Proteomics for cancer diagnostics

According to the University of Sydney's Professor Richard Christopherson, the era of genomics and the Human Genome Project is 'old hat', with all 23,000 or so human genes sequenced.

Proteomics is where it's at and Australia is at the forefront of this science, which seeks to separate, identify and characterise cellular proteins.

Proteomics, a term coined by Australia's Professor Marc Wilkins in 1995, is the crucial next step for understanding the fundamental mechanisms of disease, particularly cancers, and how the drugs used to treat such conditions work. This is the future, Christopherson says.

"We now understand quite a lot about the mutations that cause cancer, but proteomics is really at the coal-face of working out why we have uncontrolled growth, what the underlying mechanism is that causes these human cells to grow out of control," he says.

In his own work, Christopherson is using proteomics to phenotype cancers, assaying protein markers to classify or diagnose a particular disease or affected individual, and ultimately to predict an individual's response to therapy. In other words, find protein signatures for cancer cells to improve both diagnosis and treatment.

The concept of a proteomics approach to cancer was developed with Professor Cris dos Remedios some years ago when a mutual friend was diagnosed with acute myeloid leukaemia (AML). Both agreed that what was really needed in her case, and indeed for all other leukaemia sufferers, was to know the exact repertoire of proteins expressed on the surface of the patient's leukaemia.

This information would inform both a better classification of the leukaemia and a strategy for treatment by identifying potential targets for therapeutic antibodies. The idea was subsequently implemented in Christopherson's research group about seven years ago, culminating in DotScan - a solid-phase cell-capture assay to immunophenotpye cancer cells commercialised by Sydney-based company Medsaic in 2003.

(Medsaic was spun out of the University of Sydney and has a facility at the Australian Technology Park in Eveleigh, in its time winning a BioFirst Commercialisation Award from the NSW Government. Christopherson retains a position on the scientific advisory board of the company and gives science advice when relevant, but has not been involved with the commercial activities.)

DotScan is a novel antibody microarray containing a number of monoclonal antibodies to CD antigens expressed on cell surfaces. CD or "cluster of differentiation" molecules were first identified on the surface of white blood cells or leukocytes, but we now know that CDs are expressed on all human cells in cell-specific subsets.

The assay was initially focused on diagnosing or classifying leukaemias from peripheral blood, when the predominant white blood cell type is a mononuclear leukaemia.

Technically, the assay involves centrifuging peripheral blood leukocytes from samples through Histopaque, then placing a suspension of three million mononuclear leukaemia cells or normal leukocytes onto a solid-phase carrying a microarray of up to 147 different CD antibody dots laid down in 10-nl volumes over an area of approximately 0.5 cm square.

During a 30-minute incubation, the immobilised antibodies capture live cells expressing the corresponding surface molecule or CD antigen. The end result is a dot pattern that represents the surface-expression profile or immunophenotype of the particular high-level leukaemia in the patient's blood.

Leukaemia is currently diagnosed by multiple criteria. Flow cytometry is the workhorse of the diagnosis and remains the gold standard for identifying surface molecules. In a routine haematology pathology lab, approximately15 CD antigens are screened using flow cytometry, with machine and labour costs preventing more being done.

In contrast, DotScan can screen 147 CD antigens in a single assay using about1/2000 the amount of antibody needed for flow cytometry of a patient sample. Therefore, it allows the acquisition of a lot more data at a much lower cost.

"The basic hypothesis was that a dot pattern would be sufficient to diagnose or classify the leukaemia on its own without using cytochemistry, karyotyping or even cell morphology," Christopherson says.

A recently published clinical trial from the group gives hope to this aim being realised. Blood samples from 796 leukaemia patients and normal subjects were tested using the DotScan array with the results published last year in the British Journal of Haematology.

The samples came from the MD Anderson Cancer Centre in Houston, the Department of Haematology at Cambridge University, Westmead Hospital and Symbion Health in Sydney, and the Royal Melbourne Hospital. The study took four years and identified 33 different expression profiles for different leukaemias. Christopherson says it was a complex paper involving 23 authors, "quite challenging but very satisfying to have finished".

The assay produced high levels of consistency with conventional clinical and laboratory diagnostic findings, with 93.9-97.6 per cent agreement with the established classification of these leukaemias. Christopherson says that because this was a prospective study on fresh samples, comparisons of immunophenotype with patient outcome were not possible. However, such a trial using subsequently thawed samples is underway for patients with chronic lymphocytic leukaemia (CLL).

