Monitoring of multi-drug resistance and disease burden in patients with MM using liquid biopsy

Patients with multiple myeloma (MM) often experience multiple recurrent episodes of remission and relapse. The acquisition of resistance results in the lack of response to treatment or the response being diminished over time. Some tumors can become cross-resistant to a wide range of unrelated drugs. Such multidrug resistance (MDR), following exposure to a single chemotherapeutic agent, is often linked to the overexpression of plasma membrane drug efflux transporters, like P-glycoprotein (P-gp). In order to increase the chances of a positive MM treatment outcome with not only chemotherapeutic agents but also immunomodulatory drugs and proteasome inhibitors, it is important to continuously monitor for the presence and development of MDR during therapy.

One of the less invasive ways for such monitoring could potentially be testing of patients’ blood samples for the presence of circulating cancer-derived microparticles (MPs), a subset of extracellular vesicles. Levels of CD138⁺ circulating MPs have been shown to be elevated in patients with MM across all stages of the disease and correlate with plasma-cell burden and treatment response. However, currently, there is a lack of reliable tests that would allow for direct, continuous monitoring of MDR in MM.

Sabna Rajeev Krishnan and colleagues developed a novel blood test that could be used during routine follow-up to continuously monitor patients for the presence of MDR during treatment, and assess tumor burden.¹ The article below provides a summary of the data published in the Blood Cancer Journal.

Methods

• Platelet-free plasma was extracted from blood samples collected from patients with MM and age-matched adult healthy subjects
• In total, samples from 74 patients, including 12 in complete remission (CR), 30 in partial remission (PR), 14 with de novo disease, and 18 patients who relapsed were used
  • MPs phenotyping and quantitation in patient samples was performed using flow cytometry. The expression of the following markers was analyzed
    • P-gp – the MDR marker
    • CD138 – a plasma-cell marker
    • Phosphatidylserine (PS) – an MP marker and mediator of cancer spread
  • Additionally, expression of the stem-cell marker, CD34, was assessed in 11 patient samples

Results

• Compared with healthy subjects, patients with MM had on average a 5.4-fold greater number of P-gp⁺ MPs (p = 0.0071)
  • 2-fold increase in patients with de novo disease (p = 0.0032)
  • 5-fold increase in patients with progressive disease (PD; p = 0.0096)
  • No significant difference in patients in CR and PR compared to healthy subjects
• CD138 and P-gp expression on MPs was found to be mutually exclusive in MM
• Patients with MM had 4.6-fold higher levels of CD138^-P-gp⁺ MPs compared with healthy subjects (p = 0.011)
  • 5-fold increase in patients with de novo disease (p = 0.001)
  • 67-fold increase in patients with PD (p = 0.0379)
  • No significant difference in patients CR and PR compared to healthy subjects
• An increase in PS⁺ MPs was associated with disease progression and increased tumor burden
  • In patients with MM, PS⁺ MP levels were 6.4-fold greater compared to healthy controls
  • 16-fold increase in patients with PD relative to healthy subjects (p = 0.005), with smaller increase observed in patients with de novo disease (3.4-fold increase, p = 0.0026) and in PR (3.6-fold p = 0.03)
  • 78-fold increase in patients with PD compared to CR (p = 0.034)
• Overall, patients with MM had 2.4-fold increase in CD138⁺PS⁺ MPs compared to healthy subjects (p = 0.0041)
  • 3-fold increase in de novo and 3.4-fold increase in PR patients (both p = 0.007) compared to healthy volunteers but no significant difference in patients with CR and PD
• Patients with MM also had 5.1-fold increase in levels of CD138⁻PS⁺ MPs compared to healthy subjects (p = 0.001)
  • The increase for de novo PD, and PR patients was 7.6-, 8- and 2.9-fold, respectively (p = 0.004, 0.001, and 0.043, respectively)
  • There was no significant difference between patients in CR and healthy subjects
• In patients evaluated for CD34 expression, those with advanced aggressive, unresponsive, or terminal disease had raised levels of CD138⁻P-gp⁺CD34⁺ MPs relative to patients in remission or responding to therapy

Conclusion

This study demonstrated that circulating MPs can be used to detect and monitor MDR in patients with MM, acting as a surrogate marker of their cells of origin. Elevated levels of P-gp⁺ and PS+ MPs were found to correlate with disease progression and treatment unresponsiveness. Moreover, dual positive CD138−P-gp⁺CD34⁺ MPs were associated with aggressive and unresponsive disease.

The low invasiveness of this personalized liquid biopsy test makes it feasible for the continuous monitoring of resistance. The authors believe that this new test could complement existing procedures used for the management of patients with MM. The study is the first one to demonstrate the presence of P-gp on the surface of MPs in cancer.