Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

64 5 Major Histocompatibility Complex Modulation and Loss
Fig. 5.4 Different methods for determination of
the expression of MHC antigens. The schematic
diagram summarizes the different techniques
currently employed for the analysis of MHC expression.
They can be divided into nucleic acidbased
and immunological-based techniques.
Thus, the quality control of experimental approaches and reagents utilized for tumor
cell staining as well as the standardization of the interpretation of immunohistochemical
results are urgently needed. An alternative method is fluorescence-activated
cell sorting (FACS), which is more accurate, sensitive and reliable (Fig. 5.4).
For example, tumors considered to be MHC class I ± by immunofluorescence have
been shown to express low levels of MHC class Iantigens as determined by FACS
analysis [31±33]. This method can be routinely performed utilizing well-standardized
protocols. A further advantage is the simultaneous expression analysis of several
markers within a cell population and the concomitant determination of the expression
of different antigens on normal versus tumor cells using tumor-specific markers.
In addition,Western blot analysis and nucleic acid-based assays exhibiting a higher
sensitivity than immunohistochemistry and flow cytometry are also available, but
still utilized less frequently. This includes the mRNA expression profiling by Northern
blot analysis or reverse transcriptase polymerase chain reaction (RT-PCR), as
well as the gel electrophoresis of specific HLA products using Southern blotting or
genomic PCR. The PCR-based methods also allow the identification of structural alterations
by direct sequencing of the respective amplification products (Fig. 5.4).
However, a major disadvantage of these techniques is the cellular contamination of
the tumor samples by stroma cells or by tumor-infiltrating lymphocytes (TILs),
which could be minimized or even circumvented by the use of a laser dissection microscope.