and patients to stay ahead of evolving resistance.


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