By David N. Leff
Once the surgeon has cut out a cancerous tumor from the patient's body, the excised tissue is rushed to the pathology laboratory for assessing the malignancy's aggressive nature.
"The object," explained cell biologist Ronald Laskey, "is to determine how fast, and how aggressively, tumors are growing, by determining the proportion of cells that are preparing to divide, by synthesizing DNA. Only a minority of cells in the body" he pointed out, "are in the proliferating part of the cell cycle. The majority are quiescent, not dividing at all. The proliferating part of the cycle includes the S-phase, which is the only part in which DNA is being synthesized. S is flanked by two 'gap' phases - G1 and G2."
Laskey is the Charles Darwin professor of cell biology at Britain's University of Cambridge. "One thing we've found," he continued, "is that the vast majority of the cells in a tumor are in the proliferating part of the cycle - in one of those three phases: S, G1 or G2.
"There hasn't previously been a method to distinguish those phases, short of rather drastic procedures - injecting the patient with either a radioactive or a potentially fluorescent marker. But that's not something you would readily do to a patient. So to identify the cells that are actively making DNA," Laskey continued, "is something that has not been a feasible objective with patients."
He is senior author of a paper in the April 2000 issue of Nature Cell Biology, which describes," he told BioWorld Today, "a simple method that allows us to tell which cells are preparing to divide by doubling their DNA content." His paper bears the title, "Detection of S-phase cells in tissue sections by in situ DNA replication."
Timely Tumor Data Sharpens Post-Op Therapy
This information will help decide what subsequent treatment is indicated - radiation, chemotherapy, immunotherapy or (eventually) gene therapy. But usually, by the time the path lab delivers its report, the patient has been sewn up, and left the OR for the recovery room. Definitive data while the wound is still open is much to be desired, but infrequently available.
Here is how Laskey describes his method: "Quite simply, the tumor biopsy is taken and frozen, then - as would normally be done for pathological observation - cut into thin slices. But before fixing or staining it, or putting it under a microscope, we run a brief incubation to allow those cells, which were making DNA in the patient, to continue making DNA on the microscope slide. In that way we can label them by incorporating markers which we can detect fluorescently."
Laskey and his co-authors came upon this technique "as a spin-off from experiments we'd been doing on the control of DNA replication in human cells. From that work we realized that it was a small step to exploit the findings of those experiments to produce a diagnostic test, which could add to the range of tests already available."
Britain's Cancer Research Campaign (CRC), which funds his work and has applied for patents on it, acclaims the technology as "A Thirty-Minute Test For Cancer." But Laskey's reported experiments have reduced to 15 minutes the incubation time from biopsy to data.
"Fifteen minutes is the shortest," he said, adding, "We may be able to make it shorter still by a few technical modifications we've made since writing this paper. But this will require further research. There are two ways of doing it," Laskey went on, "one of which is much shorter than the other. The first and quickest is to incorporate a directly fluoroscene-conjugated nucleotide, so that you can then view immediately with no other steps. That is the quickest. We can now make it even shorter," he observed, "but in practice we think that 15 minutes is useful enough.
"The other way, which might produce more sensitivity but would add time to the protocol, is to incorporate a different ligand, which would then detect by an antibody or for instance unlabeled biotin and then fluorescent streptavidin."
Laskey and his group successfully pitted their new method against a range of cancerous tissues with known, conventionally obtained DNA diagnoses. Now, in collaboration with pathologist co-author Nicholas Coleman, they have begun clinical trials on primary tumor samples obtained at surgery.
Strategy Tailored To All Solid Tumors
"Detecting the cancer is something that could be done by less sophisticated methods, such as simple histochemical staining," Laskey pointed out. "But telling how aggressive that tumor is, or its likely outcome, is something where we feel this information can allow further generation of assessment at prognosis.
"At this stage," he concluded, "it appears to us this test should be applicable to all of the common, solid tumors. So far we've focused on three, namely, breast, colon and cervix. But it's our intention to extend it much further."
Research assistant Anthony Mills is the Nature Cell Biology paper's lead author. "We know that our test can accurately detect cells that are preparing to divide," he observed. "But we must thoroughly evaluate it in tumor samples from patients before it can be used as an everyday tool to help cancer doctors.
"The beauty of it," Mills went on, "is that it might be useful in assessing most common cancers. It is quick, and does not lead to an extra stress for the patient. This is because biopsies are taken routinely anyway, and the test happens outside the patient's body."