Download File - JOHN J. HADDAD, Ph.D.

Diagnostic Approaches to Maximize Therapeutic Effect 187
combination thereof. At least two assaying steps are carried out at different time
points during the course of disease, and comparative information is obtained
from the assaying steps. The obtained information can be used to help decide
how and when to implement, modify, or withdraw a therapy. PCR techniques
are sensitive and generally easy to implement; however, they cannot detect the
mosaicism of antigen expression within a sample. IHC (and other in situ
techniques) provides the means to observe the spatial variation of expression.
Antibody-based techniques can offer the advantage of directly detecting
protein expression at the cell surface, which is of clinical relevance, in contrast to
RT-PCR and the like, from which surface expression can only be inferred. In
general, immunohistochemical staining allows the visualization of antigens via
the sequential application of a specific antibody (primary antibody) that binds to
the antigen, a secondary antibody that binds to the primary antibody, an enzyme
complex, and a chromogenic substrate with washing steps in between. The
enzymatic activation of the chromogen results in a visible reaction product at
the antigen site. The specimen may then be counterstained and cover slipped.
Results are interpreted using a light microscope and aid in the differential
diagnosis of pathophysiological processes, which may or may not be associated
with a particular antigen. Over the past two decades, the availability of HLAspecific
monoclonal antibodies (mAb) suitable for immunohistochemical staining
and technical advancements in immunohistochemical staining techniques
have allowed extensive analysis of HLA1 expression, HLA-specific markers,
such as b2M, and TAA. However, suitable antibodies for the IHC detection of
type-specific HLA1 molecules in FFPE samples remained difficult to obtain.
We have generated highly specific monoclonal antibodies that are peptide
specific by Hybridoma technology, in house for two of our target TAA,
Prame and SSX2. Antibodies for PSMA, Melan-A, Tyrosinase, and NY-ESO-1
are available commercially.
The expression of polymorphic determinants of HLA1 requires the association
of HLA1 heavy chains with b2M. Therefore, class I expression can be
assessed by detection of b 2M. To this end, sections of formalin-fixed lesions are
stained with mAb recognizing b 2M in immunoperoxidase reactions. The b 2M
protein is a component of the MHC1. Humans synthesize three different types of
class I molecules designated HLA-A, HLA-B, and HLA-C. These differ only in
their heavy chain, all sharing the same type of b2M, which is highly conserved.
MHC1 is formed by the association of b2M and an alpha protein, heavy chain,
which comprises three domains: a1, a2, and a3. b2M associates with the a3
subdomain of the a heavy chain and forms an immunoglobulin domain-like
structure that mediates proper folding and expression of MHC1 molecules. MHC1
is found on the surface of most types of nucleated cells, where it presents antigens
derived from proteins synthesized in the cytosol to CD8 þ T cells. Two signals are
required for activation of naive CD8 þ T cells, the first provided by the interaction
of the TCR with the MHC1-antigen complex on the antigen-presenting
cell (APC) surface, and the second, costimulation, generated by the interaction of