Biomarker Discovery in Central Nervous System
on which to do research. In the early 1980s, seeing the need for fresh and snap-frozen brain tumor tis- sue for neuro-oncologic studies, Dr. Bigner established the Brain Tumor Tissue Bank at Duke, which has proven to be an invaluable source of tissue for research.
Dr. Bigner has also fostered numerous fruitful collabo- rations with cancer researchers in and outside the U.S., with the view that this would more rapidly advance the field of biomarker discovery.
This chapter is organized as an approximate chronology of the techniques we have used in our dis- covery of various biomarkers, each of which is briefly discussed to provide the reader with a sense of how we developed our approaches (increasingly in productive collaboration with other groups) to advance the search for and use of new brain tumor markers. Space limi- tations preclude our citing all of the original research papers, but these can be found within articles in the reference list.
Methods of Biomarker Discovery
Immunologic Methods: Initial Work on Neuroectodermal Tumors
Immunology was recognized early on in the twentieth century as an attractive approach to defining antigenic biomarkers of tumors, and from that came the idea of turning antibodies into weapons to use against cancer.
As far back as 1905, Paul Erlich proposed using anti- bodies to deliver chemotherapeutic agents. Multiple groups in the 1960s demonstrated immune responses to brain tumors arising naturally in human patients and showed that this was, to some extent, cell-mediated.
At Duke, Mahaley et al. (1965) used lysates of human gliomas to produce antisera in rabbits. These anti- sera localized in vivo to human brain tumor cells, as assessed by autoradiography of 125I-labeled antisera applied to tumor tissue from patients who had been treated with the antisera by intracarotid injection a few days before tumor resection. This was a power- ful demonstration of the ability of antibodies to target tumors in vivo.
At that time, it was not known whether glioma cells had quantitatively or qualitatively distinctive sur- face antigens. Westermark’s group developed antisera
in rabbits against a glioblastoma and found reactiv- ity specific for malignant glioma cell lines (Wahlstrom et al., 1974). At Duke, we carried out a systematic examination of the surface antigen profiles of human glial tumors, using a large panel of glioma cell lines (Wikstrand et al.,1977). Antisera were raised in non- human primates using multiple glioblastoma tissues and cell lines, and high-titer sera were exhaustively preabsorbed with nongliomatous material including normal adult and fetal brain tissues. These antisera were then applied to human glial tumor cells. As measured by indirect live-cell membrane immunoflu- orescence and [14C]nicotinamide release (a measure of complement activation by bound antibodies), the antisera bound to human glioma cells but not to nor- mal adult brain. Various absorbed sera did demonstrate reactivity with some nonglial tumor tissues. In addi- tion, the presence of moieties shared by fetal brain tis- sue and glial tumors, but not by normal adult brain, was demonstrated, presaging the description of oncofetal antigens in multiple tumor types.
However, the use of antisera requires multiple adsorption steps to remove non-specific antibodies, and even when purified, these polyvalent antisera react against multiple antigens and multiple portions of each antigen, which limits their use in biomarker dis- covery. The development of monoclonal antibodies (MAbs) by Kohler and Milstein 1975was a tremen- dous advance, making it possible to produce theoreti- cally endless quantities of antibodies directed against not only a single molecule, but against a specific por- tion of that molecule. Their potential for exquisite specificity means that MAbs remain a mainstay of biomarker research today, not only in discovery, but in the development of tumor-specific therapeutics.
Following the introduction of Mab technology, sev- eral groups reported the generation of Mabs directed against glioma-associated cell surface molecules. Jean- Pierre Mach, Stefan Carrel and Nicolas de Tribolet at the Ludwig Institute for Cancer Research in Lausanne, Hugh Coakham and John Kemshead from the Imperial Cancer Research Fund Laboratories in Bristol and London, and our group at Duke characterized Mabs generated by immunization of mice with glioma cell lines, fetal brain tissue, and neuroectodermal tissue (Schnegg et al., 1981; Carrel et al., 1982; Wikstrand et al.,1982; Allan et al., 1983). These studies culmi- nated in a joint publication by these groups (Wikstrand et al., 1987) comparing the in vivo localization
capacities of these Mabs, as a prelude to evaluat- ing glioma-specific Mabs as immunodiagnostic and immunotherapeutic agents (Vick et al.,1987; Lee et al., 1987).
