Using Blood Tests to Predict Treatment Response and Resistance in Breast Cancer

Table of Contents

• Introduction: The Challenge of Treatment Resistance
• Precision Oncology and Breast Cancer Treatment
• How CDK4/6 Inhibitors Work and Why Resistance Develops
• Liquid Biopsy: A Minimally Invasive Alternative to Tissue Biopsies
• Genetic Biomarkers in Blood for Predicting Treatment Response
• The Future: Epigenetic Biomarkers in Liquid Biopsies
• Conclusion and Clinical Implications
• Source Information

Introduction: The Challenge of Treatment Resistance

Breast cancer remains the most commonly diagnosed cancer in women worldwide, with approximately 70% of cases being estrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2-). While screening programs have helped detect many cases at earlier, more treatable stages, patients face a lifelong risk of metastatic recurrence. Once breast cancer spreads to distant organs, it typically becomes incurable despite available treatments.

Over the past decade, CDK4/6 inhibitors combined with endocrine therapy have transformed treatment for ER+/HER2- metastatic breast cancer. Three drugs in this class—abemaciclib, palbociclib, and ribociclib—have demonstrated significant clinical benefits in major clinical trials, improving response rates, progression-free survival, quality of life, and overall survival for patients with advanced disease.

However, treatment resistance remains a major challenge. Approximately a minority of patients experience intrinsic resistance, showing disease progression within six months of starting treatment. Even patients who initially respond well inevitably develop acquired resistance over time, with median progression-free survival ranging from 23.8 to 28.2 months in first-line metastatic treatment.

This pressing clinical problem highlights the urgent need for biomarkers that can predict which patients will benefit from CDK4/6 inhibitors and detect resistance early. While researchers have extensively investigated tissue-based biomarkers, the only clinically available predictor remains basic breast cancer subtype (ER+/HER2-). Tumor heterogeneity and difficulties distinguishing endocrine resistance from CDK4/6 inhibitor resistance have complicated biomarker discovery.

Precision Oncology and Breast Cancer Treatment

Precision oncology uses molecular information from a patient's cancer to optimize and individualize treatment approaches. This strategy helps clinicians select the most effective treatments while minimizing side effects on healthy cells. Molecular biomarkers fall into two main categories: prognostic biomarkers that predict disease course regardless of treatment, and predictive biomarkers that indicate how a patient will respond to specific therapies.

Current breast cancer treatments provide excellent examples of biomarker-directed precision oncology:

• OncotypeDx: A commercial 21-gene test for ER+ early-stage breast cancer that calculates a recurrence score indicating relapse probability without chemotherapy
• MammaPrint: A microarray assay that uses 70 genes to classify patients by recurrence risk
• HER2 status: Determines whether patients will benefit from HER2-targeted drugs like trastuzumab
• Hormone receptor status: Identifies tumors likely to respond to endocrine therapy

Resistance to endocrine therapy can develop through various mechanisms, including genetic alterations of ESR1, increased activity of cyclin-dependent kinases (CDKs), activation of signaling pathways like PI3K and RAS, or decreased levels of proteins that inhibit CDKs such as p16, p21, and p27. Many of these resistance pathways converge on the cyclin D-CDK4/6 axis, explaining why combining endocrine therapy with CDK4/6 inhibitors has proven so successful for ER+/HER2- metastatic breast cancer.

How CDK4/6 Inhibitors Work and Why Resistance Develops

The cyclin D-CDK4/6-Retinoblastoma protein (Rb) pathway regulates cell cycle progression from the G1 to S phase. In normal cell function, Rb remains unphosphorylated and bound to E2F transcription factors, keeping them inactive. When appropriate growth signals occur, cells enter the G1 phase, leading to cyclin D expression. Cyclin D then binds to CDK4/6, forming an active complex that phosphorylates Rb.

This phosphorylation causes Rb to change shape and release E2F transcription factors, which drive entry into the S phase and further cell cycle progression. The cyclin D-CDK4/6 complex also activates the FOXM1 transcription factor, promoting advancement through later cell cycle phases (G2/M). ER+ breast cancer heavily depends on this intact pathway since estrogen drives cyclin D1 expression, ultimately inducing cell proliferation through CDK4/6 signaling.

CDK4/6 inhibitors work by binding the ATP domain of CDK4/6, halting progression from G1 to S phase and preventing cancer cell division. However, resistance mechanisms remain incompletely understood, and the clinical relevance of many mechanisms identified in laboratory studies remains unconfirmed.

