Understanding of the biology of multiple myeloma (MM) is rapidly being influenced by innovative technologies that are focused on defining comprehensive genetic and protein profiles of the malignant clone. The session on innovative technologies highlighted new applications of gene microarray technologies and the genetic profiling of both myeloma cell-line model systems and patient samples. Integrating new technologies into protein profiling is another important advance, and the combination of these approaches will likely change the definition of the disease, identify novel therapeutic targets, and influence therapeutic choices.
Although MM is a single, clinically defined disease, there is considerable genetic heterogeneity among patients that impacts disease progression and response to therapy. Indeed, several years ago, the director of the National Cancer Institute issued a challenge to develop new genetic classifications of cancer. In December 2000, a progress-review group, including several members of the myeloma research community, identified several focal areas: 1) develop a genetic and protein profile of lymphoid malignancies, 2) profile expression changes associated with tumor-host interactions, 3) from these profiles generate hypotheses that can be tested in model systems, and 4) use this information to develop novel therapeutic approaches to individual patients. These challenges are being met with innovative technologies in gene and protein profiling.
In a pre-workshop symposium, two presentations highlighted the technical development and application of genetic and protein profiling. Dr. Jeff Seilhamer, cofounder of Incyte Genomics, Inc., one of the leading genomic biotech companies, presented his views on the human genome project and the development of technologies to examine genetic expression profiles, as well as analytical tools that can be used to query genetic databases. Although recent publications from both Celera and the public genome project suggested that the human genome includes a total of 32,000 genes, Dr. Seilhamer presented evidence that many genes have been missed, and discovery of rare expressed genes may significantly increase the total gene number. Dr. Reid Asbury of Amersham-Pharmacia Biotech, Inc. presented new approaches in protein profiling. Combining two-dimensional gel electrophoresis and mass spectroscopy, it is now possible to profile the expression of thousands of proteins in a tissue sample. Because genetic profiling cannot identify many of the protein modifications associated with intracellular signaling, protein profiles add significant analysis to tumor biology and may identify novel targets for therapeutic interventions. Dr. Asbury presented some initial protein profiles of myeloma cell lines, demonstrating protein changes associated with interleukin (IL)-6 signaling and ras mutations. Drs. Asbury and Van Ness are collaborating to identify such proteins in myeloma cells and correlate protein profiles with genetic expression profiles.
The scientific program included a session in which specific applications of these innovative technologies to lymphoid malignancies were presented. Dr. Louis Staudt, from the National Institutes of Health, is one of the leading authorities on genetic profiling. He presented an overview of the technology in which cDNA sequences are spotted on a glass slide and hybridized to fluorescent-tagged cDNA generated from RNA samples of lymphoid malignancies. Dr. Staudt has generated a specific "lymphochip" containing 18,000 genes, many of which are selectively expressed in lymphocytes along with genes that regulate lymphocyte function, including growth factors, receptors, and transcription factors. The goals of Dr. Staudt’s genetic profiling of lymphoid malignancies were to define genes involved in the pathogenesis of disease, establish common profiles that may offer novel prognostic indicators, and identify new targets for therapy. Dr. Staudt demonstrated through the lymphochip microarray analysis of gene expression in diffuse large B-cell lymphoma samples that this single diagnosis actually contains two different diseases that differ in expression of hundreds of genes. The two types of lymphoma resemble different types of normal B-cell lymphocytes, suggesting that these cancers have distinct cellular origins. Clinically, patients with these two types of large B-cell lymphoma had strikingly different responses to chemotherapy. Dr. Staudt presented recently obtained lymphochip profiles of myeloma patient samples and demonstrated gene expression patterns that distinguish this plasma cell malignancy from other lymphoid malignancies. Expanding this database could provide novel genetic classifications and influence therapeutic choices.
Dr. John Shaughnessy from the University of Arkansas presented the most comprehensive genetic profile of myeloma patients. Using an Affymetrix oligonucleotide-based chip containing 6,500 known genes, Dr. Shaughnessy has profiled more than 100 myeloma patients, as well as a number of normal plasma cells. One analytical tool used to evaluate the data is the clustering of genes with common expression patterns. Two major clusters were identified, one containing normal plasma cells, most of the MGUS (monoclonal gammopathy of undetermined significance) patient samples, and a group of MM patient samples. Another grouping contained all the myeloma cell lines profiled and two additional subgroupings of MM, many with particularly aggressive disease. The cluster analysis suggested that gene expression patterns can discern different subtypes of MM and MGUS. A total of 244 genes were identified that were differentially expressed in normal and malignant plasma cells (137 up regulated in MM and 87 down regulated). Genes involved in transcription represented the largest group of altered genes; genes associated with adhesion, growth control, cell cycle, signal transduction, and oncogenesis were also highly represented. In addition, gene expression patterns correlated closely to known chromosomal abnormalities (genes from chromosome 13 under represented and genes from chromosome 7 over represented).