Immunologic and Biochemical Methods: Gangliosides
The search for brain tumor biomarkers has always been intertwined with the identification of markers for normal brain cells, especially in the context of brain development because of parallels between the behavior of developing cells and cancer cells. Mabs were used in this context to exploit the variety of gangliosides, which are glycolipids characterized by their content of sialic acid (sialoglycolipids). These antigens are local- ized to the outer leaflet of the plasma membrane, so they are very accessible to antibodies.
In the mid-1980s the Duke Brain Tumor Center entered into a collaboration with Lars Svennerholm, Pam Fredman and their colleagues at the University of Gothenburg and the Karolinska Hospital in Sweden, to pursue the discovery of tumor-specific ganglio- sides. This was an attractive approach because many of the surface antigens thus far identified by MAbs had turned out to be carbohydrate structures of glyco- proteins and glycolipids, leading to the idea that car- bohydrate modifications might have specific functions in growth and differentiation. Neoplastic transforma- tion is, in fact, accompanied by changes in ganglioside patterns (Feizi,1985).
One of the first studies on fetal ganglioside anti- gens associated with human gliomas was published by Svennerholm’s group, who conducted a quantitative analysis of lipids extracted from glioma tissue to assess differences in ganglioside content between normal and tumor tissue (Fredman et al.,1986). They found that the major gangliosides GM1, GD1a, and GT1b were markedly reduced in tumor tissue; in contrast, there was an increase in the gangliosides GM3 and GD3.
They also reported structurally-unidentified ganglio- sides, both mono- and oligosialylated, which could not be detected in normal brain tissue. This work suggested that these novel gangliosides were tumor-associated and might be usefully detected by MAbs.
However, a problem with isolating gangliosides from primary gliomas is that the borders of infiltrative
tumors are so ill-defined that normal brain cells are a significant source of contaminant gangliosides. In order to reduce the contribution of normal glial cell constituents, we used a cell line, D-54 MG, which had been established as a serially-transplantable sub- cutaneous xenograft glioma line in athymic mice.
Lipids extracted from this glioma were analyzed by methods that included gas-liquid chromatography and fast-atom bombardment mass spectrometry. This work led to further characterization of glioma-associated gangliosides in the lactotetraose series: the sialyl- lactotetraosyl-ceramide 3-isoLM1 and its disialylated form 36-isoLD1 (Mansson et al.,1986).
Detailed studies of the distribution of tumor- associated gangliosides were made possible by the development of MAbs specific for different forms of tumor-associated gangliosides. For example, MAbs to 36-isoLD1 were selected from clones raised against a teratoma cell line, and screened with ganglioside fractions of D-54 MG glioma cells (Wikstrand et al., 1993). Using these MAbs, 36-isoLD1 in the CNS was found to be restricted to proliferating astroglia (fetal/neonatal, reactive and neoplastic), which indi- cated that specific MAbs against that biomarker could have clinical applications. Significantly, expression of 36-isoLD1 was not seen in cultured CNS tumor cells, even though it was increased in tumor cells of the same origin grown in vivo. This was a useful reminder that biomarker expression can be strongly influenced by the model system used for discovery.
Rolf Bjerkvig’s group showed that 3-isoLM1 was highly expressed on invasive glioma cells far from the presenting main mass of tumor cells (Hedberg et al.,2001).
In other studies, MAbs to gangliosides were used to assess the presence of fetal ganglioside antigens in newly-excised human glioma tissue and in brain adja- cent to the tumors (von Holst et al.,1997). Lipids were extracted from excised human tumor tissue and sep- arated by anion-exchange chromatography. Individual glycolipids were separated by thin-layer chromatogra- phy and analyzed with MAbs in a quantitative TLC- ELISA combined with densitometric scanning. The fetal gangliosides 3-isoLM1, 36-isoLD1, and a third ganglioside normally found in adult tissue, GD3, were frequently increased both in tumor and in the adja- cent brain, a finding that pointed to the presence of individual, grossly-undetectable tumor cells infiltrating beyond the edge of the tumor.
