Reverse phase protein arrays in acute leukemia: investigative and methodological challenges

ABSTRACT

Introduction

Acute leukemia results from a series of mutational events that alter cell growth and proliferation. Mutations result in protein changes that orchestrate growth alterations characteristic of leukemia. Proteomics is a methodology appropriate for study of protein changes found in leukemia. The high-throughput reverse phase protein array (RPPA) technology is particularly well-suited for the assessment of protein changes in samples derived from clinical trials.

Areas covered

This review discusses the technical, methodological, and analytical issues related to the successful development of acute leukemia RPPAs.

Expert commentary

To obtain representative protein sample lysates, samples should be prepared from freshly collected blood or bone marrow material. Variables such as sample shipment, transit time, and holding temperature only have minimal effects on protein expression. CellSave preservation tubes are preferred for cells collected after exposure to chemotherapy, and incorporation of standardized guidelines for antibody validation is recommended. A more systematic biological approach to analyze protein expression is desired, searching for recurrent patterns of protein expression that allow classification of patients into risk groups, or groups of patients that may be treated similarly. Comparing RPPA protein analysis between cell lines and primary samples shows that cell lines are not representative of patient proteomic patterns. 

KEYWORDS:

• Acute leukemia
• RPPA
• proteomics
• sample handling
• network-based approach
• classification
• acute leukemia
• methodology

Article highlights

• Reverse phase protein array proteomic technology is particularly well-suited for the assessment of protein expression within the context of a clinical trial;

• Variables such as sample shipment, transit time (≤72 hours), and holding temperature have minimal effects on protein expression in acute leukemia samples;

• Incorporation of standardized guidelines for antibody validation used for RPPA is recommended;

• Recurrent patterns of protein expression allow for classification of leukemia patients into risk groups independent of molecular classification. RPPA also identifies subgroups of patients that could be treated similarly;

• Protein expression in acute leukemic cell lines is not representative of patient leukemia cell protein expression.

Disclosure statement

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose