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Italian Researchers Validate CastPCR as Companion to TKI Treatment for BRAF Mutated Melanoma


NEW YORK (GenomeWeb) – Investigators at an Italian research hospital have demonstrated Thermo Fisher Scientific's competitive allele specific TaqMan PCR (castPCR) is a sensitive and specific method to detect BRAF mutations in melanoma.

Mutation testing can have an impact on treatment decisions for certain kinds of cancer drugs, and, in the case of BRAF gene mutations, status can predict treatment response to tyrosine kinase inhibitors (TKIs).

In a Scientific Reports study led by Paola Parrella in the the Laboratory of Oncology at IRCCS “Casa Sollievo della Sofferenza" Hospital, researchers evaluated castPCR using 54 patient samples. They also determined the limit of detection by diluting mutated clinical samples with non-mutated ones, and analyzed the effects of tumor heterogeneity and melanin content.

The study found 100 percent negative agreement between castPCR and Sanger sequencing, and the castPCR method also identified four additional mutated cases and demonstrated presence of mutation in two cases that had been uncertain by Sanger sequencing.

The group tested the method on dilutions of BRAF-mutated and non-mutated clinical samples instead of using DNA obtained from cell lines or synthetic oligonucleotides, which was, to the best of Parrella's knowledge, the first time this has been done.

This approach was hypothesized to better reflect the limits of BRAF testing in melanoma, due to the fact that the lab usually processes formalin-fixed paraffin-embedded specimens with a variable percentage of cancer cells that often show marked intratumour heterogeneity.

With this method, the group found a 1:100 mutated to non-mutated ratio as the limit of detection of both the BRAF V600E and BRAF V600K castPCR probes, which led to a sensitivity in clinical samples of 0.5 percent. The only other previous study to report a limit of detection using castPCR in melanoma found a sensitivity of 2 percent.

The authors also evaluated castPCR before and after melanin removal from the samples. Melanin can inhibit PCR, but castPCR showed less of an inhibition effect than Sanger sequencing.

CastPCR is a method to suppress amplification of wild-type genomic DNA such that rare mutations are more easily detected. It was originally developed by Life Technologies, and is now marketed by Thermo Fisher Scientific. The firm sells detection kits for hundreds of different mutations, as well as thousands of CNVs and SNPs, and these can all be run using Thermo Fisher Scientific brand Applied Biosystems Real-Time PCR systems. Parrella's group used the 7900HT Fast Real-Time PCR System, for example.

"CastPCR assays are optimized for highly sensitive detection of somatic mutations and it is exciting that customers are seeing these results in their experiments," Thermo's director of product management Sundiep Phanse and senior market development manager Karen Cormier told GenomeWeb in an email.

However, Phanse and Cormier also noted that while standard Sanger sequencing has lacked sensitivity at low mutation percentages, the firm's Minor Variant Finder software, which launched last week, can improve the sensitivity of detection down to 5 percent. "Researchers can feel confident that either platform they use from [Thermo Fisher brand] Applied Biosystems will give them high-quality results for somatic mutations.”

Parrella noted that her institution had been performing BRAF testing for clinical purposes since 2013, and the objective of the current study was to validate the castPCR method in the clinical setting.

The group chose castPCR because it liked the presence of the probe blocking normal allele amplification, she said. "In addition, the 7900HT sequence detection system is an open instrument allowing us to see the shape of the amplification curves."

Mutation testing is required by the US Food and Drug Administration, the European Medicines Agency, and the Italian Medicines Agency (AIFA), Parrella noted.

In the US, the cobas 4800 BRAF V600 mutation test from Roche was approved by the FDA in 2011 as a companion diagnostic for the firm's drug Zelboraf (vemurafenib). The THxID BRAF test from BioMérieux was cleared in 2013 as a companion to GlaxoSmithKline drugs Tafinlar (dabrafenib) and Mekinist (trametinib). Biocartis also signed a deal with Merck earlier this month to develop and commercialize a RAS and BRAF V600 mutation test on its Idylla platform.

However, clinical labs must also test out technologies in their own hands. Parrella's lab follows recommendations for BRAF testing — from the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytodiagnostics — which do not make restrictions on methods as long as they have been validated in the laboratory, she said.

"To the best of my knowledge there is only one other study evaluating BRAF in melanoma by castPCR," Parrella said, referring to a 2013 Australian study of 93 melanoma cases which also suggested that the method minimized the risk of false negative results.

A similar study last year evaluated castPCR for KRAS mutation detection in patients with metastatic colorectal cancer, showing essentially equivalent results to the Therascreen KRAS RGQ PCR kit from Qiagen.

Importantly, Parrella's study also provided case information for the patients whose BRAF status might have been falsely negative or undetermined by Sanger sequencing.

For example, "case 6" was a patient whose sample was classified as uncertain by Sanger sequencing, containing 25 percent neoplastic cells. Mutation was detected with castPCR and the patient showed significant benefits from subsequent TKI treatment. "Case 15" had detectable mutation levels only after treatment to remove melanin from the sample, and the patient showed stable disease after TKI treatment was initiated.

"At last follow up, [these patients] were still responding," Parrella said. Her lab is now implementing the test in the clinic.