Dr. Diane Jelinek of the Mayo Clinic has been using a commercially available high-density membrane-based microarray from Research Genetics. This system has required a significant effort to address controls and potential artifacts in experimental results, and Dr. Jelinek provided a thorough characterization of approaches to recognize and minimize these artifacts. Using these microarrays, Dr. Jelinek has focused on analysis of the IL-6 response in cell lines, well known to provide important growth stimulation in myeloma. Interestingly, there are a number of genes she sees upregulated in common with other investigators who profiled patient samples reported in this session. In addition, Dr. Jelinek is taking a novel approach to identify a class of important signaling proteins. She has developed a polymerase chain reaction (PCR)-based technique to examine expression of all tyrosine kinases, and she presented preliminary anlaysis of this class of important signaling proteins in a number of myeloma patient samples. Identification of genes that are IL-6 responsive in myeloma cells is an important key to understanding tumor growth, as is identification of the key downstream tyrosine kinases that are likely to impact atypical malignant plasma cell proliferation.
Dr. Keith Stewart, from the Toronto General Research Institute, has generated a "myeloma chip" analogous to the lymphochip developed by Dr. Staudt. Dr. Stewart’s Myeloma Gene Database consists of 4,660 nonredundant genes, many of which appear unique to myeloma cells and represent potentially novel genes. His group has classified these expressed genes according to putative function. This ongoing effort is being used to develop disease-specific microarrays that may provide the basis for more clearly defining the molecular portrait of myeloma.
Dr. Van Ness presented several approaches to gene and protein profiling, highlighting the importance of the integration of genetic and protein data sets to fully understanding the functional consequences of gene deregulation in the disease. One area of genetic analysis that should be considered is the genetic variation in the population of detoxification genes and drug-metabolizing genes. There are a number of significant variations within the normal population of such genes that likely influence both risk for developing malignancy and response to the highly toxic effects of chemotherapeutic agents. Dr. Van Ness has been correlating some of the frequent genetic polymorphisms with chemotherapy-associated toxicities. Preliminary evaluation of the data suggests that there may be some forms of the glutathione-S-transferase gene that correlate with the risk for development of secondary malignacies often associated with cumulative doses of DNA-damaging agents used in therapy. Employing cell line model systems to examine gene expression variation, Dr. Van Ness has been using the Affymetrix 12,000 gene-detection platform to examine alterations in gene expression associated with ras mutations and with myeloma interactions with the stromal microenvironment. In the collaborative work by Drs. Asbury and Van Ness, preliminary data showed that 750 unique proteins could be separated, with about 15% differential expression as a result of IL-6 stimulation and about 12% differential expression resulting from the introduction of a mutant ras gene. Specific identification of these different proteins is awaiting mass spectroscopic analysis by Dr. Asbury.
In the application of microchip devices, Dr. Christopher Backhouse of the University of Alberta presented novel applications of microelectronic fabrication technologies to produce microchannel networks in glass slides. Within these microchannels, reagents can be manipulated by applying electric fields and can be detected by optical means. These microfluidic systems allow the integration of several functions on a chip and the analysis of single cells. Recent developments have shown the use of microfluidic chips in the application of PCR, electrophoresis sizing, cytometry, and gene-sequence detection. The signature sequence of the heavy-chain immunoglobulin VDJ sequence in myeloma is a clonotypic marker of the malignancy. In collaboration with Dr. Linda Pilarski, Dr. Backhouse demonstrated an on-chip microfluidic analysis of the myeloma clonotypic sequence, combining PCR cycling on the chip and electrophoretic separation to distinguish polyclonal products of the normal population from the monoclonal "spike" produced by the malignant clone. Drs. Backhouse and Pilarski also demonstrated a microchip analysis of RHAMM, an oncogene detected in myeloma patients, with various splice variants that may differentially influence the movement of the malignant cells. This application of microchip technology provides rapid throughput and accurate detection of specific sequences from the malignant clone. Such an approach allows analysis of unique properties of each malignant clone, detection of minimal residual disease, and a rapid analysis of therapeutic interventions.
It was clear from this session that new technologies are quickly redefining important characteristics of the disease that were not previously available. The comprehensive profiling of gene expression and protein expression, as well as the rapid analysis of multiple genes that influence disease progression and therapeutic response, is changing the clinical picture. The goal is to identify the unique properties and vulnerabilities of each patient’s malignant population so that a more individualized treatment strategy can be developed.