Personalized Medicine

Personalized medicine aims to identify patients who require treatment and subsequently select the right therapy based on individual factors and tumor characteristics, i.e. personalized medicine targets to identify the right patients for the right therapy. Prognostic biomarkers provide information on the risk of disease recurrence whereby predictive ones will inform the physician of the potential clinical response of the patient to a given cancer therapy.

“In personalized medicine biomarkers are used to predict prognosis (prognostic biomarkers) and treatment success (predictive biomarkers)”.

Picture: Personalized Medizine

High risk breast cancer

One in eight to ten women will get breast cancer during their lifetime. More than 460,000 women in Europe are newly diagnosed with breast cancer each year, with about 50% characterized as high-risk.

Therapy concepts for early breast cancer depend on prognostic risk classification, i.e. pending on the risk for disease recurrence patients will be treated more or less aggressively.

Prognostic risk classification by clinico-pathological assessment includes risk factors such as axillary lymph node status, tumor size, tumor grade, hormone-receptor status, patient age and Human Epidermal Growth Factor Receptor 2 (HER2)-status. While for patients at low and at high risk treatment recommendations are straightforward, the risk-benefit analysis for chemotherapy in patients at intermediate risk is challenging. Multigene signatures help to further stratify the intermediate risk group into low-risk (endocrine therapy, no systemic chemotherapy needed) and high-risk (indication for systemic chemotherapy) categories.

Picture: Risk categories and response prediction in early breast cancer

Patients with early breast cancer at high risk for disease recurrence require systemic chemotherapy. Anthracycline-based chemotherapy is considered standard of care according to national and international guidelines. As for any systemic chemotherapy, anthracyclines are associated with considerable side effects such as cardiac toxicity as well as secondary hematological malignancies (Azim et al., Annals of Oncology (2011) 22, 1939-1947).

Selection of the right patients for anthracycline-based chemotherapy vs. alternative chemotherapy (e.g. DC or P or CMF) is now supported by the PITX2-assay. The assay provides information to the treating physician, which patients are likely to benefit from anthracycline-based chemotherapy and which are not.

„Response prediction by PITX2 DNA-methylation provides valuable information on who has a high probability to benefit from anthracycline-based chemotherapy versus who should be considered for alternative chemotherapy regimen.“

PITX2 DNA-methylation, a prognostic and predictive biomarker

DNA-methylation is a common and early event in cancer indications, including breast cancer. DNA-methylation is observed in regulatory regions of specific genes and affects gene expression. The pituitary homeobox gene 2 (PITX2) is one of these regulatory genes and the methylation status of the PITX2 promotor has been shown to be prognostic for disease-free survival (DFS) and overall survival (OS) in various indications (see literature list PITX2).

Significant evidence has accumulated about PITX2 serving as prognostic and predictive biomarker in breast cancer, especially in hormone-receptor positive disease (Aubele et al., Disease Markers, 2017). In the field of breast cancer treatment, there is still an unmet need to predict the necessity, efficacy, and potential side effects of drugs and the outcome of breast cancer patients under therapy – and thus quality of life – while also avoiding high treatment-related costs and unnecessary chemotherapy or endocrine therapy. Diagnostic tests, which can predict response of a breast cancer patient to a certain cancer drug, are highly needed and yet still in short supply (Schmitt et al, Breast Care, 2018).

The therascreen PITX2 Assay

The therascreen PITX2 RGQ PCR Kit has been developed by Therawis Diagnostics together with its development partner Qiagen using reliable RGQ PCR technology and routinely available FFPE tissue (Napieralski et al., Protocol Exchange, 2018, Schricker et al., Sci Rep, 2018).

The therascreen PITX2 RGQ PCR Kit is CE-certified and commercially available since February 2018 (Link to QIAGEN product site).

The assay is a unique DNA methylation test that determines the percent methylation ratio (PMR) in promoter 2 of the pituitary homeobox transcription factor 2 (PITX2) gene as a novel biomarker. The kit has been optimized to detect and differentiate between methylated and unmethylated areas within the PITX2 promoter and is characterized by the following properties.

  • Clinically validated CE-certified IVD (in-vitro diagnostic)

  • Limit of Detection (LOD): PMR 4 to PMR 92

  • Range of linearity: PMR 5 to PMR 50

  • Simple and efficient workflow with Ready-to-use solutions and reaction mixes

  • Sample to Insight in <48 hours

  • Automated software using Rotor-Gene AssayManager v2.1 for quick and easy results interpretation

Picture: therascreen PITX2 RGQ PCR Kit – workflow

therascreen PITX2 RGQ PCR Kit – workflow

„The therascreen PITX2 RGQ PCR assay is a CE-certified test. The whole workflow from sample to insight is performed using readily available kits within 48 hours.“

Kits can be ordered at manufacturer’s homepage (for further details klick here).

therascreen PITX2 RGQ PCR requirements:

For test performance the following sample material is required:

  • 5 µm unstained slides of formalin-fixed, paraffin-embedded breast cancer tissue material

Surface area of at least 100 mm²

Clinical validation

Overall 205 patients (ER+, PR+, N+, HER2-) were included in a prospective analysis using archived formalin-fixed paraffin-embedded (FFPE) tissues. These tissues were analyzed with the therascreen PITX2 RGQ PCR assay to determine the percent DNA-methylation ratio (PMR; Schmitt et al., Breast Care, 2018).