Known resistance mechanisms include:

• Amplification of cyclin D-CDK4/6 pathway members
• Downregulation of CDK4/6 repressor proteins like p21 and p27
• Alterations in RB1, FAT1, or signaling pathways like PI3K/AKT/mTOR and KRAS that bypass the G1/S checkpoint

These alterations allow cancer cells to continue dividing despite CDK4/6 inhibitor treatment, ultimately leading to treatment failure.

Liquid Biopsy: A Minimally Invasive Alternative to Tissue Biopsies

Liquid biopsy approaches have emerged as promising alternatives to traditional tissue biopsies for obtaining molecular information about cancers. While tissue biopsies require invasive procedures that aren't always feasible depending on tumor location and patient health, liquid biopsies can bypass these limitations by detecting tumor-derived material in easily accessible body fluids.

For most applications, peripheral blood plasma serves as the preferred fluid for liquid biopsy. While this previously presented challenges for detecting brain tumors due to the blood-brain barrier, newer technologies have improved detection sensitivity. Other fluids may be more informative for specific cancers, including saliva for oral cancer, urine for bladder cancer, and cerebrospinal fluid for brain tumors.

Liquid biopsies can analyze various tumor components, including:

• Circulating tumor cells (CTCs) - intact cancer cells in blood
• Cell-free DNA (cfDNA) - DNA fragments circulating in blood
• Extracellular vesicles - small particles released by cells
• Cell-free RNA - RNA molecules in circulation

This review focuses on circulating tumor DNA (ctDNA), which contains tumor-derived genetic and epigenetic information released into the bloodstream as cancer cells die. ctDNA typically represents a small fraction (sometimes <0.01%) of the total cell-free DNA pool, with most remaining DNA coming from blood cells and other tissues.

ctDNA levels vary based on tumor size, stage, location, treatment status, and how readily tumor cells release DNA into circulation. Once in the bloodstream, ctDNA clears quickly with a half-life between 16 minutes and 2 hours through various mechanisms including enzyme degradation, immune cell consumption, and kidney filtration. This rapid turnover enables ctDNA to provide a "real-time" snapshot of a patient's disease status.

Analysis of ctDNA through liquid biopsy could prove valuable at multiple stages of CDK4/6 inhibitor treatment:

1. Prognostication - predicting disease outcomes
2. Treatment personalization - determining which patients should receive CDK4/6 inhibitors or additional drugs
3. Treatment monitoring - tracking response to therapy
4. Resistance detection - identifying emerging treatment resistance early

Clinicians can analyze ctDNA using polymerase chain reaction (PCR), which examines single genes, or next-generation sequencing (NGS), which profiles dozens to hundreds of genes simultaneously. For ER+/HER2- advanced breast cancer, the therascreen PIK3CA RGQ PCR kit already detects PIK3CA mutations to guide treatment with PI3Kα inhibitors like alpelisib. NGS-based liquid biopsy assays typically include PIK3CA along with other genes relevant to endocrine therapy resistance, such as ESR1 and PTEN.

Genetic Biomarkers in Blood for Predicting Treatment Response

Currently, no clinically validated liquid biopsy biomarkers can distinguish which patients will benefit most from CDK4/6 inhibitor therapy. Research has primarily focused on genetic alterations in cell cycle regulation genes detected through ctDNA analysis and their relationship to treatment outcomes.

RB1 Alterations: Since Rb represents the central target of CDK4/6 in controlling cell cycle progression, genetic changes to RB1 that cause its inactivation may confer resistance to CDK4/6 inhibitors. In the PALOMA-3 trial, loss of RB1 detected in baseline ctDNA associated with worse progression-free survival for patients receiving palbociclib plus fulvestrant. Analysis of ctDNA from MONALEESA 2, 3, and 7 trials found that patients with RB1 mutations didn't experience significant improvement in median progression-free survival with ribociclib plus endocrine therapy. Additional studies identified loss-of-function RB1 mutations exclusively acquired during CDK4/6 inhibitor treatment, suggesting they were selected for as resistance mechanisms. However, the low prevalence of these alterations (4.7% in one study) indicates other resistance mechanisms likely play important roles.

ESR1 Mutations: Research shows mixed results regarding ESR1 mutations as biomarkers for CDK4/6 inhibitor response. In the PALOMA-3 trial, patients receiving palbociclib had similarly improved median progression-free survival regardless of ESR1 status (9.4 vs. 9.5 months for mutant vs. wild-type). Interestingly, some patients acquired ESR1 Y537S mutations during treatment in both arms, and these were associated with improved median progression-free survival compared to patients who didn't acquire this mutation (13.7 vs. 7.4 months). In the MONARCH-2 trial, patients receiving abemaciclib showed improved progression-free survival regardless of ESR1 status but had higher numerical median progression-free survival with mutant tumors (20.7 vs. 15.3 months).