This work suggested that gangliosides could be used as monitoring agents in combination with PET scans to determine the extent of glioma infiltration, and that they might also be targets for immunother- apy. Gangliosides, however, are not T-cell-dependent antigens, so immunization with them generally results in the induction of IgM antibodies of low or moder- ate affinity. Monoclonal IgM antibodies to GD3 can inhibit the proliferation of human malignant glioma cells in vitro (Hedberg et al.,2000), but the large size of the IgM molecule precludes its use in therapeutic approaches. IgG3 antibodies to gangliosides GD2 and GD3 have been produced, presumably by the activa- tion of γδT-cells (Honsik et al., 1986), and a phase I trial in patients with neuroblastoma and malignant melanoma using an IgG3 anti-GD2 antibody gener- ated in mice showed some efficacy (Cheung et al., 1987). The Bigner laboratory is currently using LC3 synthase knockout mice and naïve human libraries to prepare IgG antibodies and single fragment chain anti- body fragments, specifically reactive with 3-isoLM1 and 36-isoLD1 (unpublished data).
Immunologic Methods: Tenascin
Biomarkers do not have to be of the tumor cells them- selves: by the late 1970s it was becoming increasingly clear that the development and invasion of tumors might be influenced, and perhaps directed, by the nonneoplastic microenvironment in which the tumor develops. An example of a novel tumor-associated stromal biomarker discovered by immunologic means is tenascin, a protein discovered independently by sev- eral laboratories in the 1980s (Erickson and Bourdon, 1989). Bourdon et al. (1983) in our laboratory exploited the specificity of monoclonal antibodies to define new antigens of interest in glial tumors. They immunized mice with intact cells from the human glioblastoma line U-251 MG, generating MAbs which were tested by CS-RIA for reactivity to the glioma cell line and an osteogenic sarcoma line. Only those with restricted reaction to the glial line were selected for further screening.
The MAb 81C6 was chosen for full screening because it had a high binding ratio against U- 251 MG, and did not react to carcinoma cell lines
in CS-RIA. A large panel of glioma cell lines and other neuroectodermal tumors, as well as other nonneuroectodermal tumors and normal human tis- sues, were then used to screen for specificity of the new Mab. The antibody did not bind to normal brain or tumors of non-neuroectodermal origin. Antigen local- ization was examined by immunofluorescence on cell monolayers and suspended layers, revealing that 81C6 does not react with the glioma cells themselves, but with a component of their extracellular matrix (ECM).
Analysis of frozen tissue sections with peroxidase- antiperoxidase immunohistology showed that 81C6 localizes to basement membranes of abnormal blood vessels associated with gliomas.
This staining pattern was not that of any of the known ECM antigens for which antibodies were avail- able, and 81C6 immunoprecipitated an extracellular matrix protein of Mr 230,000. We referred to the new tumor-stromal antigen as glioma-mesenchymal extracellular matrix (GMEM) antigen. Another group (Chiquet-Ehrismann et al., 1986) used a polyclonal antibody raised against chicken myotendinous anti- gen (which they had discovered a few years earlier in normal chick embryos) on ENU-induced mammary adenocarcinomas in mice. They found a prominent reaction in the stroma of these tumors, and named the antigen tenascin (a combination of the Latin words tenere (to hold) and nascere (to be born)) to describe its location and developmental expression pattern.
The fact that this protein is expressed in develop- ment, and again in wound healing and oncogenesis, makes it a very interesting molecule. It is expressed in many tumors, and seems to be more prominent in those that are anaplastic, or less-differentiated.
For example, it is almost always found in glioblas- toma multiforme (GBM), a WHO grade IV glioma, but is infrequent in more differentiated astrocytomas (Erickson and Bourdon, 1989). Currently, clinical trials are testing the safety and efficacy of antite- nascin antibodies labeled with radioactive iodine in the treatment of glioblastomas (http://clinicaltrials.
gov/ct2/show/NCT00615186). The MAb used in the commercial preparation is the same one developed by Bourdon et al. (1983): 81C6. Data from the phase II trials of this agent are promising, reaffirming the potential value of immunologically-defined tumor- associated stromal biomarkers.