Thereof, 60 FFPE samples were used to confirm a pre-defined PMR cut-off value of 12 which separates patients who benefit more versus less likely from anthracycline based chemotherapy with or without endocrine therapy. In these 60 patients, the cut-off value of PMR = 12 was confirmed by log-rank statistics when tested over a PMR range from 0 to 50 (Fig 1).

Picture: Cut-off value of PMR = 12 was confirmed

Statistical determination of PITX2 DNA-methylation PMR cut-off points (n=60); competing risk analysis, 5-year time of follow-up. y-Axis: standardized log-rank statistics; x-axis: PMR.

The remaining 145 samples were used to clinically validate the PMR cut-off of 12. Applying 10-year follow-up as primary study endpoint, Patients with low PMRs (≤ 12) demonstrated increased disease-free survival, patients with high PMRs (≥12) showed a statistically significant shorter disease-free survival.

Picture: PITX2 hypermethylation predicts poor patient outcome

Legend: Survival analysis. The high-risk breast cancer patient groups were subjected to Kaplan-Meier survival analysis with 10-year time of follow-up. Left panel: Clinical Analysis Study Group (n=205), patients treated with anthracycline-based chemotherapy plus endocrine therapy. Right panel: Clinical Analysis Study Group (n=65); patients treated with anthracycline-based chemotherapy only (Source: Schmitt et al. 2018).

Kaplan-Meier survival analysis with 10-year time of follow-up. The Clinical Validation Study Group (n=145) contains patients treated with anthracycline-based chemotherapy with and without endocrine therapy (Schmitt et al, Breast Care, 2018). The Kaplan-Meier analyses resulted in HR = 2.74 with pronoounced statistical significance (p < 0.001; 955 CI 1.65-3.54).

In conclusion, patients with a low PITX2 DNA-methylation status are sufficiently treated with anthracycline-based chemotherapy with or without additional endocrine therapy, proven by an extended disease-free survival interval. In contrast, patients with high PITX2 DNA-methylation status have a worse outcome, while on anthracycline-based chemotherapy, and should be considered for alternative chemotherapy regimens or the addition of taxanes to the regimen.

“The PMR cut-off of 12 has been validated in high-risk luminal B-patients. This cut-off is optimized to identify hormone receptor-positive breast cancer patients with high versus low probability to benefit from anthracycline-based chemotherapy.”

Triple-negative breast cancer

PITX2 DNA-methylation has shown to predict outcome in high risk breast cancer patients after anthracycline-based chemotherapy. To determine the prognostic versus predictive value, the impact of PITX2 DNA-methylation on outcome was studied in an untreated versus an anthracycline-treated triple negative breast cancer (TNBC) cohort.

The percent DNA-methylation ratio (PMR) of paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time PCR test. Patient samples of routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue and clinical data from 144 TNBC patients of two independent cohorts were analyzed: 66 untreated patients and 78 patients treated with anthracycline-based chemotherapy.

Risk for 5- and 10-years overall survival (OS) increases continuously with rising PITX2 DNA-methylation in the anthracycline-treated population, but only slightly during 10-years follow-up time in the untreated patient population. PITX2 DNA-methylation with a PMR cut-off of 2 did not show significance for poor versus good outcome (OS) in the untreated patient cohort (HR=1.55, p=0.259). In contrast, the PITX2 PMR cut-off of 2 identifies patients with poor (PMR >2) versus good (PMR ≤2) outcome (OS) with statistical significance in the anthracycline-treated cohort (HR=3.96, p=0.011). Results in the subgroup of patients who did receive anthracyclines only (no taxanes) confirmed this finding (HR=5.71, p=0.014).

In this hypothesis-generating study PITX2 DNA-methylation predominantly demonstrates predictive value for anthracycline treatment in TNBC patients. The risk for poor outcome (OS) correlates with increasing PITX2 DNA-methylation.

Legend: Increasing PITX2 DNA-methylation results in increasing risk for OS. A) OS, 5 years follow-up B) OS, 10 years follow-up. The blue line depicts the untreated patient population and the red line anthracycline-treated patient population (Source: Napieralski et al. 2020).

Legend: Kaplan-Meier analysis applying the PITX2 cut-off value of PMR 2. A) untreated patient cohort (n=66) B) anthracycline-treated patient cohort (n=78), and c) anthracycline-treated patient who received anthracyclines only (no taxanes added) in their chemotherapy regimens (n=50).

Literature list