Protein Biomarker Identification
Current diagnostic methods for lung cancer include CT or PET followed by biopsy and surgery. However, sporadically discovered small nodules in lungs are typically inflammatory in character. Therefore, the standard of care often involves simply waiting to see if they grow in size.
This strategy seems to run contrary to the fact that survival rates are considerably better when lung cancer is diagnosed at early stages. “A blood biomarker that is able to detect lung cancer at early stages would guide the treatment decision at the time when the patient’s chances for survival are high,” comments Laszlo Takacs, M.D., Ph.D., CSO of Biosystems International (BSI).
While many approaches include finding a blood biomarker by mass spectrometry based on differences between affected and healthy individuals, Biosystems International decided on a different strategy. Its approach is based on generating antibodies against the whole plasma proteome of lung cancer patients.
BSI prepares an immunogenic fraction of whole blood plasma by removing the most abundant proteins and then normalizing concentrations of the remaining proteins. The fraction is then injected into mice to generate nascent hybridomas.
After multiple rounds of screening, 3,000 hybridomas were narrowed down to 13 clones with good discriminatory power between patient and normal samples. “The preliminary data shows that our hybridomas recognize five proteins specific for lung cancer,” says Dr. Takacs.
“Moreover, some of these are cancer-specific isomers, also detected by immunohistology. We see significant potential for our diagnostic assay in guiding treatment decisions at the initial encounter of the stage I cancer patient with a primary-care physician or pulmonologist. Definitive management of the disease could be introduced earlier and in more specific ways.” The company is preparing for the first clinical trials in symptomatic patients with an ELISA-based diagnostic kit.
Blood-based protein biomarkers would make a significant difference in clinical practice. Even though proteomics is expected to play a leading role in clinical biomarker discovery, translation of MS-based assays into clinical diagnostic tests has been lagging.
The human proteome content varies significantly even within a single individual based on food intake, rest, and other physiological factors. Finding differences between blood proteomes of healthy individuals and cancer patients that could indicate the presence of cancer rather than merely a physiological bias has been problematic.
The strategy proposed by Josip Blonder, M.D., head of clinical proteomics at the National Cancer Institute, utilizes combined plasma/tissue proteome analysis from a single patient. This strategy relies on the assumption that salient proteins present in the tumor are detectable in blood. A three-way comparison of plasma, tumor, and normal tissue would help to narrow down the selection of relevant biomarker candidates.
Comparison of samples from the same patient minimizes the effect of normal biological variances. First, Dr. Blonder’s team identified tumor-specific proteins by discounting common proteins found in renal cell carcinoma (RCC) and adjacent normal tissue. Next, the tumor-specific proteins were compared with the plasma, looking for overlapping sets.
“This strategy is not devoid of typical proteomics deficiencies, such as the mismatch between the dynamic ranges of MS instrumentation and the human proteome. This issue is significantly alleviated, however, by the use of shotgun proteomics coupled with high-resolution and high-accuracy mass measurements,” says Dr. Blonder.
“Analysis resulted in the identification of 202 proteins that belong only to the tumor. This set was compared with 179 proteins from plasma, revealing 8 overlapping proteins, whose spectral count was higher in the tumor than in the plasma.” All eight proteins were specific to the tumor tissue, and four of these proteins were cross-validated in the blood of this patient and in the blood of four additional patients diagnosed with RCC.
“This pilot study was a proof of concept demonstrating that the comparative analysis of blood and tissue may result in the identification of tumor proteins in blood and may yield a realistic biomarker panel,” continues Dr. Blonder. “We hope this schema will be adopted and popularized by other laboratories.
“I do believe that current state-of-the-art clinical proteomics has matured enough for comprehensive and coordinated effort focused on creation of the cancer proteome atlas by relying on shotgun proteomics to sequence human cancers individually. The resulting atlas will be used as a complement to the cancer genome atlas and accelerate our understanding of the molecular basis of cancer.”