Cytogenetic Methods: Karyotypic Analysis
Bigner et al. (1984), working in the Duke Brain Tumor Center, began to explore the cytogenetic abnormali- ties of astrocytic gliomas using karyotypic analysis.
One of the first applications of this method of genetic analysis was in human malignancies, and it provided a means of identifying tumor markers that is still widely used in diagnosis and monitoring. Metaphase spreads stained with orcein demonstrated the gross structure of the chromosomal material in cells and showed abnormalities in some tumor cells, including changes in ploidy, gain or loss of whole chromo- somes, or structural abnormalities of chromosomal arms, including losses or gains of chromosomal mate- rial. More subtle alterations began to emerge when the trypsin-Giemsa banding technique was developed:
homogenously-staining regions (chromosomal regions that do not exhibit the usual banding pattern), translo- cations, insertions, inversions, extra bands, and struc- turally abnormal marker chromosomes. These aberra- tions were often characteristic of particular tumors and suggested that the changes were markers of malignant transformation.
Cytogenetics: Double-Minute
Chromosomes and EGFR Amplification
Double-minute (DM) chromosomes were first demon- strated in a human tumor cell by Spriggs et al. (1962).
These structures resemble minute chromosomes, but they appear as paired dots without a centromere. They are unstable and segregate randomly during cell divi- sion, so one tumor cell may receive many copies and another, few. Following this observation, many groups demonstrated the presence of double-minute chromosomes in different human tumors, including neurogenic tumors (Mark1971).
Bigner et al. (1984,1986) thus began a long-term study in collaboration with the Central Hospital in Skovde, Sweden, in which karyotypic profiling was used to determine if there were any patterns in the gross chromosomal changes of malignant gliomas.
In the first part of this work, using both direct preparations and short-term culture of human gliomas
(9 glioblastomas, 2 anaplastic astrocytomas and a gliosarcoma), they generated G-banding, C-banding and standard metaphase spreads, and compared them to preparations made from blood cells of the same patients (Bigner et al., 1984). They found that a few nonrandom numerical changes were the earliest changes in this tumor type, and could be grouped into three subgroups: those with gonosomal (sex chromo- somal) losses, gains of whole copies of chromosome 7 and losses of chromosome 10, or losses of chromo- some 22. They also found that approximately half of the gliomas exhibited DM chromosomes. Other prefer- ential chromosomal alterations were also seen, and in examination of an additional 15 gliomas (Bigner et al., 1986), they were able to confirm that deletions and translocation involving 9p were common in high-grade gliomas, as well as rearrangements of chromosomes 1, 6 and 13, and less frequently 7, 11 and 16.
EGFR is encoded by the c-erbB1 proto-oncogene and is a transmembrane receptor with an extracellu- lar ligand-binding domain and an intracellular tyrosine kinase. Kondo and Shimizu (1983) established that the locus for the human gene is on chromosome 7.
Libermann et al. (1985) had shown elevated EGFR expression in human glioma biopsies as assessed by kinase activity, and demonstrated amplification of EGFR in some of these tumors using densitomet- ric quantification of Southern blots. Following up on some early evidence that suggested a correlation of DM chromosomes with oncogenes, we began a col- laboration with Bert Vogelstein in the mid-1980s in order to better characterize the genetic abnormali- ties noted in gliomas. Structural abnormalities of 7p seemed to be associated with some EGFR abnormali- ties, but polysomy of chromosome 7 (present in over half of the gliomas) seemed to be unrelated to EGFR amplification. In 33 gliomas, we found the majority of DMs contained markedly increased copy numbers of the oncogene, EGFR, and concluded that DMs are the usual location for amplified genes, usually EGFR (Bigner et al.,1987). At the same time, analyzing 63 primary gliomas using slot-blot and in situ hybridiza- tion of an EGFR cDNA probe to tumor samples, Wong et al. (1987) showed that large increases in expression of EGFR are invariably associated with alterations in gene structure, i.e., amplification. It is now known that EGFR expression is found in up to 90% of high-grade gliomas and is an indication of shorter survival time
in patients with gliomas (Yuan et al.,2001), so this is a biomarker useful for both diagnostic and prognostic purposes. This strong association of the EGFR gene to chromosomal abnormalities in gliomas made it of great interest and fueled further investigation.
