Issues And Caveats With Pbmc Profiling In Oncology Studies

PBMC profiling represents a difficult task, both in terms of logistics and interpretation. Our own internal studies and more recent reports have documented significant alterations in select subsets of transcripts in PBMCs following overnight incubation of whole blood at room temperature under conditions that mimic those encountered in clinical trials, where blood samples are drawn at clinical sites and then shipped overnight to a central processing laboratory.3940 Debey et al.39 reevaluated the transcripts our laboratory identified as disease associated in patients with RCC32 and identified only 12 of the 132 disease-associated transcripts as belonging to the group of transcripts subject to significant fluctuation following overnight incubation. This was an expected result, since our original analysis involved PBMCs from disease-free individuals and patients with RCC that were handled under identical conditions of overnight storage of whole blood prior to processing to PBMCs. However, the authors correctly opine that, while the majority of disease-associated transcripts in RCC PBMCs do not appear to be subject to significant alteration during ex vivo incubation, it is possible that our laboratory missed transcripts that might have been truly differentially expressed in patients with RCC relative to healthy controls, but were missed following an overnight incubation that resulted in severe artifactually induced downregulation of a subset of transcripts in both populations of blood samples. It is clear that immediate processing is the optimal approach, but for large multicenter trials this remains a practical impossibility since many of the sites lack the necessary staff or equipment to execute these purifications. In this instance, then, overnight shipping of collected blood samples and processing by a central laboratory is the method of choice, since this method treats all samples similarly. Nonetheless, our laboratory and others are constantly evaluating alternative approaches to blood collection, stabilization, and preparation for the purposes of minimizing artifactual ex vivo changes in gene expression profiles determined from in vivo samples. The issues and caveats associated with blood profiling are presented in this book in greater detail in Chapter 2.

In addition to the ex vivo effects of storage prior to processing, depending on the disease state of the individual in question, activated neutrophils and other poly-morphonuclear leukocytes can differentially migrate through density gradients designed to enrich for mononuclear cells, further confounding analyses of PBMC transcriptional profiles.41 While these alterations may be very relevant to the overall disease process rather than a simple technical artifact (neutrophil activation may be correlated with tumor aggressiveness, etc.), these variabilities in cellular composition nonetheless present difficulties associated with analysis and interpretation of tran-scriptional profiling data in PBMCs.

Our laboratory now routinely employs ANCOVA approaches to account for variations in cell populations, and this approach greatly assists in delineation of transcriptional differences in PBMCs that appear related to differences in cell populations vs. differences that are independent of cell populations and therefore likely represent bona fide alterations in transcriptional regulation. Nonetheless, newer technologies like PaxGene (Qiagen, Valencia, CA) and other whole-blood stabilization technologies afford an opportunity to minimize variation due to sample handling and processing. While they are associated with their own challenges (for instance, PaxGene-purified RNA contains excess hemoglobin RNA that can dramatically reduce the sensitivity of RNA profiles measured on oligonucleotide arrays), these advances in technology are continuing to evolve and striving to provide a more reliable alternative to enable surrogate tissue profiling with greater reproducibility. For example, globin reduction protocols have been developed, and other non-RNAse-based methods for globin reduction are currently under development, for the removal of globin from whole-blood samples prior to expression profiling. Further studies are required to determine whether profiling of surrogate tissues such as PBMCs will provide transcriptional markers with meaningful applicability in solid tumor diseases, but given the abundant evidence for the interaction between the immune system and tumor cells trying to evade it, the possibility remains an attractive hypothesis for exploration.

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