PIK3CA Mutations: Studies consistently show that PIK3CA status doesn't predict differential benefit to CDK4/6 inhibitors. In PALOMA-3, patients receiving palbociclib had similarly improved progression-free survival regardless of PIK3CA status (9.5 vs. 9.9 months for mutated vs. wild-type). The MONARCH-2 trial found similar results, though patients in the placebo group with PIK3CA mutations had worse median progression-free survival compared to wild-type (5.7 vs. 12.3 months). Some smaller studies suggested PIK3CA mutations might predict worse outcomes, but larger trials haven't confirmed this finding.

FGFR Alterations: Limited evidence suggests FGFR alterations might influence CDK4/6 inhibitor response. In MONALEESA-2, patients with FGFR1 alterations receiving ribociclib showed worse median progression-free survival (10.61 vs. 24.84 months), though statistical significance wasn't achieved due to small sample size. PALOMA-3 found patients with FGFR1 amplifications had worse progression-free survival in both treatment arms, but acquired FGFR2 alterations showed no apparent difference between treatment groups.

The Future: Epigenetic Biomarkers in Liquid Biopsies

While genetic-based liquid biopsy approaches have made limited progress in identifying clear predictive biomarkers for CDK4/6 inhibitor response, emerging epigenetic methodologies show significant promise. Epigenetics refers to modifications that regulate gene expression without changing the underlying DNA sequence, including DNA methylation, histone modifications, and chromatin remodeling.

Epigenetic profiling of ctDNA may reveal novel biomarkers that better predict treatment efficacy and resistance mechanisms. Unlike genetic alterations, which are relatively stable, epigenetic changes can dynamically respond to treatment pressures and environmental factors, potentially providing more sensitive indicators of emerging resistance.

Research in this area is still developing, but early studies suggest epigenetic markers in ctDNA could:

• Detect resistance earlier than genetic changes
• Provide insights into multiple resistance mechanisms simultaneously
• Offer information about tumor heterogeneity and evolution under treatment pressure
• Identify patients who might benefit from combination therapies targeting epigenetic mechanisms

As epigenetic technologies continue advancing and becoming more accessible, they will likely play an increasingly important role in personalizing CDK4/6 inhibitor treatment and managing resistance in breast cancer patients.

Conclusion and Clinical Implications

This comprehensive review highlights both the current state and future potential of liquid biopsies for predicting CDK4/6 inhibitor efficacy and resistance in breast cancer patients. While genetic approaches to ctDNA analysis have yet to yield clinically validated predictive biomarkers, the field continues to evolve rapidly with promising developments in epigenetic methodologies.

For patients, this research means that simple blood tests may soon help oncologists:

1. Predict who will benefit most from CDK4/6 inhibitors before starting treatment
2. Monitor treatment response through regular blood tests rather than scans
3. Detect emerging resistance earlier, allowing for timely treatment adjustments
4. Personalize combination therapies based on individual resistance mechanisms

The studies reviewed demonstrate that while specific genetic alterations (particularly in RB1) show promise as resistance biomarkers, their relatively low prevalence suggests multiple mechanisms contribute to treatment failure. This complexity underscores the need for comprehensive approaches that capture the full spectrum of resistance pathways.

As liquid biopsy technologies continue improving in sensitivity, cost-effectiveness, and clinical accessibility, they will likely become integral components of breast cancer management. The ability to repeatedly assess tumor biology through simple blood draws represents a significant advance over traditional tissue biopsies, particularly for monitoring treatment response and detecting resistance in real-time.

Patients should discuss with their oncologists whether participating in clinical trials involving liquid biopsy analysis might be appropriate for their situation, as this rapidly evolving field continues to generate new insights that may directly impact treatment decisions and outcomes.

Source Information

Original Article Title: Liquid biopsies to predict CDK4/6 inhibitor efficacy and resistance in breast cancer

Authors: Sasha C. Main, David W. Cescon, Scott V. Bratman

Publication: Cancer Drug Resist 2022;5:727-48

DOI: 10.20517/cdr.2022.37

This patient-friendly article is based on peer-reviewed research and aims to make complex scientific information accessible to educated patients and their families. It preserves all key findings, data points, and clinical implications from the original research while translating technical language into more understandable terms.