Biomarker Testing in Advanced Breast Cancer

PIK3CA, AKT, and PTEN mutations

The PI3K/AKT/PTEN pathway is a key signaling pathway involved in cell proliferation and survival. Activating mutations in PIK3CA and AKT1 and inactivating mutations in PTEN can lead to hyperactivation of this pathway in up to 50% of HR-positive breast cancer samples. While polymerase chain reaction (PCR) can be used to evaluate alterations in single genes like PIK3CA, next-generation sequencing (NGS) would enable the simultaneous assessment of alterations in PIK3CA, AKT, PTEN, and more.

ESR1 mutations

The estrogen receptor 1 (ESR1) gene encodes estrogen receptor alpha (ERα) and plays a critical role in breast cancer by regulating the cell’s response to estrogen. Mutations in ESR1 are commonly associated with resistance to endocrine therapy and are more likely found after cancer progression in HR-positive, HER2-negative breast cancers.

Since ESR1 mutations are acquired over time, repeat testing with liquid biopsy may be preferred to determine the relevant ESR1 mutations, which are typically located in the ligand-binding domain. Although both PCR and NGS can be used to assess for ESR1, NGS allows for the detection of ESR1 and other relevant biomarkers at the same time from one sample. In contrast, PCR offers a relatively simpler and faster method but may be more limited in coverage.

BRCA1 and BRCA2 mutations

Breast cancer early onset gene 1 (BRCA1) and 2 (BRCA2) were initially discovered as major hereditary breast cancer susceptibility genes. BRCA1/2 are tumor suppressor genes that play an important role in double-strand break repair through the homologous recombination repair (HRR) pathway. Inherited mutations in BRCA1/2 are associated with an increased risk of breast and ovarian cancer because pathogenic germline BRCA1/2 mutation carriers generally follow a two-hit hypothesis, where the first “germline hit” followed by the second “somatic hit” drives tumorigenesis.³

A robust assay to identify pathogenic BRCA1/2 mutations, including large rearrangements, is therefore critical to gain important insights into this biomarker. NGS testing can be used to detect both somatic and germline variants in BRCA1/2, and if a mutation is detected, a subsequent germline test may be needed to determine the mutation origin.

NGS for biomarker testing in advanced breast cancer research

Breast cancer molecular profiling includes various methods such as PCR, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and NGS. However, NGS enables comprehensive evaluation of tumors for genomic alterations across hundreds of genes and for complex biomarkers or genomic signatures like TMB, MSI, and homologous recombination deficiency (HRD).

Because NGS can identify multiple biomarkers in a cost- and time-efficient manner while conserving tissue, traditional methods are gradually being replaced by high-throughput technologies, though they remain useful in certain contexts. For instance, digital PCR (dPCR) may be used to verify NGS results from the same sample or to monitor specific genes over time at lower cost than with NGS. While tissue NGS is the gold standard for gaining insights into the biology of a tumor, liquid biopsy–based NGS is emerging as a highly sensitive method to understand resistance mechanisms as a tumor evolves. Liquid biopsies are minimally invasive and may be used as an alternative when tissue samples are not available. Moreover, liquid biopsy testing may complement tissue testing to better understand inter-and intra-tumor heterogeneity.

Figure 1. NGS uses a single assay to assess for relevant and emerging biomarkers in breast cancer research.

As a single vendor of sample-to-report solutions, including instruments, consumables, analysis, and support, Thermo Fisher Scientific simplifies the implementation of in-house NGS. The breadth of Ion Torrent Oncomine assays, from focused to comprehensive panels for tissue and liquid biopsy applications, enables researchers to select the optimal solution for their needs.

Key benefits of Oncomine assays include ease of use with minimal hands-on time to improve efficiency, fast turnaround times for timely insights and decisions, and high success rates for reliable testing of more samples, including those typically considered quantity not sufficient (QNS) with other technologies.

Table 2: Oncomine assays for advanced breast cancer research testing

Table 3: Oncomine assay variant coverage in PIK3CA, AKT1, PTEN, and ESR1