Molecular Biology, Biochemical, and Immunologic Methods: EGFRvIII
As discussed, increased expression of EGFR in gliomas is often accompanied by gene rearrangements.
It was already known that the viral homolog of EGFR, v-erbB, encodes a truncated version of the receptor that lacks the extracytoplasmic domain. This led to speculation that these proteins are oncogenic because their intracytoplasmic domain is unregulated and con- stitutively active (Downward et al.,1984). Libermann et al. (1985) had noted a possible rearrangement of the EGFR based on novel restriction fragment polymor- phisms on Southern blots and abnormal transcripts on Northern blots. In work done in collaboration with Bert Vogelstein’s laboratory at Johns Hopkins, Humphrey et al. (1988) found a structurally-altered version of EGFR using a combination of enzyme affinity assays, immunoprecipitation, western blots, and immunohis- tochemistry to determine expression of EGFR in xenografts from eight human gliomas. They then char- acterized the genetic alterations associated with ampli- fied EGFR using DNA from human glioblastoma lines propagated as xenografts (Wong et al.,1992). The gene was cloned from a library made from a glioma without EGFR rearrangements and a restriction map was gen- erated. Southern blot hybridization with phage clones was done using restriction enzyme-digested DNA from different glioma xenografts. RNAase protection assays were used to determine structures of mRNA tran- scripts, and then the altered regions were sequenced.
They found that the same deletion mutation of EGFR protein (loss of extracytoplasmic domains) occurred in tumors from different patients. That the same alteration was seen in all five of the gliomas studied strongly suggested a role for this mutant receptor (later called EGFRvIII) in tumorigenesis.
In order to exploit this tumor-specific alteration, we generated site-specific anti-peptide antibodies against the mutated region of EGFRvIII (Humphrey et al., 1990). A polyclonal antiserum raised against
a 14-amino acid synthetic peptide corresponding to the junction formed by this deletion was found to recognize mutant, but not normal EGFR. The pro- duction of this new polyclonal antibody demonstrated that a reagent could be generated that was abso- lutely specific to the altered sequence resulting from the in-frame deletion. Subsequently, monoclonal ver- sions were developed that were also tumor specific (Wikstrand et al.,1995). The expression of EGFRvIII was also observed in breast and lung cancers, and the antibodies were also specific for these tumors (ibid).
Another advantage of this biomarker is the extracel- lular location of the EGFRvIII mutation, making it theoretically more accessible in vivo for localization and therapeutic targeting. EGFRvIII expression has now been demonstrated in numerous other cancers, including ovarian and prostate (Kuan et al.,2001) and head and neck squamous cell carcinoma (Sok et al., 2006).
It is now known that EGFRvIII is the most com- mon mutation of this receptor in human glioblastomas, and we and other groups continue to develop thera- pies directed against that target, because it holds the promise of being completely specific for tumor cells.
In addition to the use of MAbs to EGFRvIII as ther- apy, mutant peptide is also being exploited as a vac- cine. Phase II clinical trials and continuation studies of a commercial peptide vaccine preparation showed that EGFRvIII-specific CD3+CD8+gamma-IFN pro- ducing T-cells were induced even with concurrent temozolomide therapy, and improved median survival.
Historically-controlled Phase II data suggest that vac- cination against EGFRvIII after gross total resection, chemotherapy, and radiation can approximately double progression-free survival and overall survival (http://
clinicaltrials.gov/ct2/show/NCT00458601).
Molecular Biology Methods: GLI
In our research we have frequently evaluated brain tumor markers previously identified in other tumor sys- tems, but one of the biggest challenges is to identify novel markers. A logical approach to this is to look for differences between normal and brain tumor cells, and an example of the successful use of this approach is the discovery of the GLI gene (Kinzler et al.,1987) using the precision of molecular biology.