Historically, many tumor targets and markers were discovered by serology. With the advent of hybridoma technique, whole tumor cells were used to immunize lymphocytes, and clone selection was based on their differential expression of tumor over marrow/blood, or tumor over normal tissues. However, this marker discovery approach has major limitations. Lymphocytes, regardless of its being in vivo or in vitro, may be anergic or tolerized to certain antigens. Depending on the types of antigens studied, the immune repertoire of these lymphocytes can be limited, or skewed by dominant clones.
Tumor marker discovery using genome-wide expression array approach is an attractive alternative. Screening of expressed genes overcomes the issues of clonal frequency and lymphocyte restrictions. While the whole tumor approach identifies antibody before the antigen, the expression profiling approach pinpoints known genes, which are likely to have web-based tissue expression information available to filter out rapidly false leads. This marker discovery strategy was successfully applied to identify three novel markers of Ewing family tumors (8
). The detection of STEAP1, CCND1, and/or NKX2-2 at diagnosis was informative and was predictive of whether patients had a higher likelihood to eventually develop new metastases and to die of this cancer. When applied to other tumor expression arrays, novel gene lists have recently been generated (data not shown). Besides genomics, proteomics and glycomics can take advantage of this approach.
The marker discovery outlined in this report was based on profiling a substantial number of stage 4 NB tumors, ensuring a representative spectrum of this metastatic cancer. The differential gene expression of NB tumors to remission marrow ratios as well as expression level was taken into account. Selection was based on tumor to marrow expression ratio superior to that of TH, a widely accepted NB marker, which serves as the gold standard. Based on these criteria, ~16,000 genes were filtered to a list of 34 candidate genes as potential markers. However, there was no one-to-one corresponding correlation between gene expression array ranking and the ranking derived from the sensitivity experiments (). This is likely because the expression level on the array depends on the target sequences of the specific gene chips. For example, even though 18/48 tumors in the array had genomic MYCN amplification, MYCN expression level did not meet the criteria we set, and was rejected in our final list of candidate genes. Additionally, glycolipid markers including beta-1,4-N-acetyl-galactosaminyl transferase (GD2 synthase) and sialyltransferase STX (ST8SiaII) were not detectable by the Affymetrix U95 Chip. This was most likely due to the substantial difference between the Affymetrix target sequence from the sequence deposited at GenBank. These findings reinforced our continual effort to discover novel molecular markers using a multipronged approach, be it genome-wide expression screen, or focusing on aberrations (e.g. MYCN) and pathways specific for tumor or metastases. We believe that both global screening and pathway-based approaches can complement the MRD marker discovery process.
Besides being highly sensitive with detection in 10−6
frequency, the 8 top ranking markers were also specific with high expression in stage 4 NB tumors (). CCND1 (Cyclin D1) has a pivotal role in controlling cyclin-dependent kinases during cell cycle progression (15
), and it is over-expressed and has adverse prognostic impact in human cancers including NB (16
). In addition to CCND1, DDC (dopa decarboxylase) and PHOX2B (paired-like homeobox 2b) are also associated with NB. DDC, an enzyme involved in the pathway of catecholamine synthesis, was shown to have utility as a tumor marker for NB (17
). PHOX2B is a gene involved in the development of several major noradrenergic neuron populations. It is highly expressed in NB, and its germline mutation may be linked to hereditary NB (18
The rest of our novel markers, namely CRMP1, GABRB3, KIF1A, ISL1, TACC2 have not been previously reported to be associated with NB. CRMP1 (collapsin response mediator protein 1) belonging to a family of cytosolic phosphoproteins expressed exclusively in the nervous system is involved in signal transduction pathway during neural development. GABRB3 (gamma-aminobutyric acid A receptor, beta 3), which encodes a member of the chloride ionic channel family, serves as the receptor for gamma-aminobutyric acid, the major inhibitory transmitter of the nervous system. KIF1A (kinesin family member 1A) belongs to the microtubule family involved with ATP binding. ISL1 (ISL LIM homeobox 1) is a zinc finger transcription factor, whereas TACC2 (transforming, acidic coiled-coil containing protein 2) belongs to a conserved family of proteins that are implicated in tumorigenesis by encoding a protein that concentrates at centrosomes throughout the cell cycle.
As part of our discovery algorithm, we tested these 8 candidate markers on the post-cycle 2 bone marrows of a cohort of stage 4 patients treated uniformly with an immunotherapy protocol. In addition to CCND1, a novel MRD response marker of NB reported previously (9
), 5 additional markers, namely DDC, GABRB3, ISL1, KIF1A, and PHOX2B, were found to be useful in predicting patient outcome. These markers will need to be tested using an independent set of patient samples collected prospectively to confirm clinical utility. Moreover, a further fine-tuning will be required to determine which markers should be combined with GD2 synthase or TH in order to optimize the detection of marrow MRD in metastatic NB patients.