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184 Chiang et al.
technologies are enabling the stratification of cancers, the ability to follow
cancer progression, the ability to stratify patients as responders or non-responders
to therapy, and the ability to monitor in vivo cancer biology and therapeutic
responses.
Demonstrating the presence of targeted antigens in cancer cells is an
important first step for selecting patients most likely to respond. A number of
methods have been used to demonstrate that the targeted antigens are present in
the cancer cells. The methods include traditional methods such as immunohistochemistry
(IHC) and cytogenetics as well as newer molecular methods
such as RTPCR, PCR, and in situ hybridization. PCR and RTPCR are highly
sensitive methods; under favorable conditions, the presence of a few molecules
of nucleic acids in the sample can be detected. However, these methods lack
the ability to indicate the spatial distribution of the target molecules in reference
to morphological features. For example, the detection of PSMA mRNA in
a tumor sample cannot indicate whether PSMA is expressed by the tumor cells
or neovasculature surrounding the tumor cells. Furthermore, the detection or
quantitation of mRNA would not provide direct information on the amount of
the protein antigen in tumor cells or its subcellular location. In addition, the
detection of mRNA in formalin-fixed paraffin-embedded (FFPE) tumor samples
(the most common type of archived samples) is difficult because of RNA
degradation during the processing steps and storage of the FFPE samples
(Table 2).
IHC (and other in situ techniques), though potentially more labor intensive,
allow spatial variation of expression within a sample to be observed. Distinctions
can be made such as coexpression of antigens within the same cells providing for
greater redundancy of targeting and reduced likelihood of escape mutants arising
by antigen loss, and coexpression within different cells within the same sample,
revealing how a greater proportion of the total tumor tissue can be directly
targeted. Such information is also relevant to the use of antigens with more
complex expression patterns. For example, PSMA, which can be expressed by
Table 2 Assays for Patient Stratification in Targeted Immunotherapies
Name Use
IHC Ascertain tumor tissue expresses targeted antigen
RTPCR Ascertain tumor tissue expresses targeted antigen
Immune competence a
Ascertain patients’ immune function
HLA genotyping b
Make sure patient has targeted HLA type
Flow cytometry c
Ascertain tumor cells expresses targeted antigen
a
for active immunotherapies only.
b
for T-cell–based active immunotherapies and passive immunotherapies relying on
presence of HLA-peptide epitope complexes.
c
for leukemia.