Quantitative Measurement of the mRNA Expression
of the Tumor-Associated Antigen PRAME by Real-Time
RT -PCR Using LightCycier and SYBR Green I Technology
JOCHEN GREINER, MARK RING HOFFER, ANITA SZMARAGOWSKA,
SANDRA HUBSCH, HARTMUT DOHNER, MICHAEL SCHMITT*
Introduction
Characterization of immunogenic tumor-associated antigens (TAA) is mandatory for the design of cancer vaccines. Cancer antigens eliciting an immune
response in the tumor-bearing host are targets for specific immunotherapy. In
recent years, many TAA have been identified by methods using cytotoxic T-lymphocytes or by serological screening of recombinant expression cloning. TAA can
be classified into three major categories:
1. Overexpressed genes are known to elicit immune responses by overriding
thresholds critical for the maintenance of tolerance [1]. These antigens are not
strictly tumor-specific, but they are overexpressed in different tumor tissues.
2. Antigens encoded by mutated genes induce immune responses by changing
the expression and/or conformation of proteins [2].
3. Cancer/testis (CT) antigens are a family of tumor genes expressed exclusively
in cancer cells and in testis tissue [3].
The TAA PRAME (Preferentially expressed fl,ntigen of melanoma) is such an antigen with high expression in tumor and testis. It is also expressed at a low level in
the endometrium and ovary [4]. PRAME has been characterized in a melanoma
cell line derived from a patient (LB33) [4]. The cellular function of PRAME
remains to be elucidated. PRAME is frequently expressed in melanoma (91 %),
lung squamous cell carcinoma (78%), sarcoma (39%), and renal cell cancer (41 %)
[4,5]. It is also expressed in hematological malignancies such as acute and chronic myeloid leukemias and in different lymphoma subtypes [6, 7]. Approximately
47% of acute myeloid leukemia (AML) samples obtained from patients diagnosed
at our institution expressed PRAME at a high mRNA level [7], while no mRNA
expression was found in peripheral blood, bone-marrow cells and CD34-positive
separated cell samples of healthy volunteers by RT-PCR [6, 7]. The fact that
PRAME is not expressed in most other normal tissues [4,6] reduces the risk for
negative effects of a vaccination with the antigen PRAME. It is therefore an inter* Michael Schmitt (~) (e-mail: michael.schmitt@medizin.uni-ulm.de)
Jochen Greiner, Mark Ringhoffer, Anita Szmaragowska, Sandra Hiibsch, Hartmut Dohner
Third Department of Medicine, University of Ulm, 89070 Ulm, Germany
of the Tumor-Associated Antigen PRAME by Real-Time
RT -PCR Using LightCycier and SYBR Green I Technology
JOCHEN GREINER, MARK RING HOFFER, ANITA SZMARAGOWSKA,
SANDRA HUBSCH, HARTMUT DOHNER, MICHAEL SCHMITT*
Introduction
Characterization of immunogenic tumor-associated antigens (TAA) is mandatory for the design of cancer vaccines. Cancer antigens eliciting an immune
response in the tumor-bearing host are targets for specific immunotherapy. In
recent years, many TAA have been identified by methods using cytotoxic T-lymphocytes or by serological screening of recombinant expression cloning. TAA can
be classified into three major categories:
1. Overexpressed genes are known to elicit immune responses by overriding
thresholds critical for the maintenance of tolerance [1]. These antigens are not
strictly tumor-specific, but they are overexpressed in different tumor tissues.
2. Antigens encoded by mutated genes induce immune responses by changing
the expression and/or conformation of proteins [2].
3. Cancer/testis (CT) antigens are a family of tumor genes expressed exclusively
in cancer cells and in testis tissue [3].
The TAA PRAME (Preferentially expressed fl,ntigen of melanoma) is such an antigen with high expression in tumor and testis. It is also expressed at a low level in
the endometrium and ovary [4]. PRAME has been characterized in a melanoma
cell line derived from a patient (LB33) [4]. The cellular function of PRAME
remains to be elucidated. PRAME is frequently expressed in melanoma (91 %),
lung squamous cell carcinoma (78%), sarcoma (39%), and renal cell cancer (41 %)
[4,5]. It is also expressed in hematological malignancies such as acute and chronic myeloid leukemias and in different lymphoma subtypes [6, 7]. Approximately
47% of acute myeloid leukemia (AML) samples obtained from patients diagnosed
at our institution expressed PRAME at a high mRNA level [7], while no mRNA
expression was found in peripheral blood, bone-marrow cells and CD34-positive
separated cell samples of healthy volunteers by RT-PCR [6, 7]. The fact that
PRAME is not expressed in most other normal tissues [4,6] reduces the risk for
negative effects of a vaccination with the antigen PRAME. It is therefore an inter* Michael Schmitt (~) (e-mail: michael.schmitt@medizin.uni-ulm.de)
Jochen Greiner, Mark Ringhoffer, Anita Szmaragowska, Sandra Hiibsch, Hartmut Dohner
Third Department of Medicine, University of Ulm, 89070 Ulm, Germany