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Colorectal cancer

More recently, the DotScan assay was tested in a small trial of patients with colorectal cancer (CRC). "In the case of CRC the cancer cells may not be predominant as in the leukaemias we studied in earlier work, so we had to develop a technique of fluorescence multiplexing to enable detection and immunophenotyping when the cancer cells are in the minority," Christopherson says.

"We made a suspension of live cells from a surgically resected cancerous polyp and from normal mucosal tissue from the base of the polyp." These live cells were then captured on the antibody microarray as for the leukaemia cell preparations. The resulting optical dot pattern represented a heterogeneous mixture comprising CRC cells, normal colon cells and a whole range of leukocytes.

"What we wanted though was a dot pattern just for the cancer cells. To do this, we devised a cocktail of three different, fluorescently labeled antibodies to recognise specific cell types - chorio-embryonic antigen (CEA) or epithelial cell adhesion molecule (EpCAM, both upregulated on CRC), CD4 (a T-lymphocyte antigen) and CD20 (a B-lymphocyte antigen). We could then visualise the spots with a three-laser scanner, and so get three dot patterns from each array (see Figure)."

This multiplexing procedure produced dot patterns from colorectal cancers that were distinct from those of adjacent normal mucosa. Subtraction of the expression levels for each antigen from normal tissue from those for the cancer showed differential expression for a number of CD antigens to give a phenotype of the particular cancerous cells in that polyp.

"It was great to get the expression profile or disease signature of the CRC, but the real bonus was that using the anti-CD4 antibody allowed us to also get a dot pattern for activated tumour-infiltrating lymphocytes (TILs) in the cancerous polyp, and this signature is a possible indicator of prognosis," Christopherson says.

TILs have been used for treating metastatic melanoma by growing large numbers from a patient's tumour in vitro and infusing them back into the patient for immunotherapy. Christopherson's study revealed a major difference in expression profiles of the T-lymphocytes between the cancer and surrounding normal mucosa.

"This was a fortuitous finding ... you might even say it was serendipity." This preliminary trial involved 14 patients and the findings were also published last year, in Proteomics.

The primary use of DotScan is to classify or diagnose leukaemias and other cancers. The second potential use of the array is identification of targets for therapy. "We all hope that cytotoxic drugs, like methotrexate, are on the way out to be replaced with more specific therapies, one of which would involve therapeutic antibodies," he says.

"Therapeutic antibodies are one of the most rapidly expanding areas of pharmaceuticals, particularly in the US, and if you have a microarray you have a much better chance of identifying a uniquely expressed target on the leukaemia cells not commonly found on other cells in the body."

For example, CRCs are not usually profiled and the methods of classification are currently quite unsatisfactory, based largely on how long the tumour has been there and how big it has grown rather than what the genetic changes and consequent repertoire of surface proteins might be.

"We need a classification system that relates directly to the mutations, and then we will be able to accurately predict prognosis and drug responses. We really need such a simple in vitro test in this area."

What Christopherson hopes to see realised in the not-too-distant future is the introduction of personalised medicine, where treatments will be tailored to the patient's particular cancer.

"We still need to amass a lot more information about particular cancers to have these types of therapies, and microarrays are one of the simplest ways to get a lot of data quickly, practically (at the bedside) and at reasonable cost. They would provide a routine method of analysis - the dot patterns give you the diagnosis and, at the same time, identify targets specific for that patient's disease to be matched with particular therapeutic antibodies. That is how it will go."

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Proteome and cancer research

Richard Christopherson has pretty much always worked in cancer research. He completed his PhD at the University of Melbourne, before heading to Los Angeles on a fellowship from the Damon Runyon-Walter Winchell Cancer Fund held at the University of Southern California Medical School.

After two years, he moved with Dr Mary Ellen Jones to the Department of Biochemistry at the University of North Carolina Medical School in Chapel Hill, where she became head of department and he took up a special fellowship of the Leukemia Society of America.

Christopherson returned to Australia as a research fellow in the John Curtin School of Medical Research and was then appointed as the CR Roper Fellow in Medical Research at the University of Melbourne where he started his own research laboratory. He has now worked at the University of Sydney for 21 years, where he was the foundation chair of the School of Molecular and Microbial Biosciences from 1998-2003 and now holds a personal chair.

Christopherson has held a long-term interest in the cytotoxic mechanisms of anticancer drugs. In the early 1990s, his group elucidated the antipurine mechanism for methotrexate, an antifolate drug used to treat a variety of cancers and autoimmune diseases. This was a significant discovery that went against the model at the time developed by the US National Cancer Institute, paving the way for widespread use of methotrexate at different dosages.

In 2003, Christopherson and his school received a grant of $1.8 million to establish the Sydney University Proteome Research Unit, of which he is director.