FAQs

ESR1 FAQs

What are tumour mutations/variants?
Tumour mutations (also referred to as variants) are changes in the DNA of cancer cells. Some mutations are more common in breast cancer, such as PIK3CA or TP53 mutations. These mutations can affect how cells grow and respond to treatment. Some mutations are drivers of tumour growth, while others may have little impact. Understanding these mutations can help inform treatment decisions.

What are ESR1 mutations?
ESR1 mutations are changes in the genetic code (DNA) of the ESR1 gene that encodes the oestrogen receptor alpha (ERα). ESR1 mutations are acquired mutations driven by selective pressure following prolonged exposure to ET, occurring almost exclusively following AI treatment in the metastatic setting. Longer exposure to ET increases the chance of acquiring ESR1 mutations in mBC. ESR1 mutations may be found in ~40% of patients with a/mBC following ET and are associated with a poor prognosis for patients.

Why are ESR1 mutations important in advanced/metastatic breast cancer (a/mBC)?
ESR1 mutations are a major driver of endocrine resistance. Patients with ESR1 mutations have significantly faster disease progression and worse survival outcomes (OS and PFS) as the accumulation of mutations can generate a more aggressive disease phenotype. Routine ESR1 mutation testing at each disease progression is, therefore, important to inform appropriate treatment decisions.

When should an ESR1 mutation test be performed?
It is important to test for an ESR1 mutation every time mBC progresses if not detected previously, since ESR1 mutations are acquired following exposure to ET and may occur at any step of the patient´s treatment journey.

Why is liquid biopsy preferred for ESR1 mutation testing?
According to international guidelines, blood-based ctDNA is the preferred testing methodology for ESR1 mutation testing. ctDNA is DNA that is shed by cancer cells into the bloodstream. Liquid biopsy is preferred due to its high sensitivity for detecting subclonal ESR1 mutations and because it can detect tumour shedding, regardless of the location or metastatic site. A liquid biopsy captures all sites, regardless of spatial distribution. A liquid biopsy is minimally invasive, highly sensitive, can be collected quickly, and can be performed at each progression.

Is ESR1 mutation testing needed at each progression in mBC?
Metastatic breast cancer changes over time and throughout treatment. As ESR1 mutations emerge
following exposure to ET, 
a test for ESR1 mutations should be performed at each progression if not detected previously. ESR1 mutations may develop at each step of the patient´s treatment journey. ESR1 mutations are rarely found in the primary tumour; however, they may develop in ~40% of patients with a/mBC following exposure to ET.

Why is archival tissue not appropriate for ESR1 mutation testing?
ESR1 mutations are acquired, and rarely present in primary tumours; testing of primary archival tumours will likely not detect the acquired mutations that occur after treatment with ET. According to international guidelines, blood-based ctDNA is the preferred methodology for ESR1 mutation testing.

How long does it take to get the result of an ESR1 mutation test?
Typically, the report for an ESR1 test with liquid biopsy is shared with the healthcare team within 14 days.¹ However, some diagnostic laboratories might have different timelines.

What technology should be used for an ESR1 mutation test?
For liquid biopsy-based ESR1 mutation testing, sensitive and validated methods like dPCR or NGS techniques should be used reaching a LoD <1% VAF.^2,3

What blood collection kits are needed and where can I get hold of them? 
Only use specific cfDNA collection tubes to collect your sample.⁴ Work with your genomic laboratory hub (GLH) to ensure you have the right tubes. Individual Trusts are required to purchase their kits locally via their own procurement and ordering processes. Further formation may be found on the GLH website.

Is the variant allele frequency (VAF) important?
The VAF, also referred to as MAF in scientific literature, should not be used to inform treatment decision-making. ESR1 mutations are subclonal, meaning they are not present in every cancer cell. This means the VAF is likely to be lower due to intra- and inter-tumour heterogeneity because they are not found in every cell, tumour, or body. Clonal mutations (e.g., PIK3CA, AKT1) are likely to have a higher VAF due to their presence in every tumour and tumour cell.

What should be done if the ESR1 mutation test result is invalid?
If a test result is invalid, a retest for ESR1 mutations is recommended. It is also recommended to discuss with the diagnostic lab the potential reason for the invalidity and how to better optimizeoptimise the test workflow from sample collection to result reporting to maximizemaximise patient care.

