Questions & Answers

Below are answers to some of the most pressing customer questions that were submitted during the webinar.

Liquid biopsy testing

Can liquid biopsy be used for BRCA gene-related cancers?

Some commercial panels have already implemented BRCA and/or BRCA-related gene analysis. Therefore, you can easily apply these products to look for alterations in BRCA-based cancers.

Do you think that the locational information obtained with HER2-PET imaging will provide useful information beyond what you get with liquid biopsy?

Liquid biopsy relies on the dimension of the lesion. The smaller it is, the more difficult it will be to detect a mutation in the bloodstream. Locational imaging like HER2-PET still remains a useful diagnostic tool to assess the striations of the tumor.

dPCR and NGS technology

Why could dPCR alone be used for patient stratification?

As shown during the presentation, we applied dPCR to track index mutations previously found by NGS analysis. In selected cases (e.g., in the case of a precise query from the oncologist), we also applied dPCR assays based on tissue findings without running a baseline NGS analysis.

Mutational insights

I have a question related specifically to TP53 mutations. Have you tested the buffy coat in these cases to rule out clonal hematopoiesis CHIP mutations?

Yes, we also collected peripheral blood mononuclear cells (PBMCs) from the enrolled patients and tested these analytes for TP53 mutations. In two cases, aged 79 and 82, we confirmed that TP53 mutations seen as ctDNAs in the bloodstream were present as gDNA in the white cells, thus being derived from clonal hematopoiesis.

Sample collection

How often were the blood samples obtained during follow up?

We monitored our patients by collecting blood every 21 days, at the occasion of chemotherapy infusion.

Sample extraction

We do recommend cfDNA or ctDNA/ctTNA isolation from body fluids, also including plasma, for different reasons, most of which are related both to research and diagnostic purposes (the latter within a molecular tumor board).

Can this kit be used to isolate cell-free DNA from frozen plasma samples right away?

There are many kits on the market based on silica or magnetic beads. We routinely extract our ctTNAs from frozen plasma using a commercially available magnetic bead-based kit.

Data analysis

How can you filter out the background DNA in your liquid biopsy samples as mutations are in a very small proportion in comparison with the total DNA in your ctDNA/cfDNA samples?

We routinely filter our NGS results by applying a preset filter in the analysis software. We also confirm every SNV found by NGS by running the specific dPCR assay. Only mutations that were called by the two platforms were considered "true" positives.