An AFFIRM Assay for Potential Biomarkers in Relapsed Breast Cancer
(2016) KIMM01 20161Educational programmes, LTH
Department of Immunotechnology
- Abstract
- Relapsed or recurrent breast cancer is the chief cause of breast cancer related deaths and it is estimated that one-third of breast cancer patients would likely suffer a relapse. Due to high mortality and incidence, recurrent breast cancer requires better means of prediction, prevention and treatment. The AFFIRM platform uses recombinant antibody technology and selected reaction monitoring (SRM) mass spectrometry (MS) and has in previous studies proven to successfully detect and measure target proteins in serum. In this work, a multiplexed AFFIRM assay for measuring potential biomarkers of interest in plasma obtained from breast cancer patients at primary and secondary diagnoses was developed. Thirty proteins were selected as target... (More)
- Relapsed or recurrent breast cancer is the chief cause of breast cancer related deaths and it is estimated that one-third of breast cancer patients would likely suffer a relapse. Due to high mortality and incidence, recurrent breast cancer requires better means of prediction, prevention and treatment. The AFFIRM platform uses recombinant antibody technology and selected reaction monitoring (SRM) mass spectrometry (MS) and has in previous studies proven to successfully detect and measure target proteins in serum. In this work, a multiplexed AFFIRM assay for measuring potential biomarkers of interest in plasma obtained from breast cancer patients at primary and secondary diagnoses was developed. Thirty proteins were selected as target proteins for the assay, based on availability of in-house antibodies and previous studies describing association with breast cancer and proven presence in plasma. Assay conditions such as working plasma concentration, addition of protease inhibitors, use of Trypsin or Trypsin/Lys-C for digestion, SRM assay development and degree of multiplexing were studied. Out of the thirty target proteins, successful assays were developed for 26 of those. 50% plasma was concluded to be a suitable working concentration. Addition of protease inhibitors was demonstrated to slightly negatively affect SRM signals and Trypsin/Lys-C use was shown to improve digestion. Most importantly, it was demonstrated that all twenty-six proteins could be analysed in a multiplexed AFFIRM setup, with a high degree of reproducibility and consistency. This greatly saves time and reduces labour and is crucial because of limited volume of samples. The successful recombinant antibodies in this study were re-cloned into a vector with a biotin-accepting domain to allow using streptavidin magnetic beads for coupling for the final measurements of the target proteins in the clinical plasma samples. (Less)
- Popular Abstract
- Breast cancer is the most common form of cancer in women. Although noteworthy efforts have been made to accomplish early detection and efficient treatment, recurrent breast cancer cannot be cured.
Breast cancer is described as recurrent or relapsed if it appears after being treated initially. It is the major cause of the breast cancer related deaths. Early prediction of breast cancer recurrence would dramatically increase treatment possibilities and survival and a blood based test to follow the patient for these purposes would be of significant importance.
A biomarker can be defined as any substance that can be measured in the body or its products in order to predict or determine the incidence of a disease.
This study attempted to... (More) - Breast cancer is the most common form of cancer in women. Although noteworthy efforts have been made to accomplish early detection and efficient treatment, recurrent breast cancer cannot be cured.
Breast cancer is described as recurrent or relapsed if it appears after being treated initially. It is the major cause of the breast cancer related deaths. Early prediction of breast cancer recurrence would dramatically increase treatment possibilities and survival and a blood based test to follow the patient for these purposes would be of significant importance.
A biomarker can be defined as any substance that can be measured in the body or its products in order to predict or determine the incidence of a disease.
This study attempted to create a test measuring the levels of a group of selected proteins that would be measured in blood plasma. This test, called AFFIRM, would then be applied to the blood plasma of patients collected when the breast cancer was first discovered and treated as well as when it was discovered the second time, i.e. the recurrent breast cancer. AFFIRM uses the major technologies recombinant antibodies and mass spectrometry (MS). The recombinant antibodies are coupled to magnetic beads and are used to target proteins that would serve as biomarkers. The targeted proteins would then be digested then analysed with MS to produce a specific readout.
The ability of the test created in this study will provide a proof-of-concept and the basis for refinements and application to a larger and more detailed clinical study that will hopefully be able to define the early detection and monitoring of breast cancer patients at risk of suffering a relapse. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8881062
- author
- Olaleye, Oladapo LU
- supervisor
- organization
- course
- KIMM01 20161
- year
- 2016
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Relapsed Breast Cancer, SRM, Biomarkers, Mass Spectrometry
- language
- English
- id
- 8881062
- date added to LUP
- 2016-06-22 14:08:33
- date last changed
- 2016-06-22 14:08:33
@misc{8881062,
abstract = {{Relapsed or recurrent breast cancer is the chief cause of breast cancer related deaths and it is estimated that one-third of breast cancer patients would likely suffer a relapse. Due to high mortality and incidence, recurrent breast cancer requires better means of prediction, prevention and treatment. The AFFIRM platform uses recombinant antibody technology and selected reaction monitoring (SRM) mass spectrometry (MS) and has in previous studies proven to successfully detect and measure target proteins in serum. In this work, a multiplexed AFFIRM assay for measuring potential biomarkers of interest in plasma obtained from breast cancer patients at primary and secondary diagnoses was developed. Thirty proteins were selected as target proteins for the assay, based on availability of in-house antibodies and previous studies describing association with breast cancer and proven presence in plasma. Assay conditions such as working plasma concentration, addition of protease inhibitors, use of Trypsin or Trypsin/Lys-C for digestion, SRM assay development and degree of multiplexing were studied. Out of the thirty target proteins, successful assays were developed for 26 of those. 50% plasma was concluded to be a suitable working concentration. Addition of protease inhibitors was demonstrated to slightly negatively affect SRM signals and Trypsin/Lys-C use was shown to improve digestion. Most importantly, it was demonstrated that all twenty-six proteins could be analysed in a multiplexed AFFIRM setup, with a high degree of reproducibility and consistency. This greatly saves time and reduces labour and is crucial because of limited volume of samples. The successful recombinant antibodies in this study were re-cloned into a vector with a biotin-accepting domain to allow using streptavidin magnetic beads for coupling for the final measurements of the target proteins in the clinical plasma samples.}},
author = {{Olaleye, Oladapo}},
language = {{eng}},
note = {{Student Paper}},
title = {{An AFFIRM Assay for Potential Biomarkers in Relapsed Breast Cancer}},
year = {{2016}},
}