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."