What should be done if the ESR1 mutation test result is invalid?
If a test result is inconclusive, retesting for ESR1 mutations may be appropriate. Please engage with your assigned GLH to understand the potential reason for inconclusiveness of the test.⁵

What should be done if the ESR1 mutation test result is negative?
If a ESR1 test is negative, a retest at the next progression is recommended, since ESR1 mutations can present at each step of the patient´s treatment journey and may develop in ~40% of patients with a/mBC following exposure to ET.

What should be done if the ESR1 mutation test result is negative?
If an ESR1 mutation test is negative, please consider retesting at the next progression on ET.⁵

What should be done if a (archival or fresh) tissue biopsy was used to perform an ESR1 mutation test and the test returned negative?
If a tissue sample was used to perform the ESR1 mutation test and the result was negative, a retest using a liquid biopsy is recommended,If a tissue biopsy (archival or fresh) was used to perform the ESR1 mutation test and the result was negative, retesting would be considered appropriate, since a liquid biopsy has a high sensitivity for detecting subclonal ESR1 mutations and can detect tumour shedding regardless of the location or metastatic site.⁶ In addition, ESR1 mutations are acquired and rarely present in primary tumours.⁷ A test performed on a primary archival tumour sample is therefore unlikely to detect these acquired mutations, which may develop in ~40% of cases following exposure to ET.⁷

Where to send an ESR1 mutation test sample for analysis?
If it is unclear where to send samples for ESR1 mutation testing, it is recommended to align with the local diagnostic lab and/or a local Menarini Stemline representative.

Where to send an ESR1 mutation test sample for analysis?
ESR1 mutation testing via liquid biopsy is reimbursed in the UK and should be sent to your assigned GLH. Contact your GLH to find out how they manage test requests and sample collection.

How should a blood sample be stored & transported?
A blood sample should be stored and transported using cfDNA collection tubes that stabilize the sample. The sample should typically be stored at room temperature (6–37°C). For further details, the manufacturer instructions for use should be consulted. Storing the sample outside this temperature range may cause blood cell lysis (the breakdown of blood cells) and the release of genomic DNA, which may lead to false, or invalid results. If a sample is stored outside this temperature range, it is recommended to take a new blood sample.

How should a blood sample be stored & transported?
A blood sample should be stored and transported using the ctDNA collection tubes. The sample should typically be stored at room temperature (10–30°C).⁸ For further details, the manufacturer instructions for use should be consulted. Storing the sample outside this temperature range may cause blood cell lysis (the breakdown of blood cells) and the release of genomic DNA, which may lead to false or inconclusive results.⁹

How should a blood sample be collected?
For blood collection, ctDNA-specific cfDNA collection tubes are recommended. cfDNA collection tubes contain a specific stabilizer for ctDNA. Examples include: Streck cfDNA BCT Tubes^®, PAXgene^® Blood ccfDNA Tubes, Roche cfDNA collection Tubes^®. For blood collection, needle sizes of 21G or greater are recommended, since smaller needles may cause blood cell lysis (the breakdown of blood cells) and the release of genomic DNA, which may lead to false or invalid results.

How should a blood sample be collected?
For blood collection, ctDNA-specific cfDNA collection should be used. For blood collection, needle sizes of 21G or greater are recommended,⁸ since smaller needles may cause blood cell lysis (the breakdown of blood cells) and the release of genomic DNA, which may lead to false or inconclusive results.

Why is it important to not shake the blood collection tubes?
It is important to only invert the blood collection tubes 8-10 times,^8,10 or according to the manufacturer instructions for use. Shaking may cause blood cell lysis (the breakdown of blood cells) and the release of genomic DNA, which may lead to false or invalid results.^9,11If a sample is shaken, it is recommended to take a new blood sample.

Why is it important to completely fill the blood collection tubes?
It is important to fill the blood collection tubes up to 10 ml according to the manufacturer instructions for use. 
If not completely filled, the concentration of the stabilizer in the tube will be suboptimal, which may impact the sample analysis. If a sample tube is not completely filled, it is recommended to take a new blood sample. 

Why is it important to completely fill the blood collection tubes?
It is important to fill the collection tubes completely, per the manufacturer instructions for use.^10,11 This is because the blood collection tubes for ctDNA contain preservatives designed to stabilise white blood cells and prevent the release of genomic DNA (gDNA). If the tube is underfilled, the ratio of preservative to blood is too high, which can cause inaccurate test results.¹¹