microsampling to measure tamoxifen for breast cancer TDM

An article by Yahdiana Harahap et al at three institutions in Indonesia, published in the June 2021 edition of Heliyon, investigated the use of Mitra^® devices with VAMS^® technology for measuring tamoxifen (TAM) and related metabolites in breast cancer treatment. The paper is entitled “Analysis of tamoxifen and its metabolites in dried blood spot and volumetric absorptive microsampling: comparison and clinical application.”

This study successfully validated both the prodrug (TAM) and the three most abundant active metabolites. Following this, the assay was tested on 30 study participants with breast cancer. The authors concluded that this method had strong potential as a therapeutic drug monitoring (TDM) application. However, the researchers determined that more work is needed to understand any hematocrit biases and also to bridge from dried capillary blood to venous plasma/serum.

Breast Cancer Rates and Treatments

According to the World Health Organization (WHO), breast cancer was reported to be the most prevalent cancer in 2020. Indeed, the report mentioned that in 2020 alone, there were 2.3 million women newly diagnosed with breast cancer and 7.8 million living with the condition worldwide. Sadly, there were also 685,000 deaths as a result of the disease.

As with many cancers, there are several options for treating breast cancer including chemotherapy, surgery, radiotherapy, targeted therapeutics, bisphosphonates for bone strengthening in types of cancer that cause bone damage, and hormonal therapy. One hormonal treatment is the selective estrogen cancer receptor modulator Tamoxifen (TAM).

History and Importance of Tamoxifen

Synthesized in 1962 by ICI (now part of AstraZeneca), compound ICI 46474 (now tamoxifen) was originally developed as an oral contraceptive. However, even though it was designed to be an anti-estrogen, the ICI compound acted to stimulate ovulation rather than prevent it. It was then repurposed as a drug to treat and prevent breast cancer. As a result, it became one of the world’s bestselling hormonal breast cancer medicines.

Indeed, a study reported in The Lancet in 2012 about the Adjuvant Tamoxifen: Longer Against Shorter (ATLAS) trial demonstrated that 10 years of tamoxifen treatment reduced mortality from breast cancer by 50% in 10-14 years after diagnosis.

Tamoxifen Mode of Action    

As reported in the paper by Yahdiana Harahap et al, TAM acts to inhibit estrogen receptor transcription activity. This, in turn, suppresses the growth of breast cancer tumors. TAM is a prodrug that is enzymatically converted to endoxifen (END), 4-hydroxytamoxifen (4-HT), and N-desmethyltamoxifen (NDT). Out of these, the vast majority is END (92%) and is reported to have 30-100 times more efficacy than TAM.

It has been reported that serum concentrations of END above 5.9 ng/mL reduce the risk of reoccurrence compared to levels below this. Typically, TAM analysis is conducted by measuring venous plasma levels. However, the research group identified that venous blood draws for plasma sampling cause discomfort for study participants and also is inconvenient due to the need to cryogenically preserve samples for transport and storage.

For these reasons, the research group decided to evaluate both dried blood spot (DBS) cards and Mitra^® devices with VAMS^® technology. Both of these dried matrix approaches eliminate the need for cold shipping and storage, require lower blood volumes, and enable simpler sampling procedures compared to venipuncture. The researchers commented that a disadvantage of DBS cards was that they were less sensitive and needed longer extraction time compared to venipuncture.

The group reported that the VAMS devices had the advantage of consistent sampling compared to DBS. Another reason for evaluating both devices was because DBS and VAMS were made from different materials and so one may perform better than the other from a sample process standpoint. It was hoped that this evaluation would show a possible way to conduct TDM more efficiently in the future for tamoxifen.

Tamoxifen Study Methods and Findings

• DBS samples were prepared by pipetting 20 µL of either spiked (TAM, END, 4-HT, and NDT) or participant blood (collected in a vial) onto PE226 paper. Mitra samples (with 20 µL VAMS tips) were sampled directly onto blood, skipping the pipetting stage. The Mitra microsamplers with VAMS tips are designed to collect a precise volume of blood so there is no need to use a pipettor. The dried blood samples were then sonicated in methanol (plus internal standard in propanol).
  
  
• The extracts were analyzed by LC-MS/MS and the method was validated based on both the Food and Drug Administration (2018) and European Medicines Agency (2011) guidelines for validation of bioanalysis. Highlights are as follows:
  
  
  • Imprecision (intra- and inter-assay) for DBS was between 2.24 – 13.07% (15.85% for LLOQ) and 5.01-12.03 respectively.
    
    
  • Imprecision (intra- and inter-assay) for VAMS was between 0.58 – 11.8% and -12.64 to 15.35%, respectively.
    
    
  • The estimated accuracy on VAMS was better (intra-assay bias 3.50-9.48% and inter-assay bias was -1.02-5.79%) compared to DBS (intra assay bias -17.29 (LLOQ) to 14.00% and inter-assay bias -19.69 (LLOQ) to 14.49%).
    
    
  • All analytes were stable for 1 month at room temperature (20-25° C) on both devices as well as 24h at -20° C and 40° C to mimic transportation conditions.
    
    
  • The group commented that recoveries from VAMS were better than from DBS and concluded that this was possibly due to the difference in the two materials and their intermolecular properties.
    
    
  • It has been historically shown that % hematocrit affected % bias so the group aims to conduct follow-up work to investigate this.
    
    
• The assay was tested on 30 patients:
  
  
  • The mean concentration measured on VAMS compared to DBS was slightly higher but statistical analysis proved this not to be significant.
    
    
  • Two patients showed low END levels.
    
    
  • The group concluded that both VAMS and DBS could be used to examine TAM and its metabolites for TDM, but they would need to conduct bridging between traditional venous (plasma / serum) and capillary samples as there may be a difference in concentrations due to the presence of the hematocrit.

Neoteryx Comments

This study demonstrates how dried blood microsamples can be used for therapeutic drug monitoring (TDM) research and potential deployment. Although the results in this study showed that both DBS and VAMS microsampling techniques could be used, it must be noted that whole spot extraction was conducted on the DBS spots from pre-pipetted samples.

This would be more of a limitation to those self-collecting or for people collecting blood without training on how to use a pipettor. In contrast, sample collection with VAMS devices can be conducted by practically anyone, anywhere.

Finally, as highlighted by the authors, it would be very important to conduct a bridging study to ensure the equivalence between wet plasma / serum and dried blood. Indeed, blood to plasma partitioning has been highlighted and discussed in much of the literature describing studies that compared traditional sampling to dried blood microsampling.

For example, in 2021 Ugo de Grazia et al, reviewed VAMS TDM literature of antiseizure medications (ASMs) where blood to plasma ratios were compared from different studies. They stated that for those ASMs where the blood/plasma ratio deviates from 1, a correction factor is needed to calculate a plasma concentration equivalent.

This article was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this blog, visit the original article in Heliyon.

For more information on how Neoteryx microsampling products from Trajan help researchers, visit our Technical Resource Library.

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