The combined use of tissue microarray and automated quantitative assessment of immunofluorescence (TMA-AQUA) has potential to serve as a translational research tool by permitting high-throughput validation of potential protein targets identified in large profiling studies leading to development of these targets as routine immunohistochemical stains that are evaluated by diagnostic pathologists. TMA-AQUA has already demonstrated this capacity in a number of solid tumor cohorts3, 4, 4-6
, but to our knowledge, its performance in lymphoid malignancies, whose cytologic and architectural distinction from carcinoma may manifest distinct challenges to TMA-AQUA, has yet to be established. Using a pilot scale TMA comprised of 15 MCL cases and appropriate control tissues, we demonstrate findings that can guide effective utilization of TMA-AQUA as a protein biomarker screening platform for non-Hodgkin lymphoma.
Heterogeneity of target protein expression becomes a concern when tumor is represented by a small core on a TMA. In MCL, expression of proliferation markers are of particular concern because not only can their expression be heterogeneous, they are also the genes whose expression is most predictive of outcome.7
We found that protein expression determined by AQUA score from duplicate cores drilled from different locations on MCL specimens correlated well with one another, including the proliferation marker Ki-67. This suggests that heterogeneity in expression may not be a significant issue in MCL, however, focal areas of increased expression may not have been adequately sampled by our methods. We do demonstrate that duplicate cores do not provide a significant advantage over a single core TMA in accounting for location-dependent expression of markers in MCL.
Using a known diagnostic biomarker for MCL, Cyclin D1, we validated both the TMA-AQUA platform and the MCL cases on the TMA by showing the AQUA score representing Cyclin D1 expression in the B-cells was significantly higher in the MCL cases than non-MCL controls. also demonstrates an advantage of continuous scale quantification. A clear difference between AQUA scores for Cyclin D1 in MCL and non-MCL cases is apparent, suggesting that for markers that need binary classification, a cut-off value for positive expression of a target can be established.
The ability to localize protein expression to the nuclear or cytoplasmic compartments is unique to AQUA and can yield particular advantages. Not only could effects of non-specific staining be reduced by confining analysis to the pertinent subcellular compartment only, but atypical localization patterns of target proteins can also be assessed for clinical significance. We found that TMA-AQUA performed on MCL cases could accurately quantify nuclear versus cytoplasmic expression for targets that have high levels of expression, but failed to do so for the targets that had low levels of expression (Ki-67 and Mcm2 in ). Accordingly, before initiating analysis of subcellular compartment specific protein expression levels, each marker, especially those with low levels of expression, needs to be validated for compartment specificity.
For most of the markers tested, AQUA scores correlated well with semi-quantitative scoring of DAB staining performed by two pathologists, supporting the idea that prognostic markers identified from TMA-AQUA screening can be successfully developed into DAB stains that are sufficiently quantifiable by diagnostic pathologists. Of the six proteins assessed, the only one that did not achieve a good correlation between AQUA score and DAB scoring was Cdc2, the protein that had the lowest range in DAB scoring (). This case is illustrative of situations where the sensitivity of AQUA to small differences in protein expression levels could not be sufficiently reproduced by semi-quantitative scoring of DAB staining.
Not surprisingly, we found that the correlation of gene expression with protein expression varied considerably (), underscoring the fact that selecting protein targets based on results of gene expression profiling studies carries few guarantees. However, without reliable protein profiling studies to turn to at the present time and left with long lists of prognostic mRNA targets that likely won’t translate into protein markers, TMA-AQUA at least offers a high-throughput approach to this problem.
In summary, we find that despite challenges posed by particular cytologic and architectural features of non-Hodgkin lymphoma, TMA-AQUA has the capability to serve as a high-throughput screening platform for the validation of potential protein biomarkers to advance their development as routine immunohistochemical stains, thereby addressing a large translational gap in risk-stratification of patients with lymphoma.