Phage Immunoprecipitation Sequencing of Tumor-Specific Autoantigens as Biomarker of Testicular Germ Cell Tumor Diagnosis and Outcome Prediction

Testicular cancer is one of the most common solid tumors encountered in active-duty military population. Some studies have shown that the incidence rates of testicular cancer have increased over the years. It is estimated that there will be 9,470 new cases of testicular cancer in 2021 and 440 deaths resulting from it. However, traditional tumor biomarkers have overall low sensitivity and specificity for testicular cancer diagnosis. In the proposed study, we are trying to develop a high throughput technique utilizing patients' immune response against tumor proteins to find testicular cancer specific antibody signatures or immunoprofiles.

Cancer cells stimulate generation of antibodies and T-cell response against immunoprivileged cancer proteins. Whether this response is effective in stopping the growth and spread of cancer or unsuccessful, the autoantibody profile generated can serve as useful cancer biomarker. Additionally, anti-tumor autoantibodies may be present in sera, potentially even before the tumors are detectable via laboratory or radiographic tests. Furthermore, they likely persist in the serum as long as the corresponding tumor-specific antigens that elicited the humoral response remains, and in some scenarios even after underlying tumor has been removed due to persistence of memory B-cells or plasma cells. This stability and persistence of autoantibodies gives them a considerable advantage over other serum tumor biomarkers.

We will use a library of bacteriophage viruses expressing cancer proteins which are not typically expressed in normal human tissues (except for immunoprivileged sites such as testes and brain) to evaluate presence of cancer specific antibodies in patients. Careful analysis of anti-tumor antibody repertoires with this novel technique provides an opportunity to identify an antibody signature for tumor detection (diagnosis) and a distinct antibody signature to inform tumor outcome or tumor immunotherapy response (prognosis). We will compare these readouts across different testicular cancer types in addition to comparisons with healthy individuals, and other cancers. We think this assay will provide a diagnostic blood test for underlying testicular cancer. This will especially be useful for patients with germ cell tumors such as seminomas in their chest (mediastinum) or abdomen (retroperitoneum), which are more difficult to biopsy or surgically remove.

Some studies have already highlighted favorable cancer prognosis among patients with onconeural antibodies such as SRY-Box Transcription Factor 1 (SOX1) in patients with small cell lung cancer and Lambert Eaton Myasthenic Syndrome. However, paraneoplastic syndromes are very rare and the frequency of onconeural antibodies among cancer patients without co-existing paraneoplastic presentations is low, limiting utilization of these antibodies for cancer prognostication. To overcome this limitation, we will be analyzing autoantibody profiles against all known tumor specific antigens (greater than 1,500) detected in a wide variety of tumors. We plan to correlate these anti-cancer antibody repertoires with International Germ Cell Consensus Classification risk categories, which is utilized to predict favorable or unfavorable cancer outcomes.

Customized tumor-specific antigen phage immunoprecipitation sequencing provides an affordable and technically robust approach for discovery of testicular cancer specific autoantibody profiles. Furthermore, our laboratory has a strong track record in tumor antibody biomarker discovery over the past two decades, and we have the necessary infrastructure, expertise, and patient samples to carry out this unique project.

The proposed grant directly impacts the military health focus of mission readiness as it pertains to early cancer detection/diagnosis and prognostication. As the proposed research can be utilized for cancer diagnosis among high-risk population, such as patients with testicular/mediastinal/retroperitoneal mass, it also directly impacts the FY21 PRCRP overarching challenge.