Eluding Capture
Hidden tumor cells that evaded removal are now detected and captured by updated screening methods.
by Alfred Kildow
For men surgically treated for prostate cancer, a looming fear is that the cancer will strike again, rising from hidden pockets of the cancer that were not detected and removed in the initial surgery.
Now,
members of an elite research team from the Keck School of Medicine of USC,
under the leadership of Richard Cote, M.D., have validated their latest method
of screening for these hidden, or occult, cells to determine whether the cancer
has spread, or metastasized, beyond the prostate gland itself. Successful
detection might enable doctors to attack the elusive cells with a second wave
of “adjuvant” therapy—chemical, radiation, hormonal or further
surgery.
“Metastasis is the most important event for determining outcome in cancer patients,” says Cote, professor of pathology and urology. His latest screening method is already updated, and he is looking at new methods to detect, as early as possible, the spread of cancer.
Cote’s research team can be likened to detectives working on cold cases, in which the criminals who perpetrated the crimes were not captured, but are hiding out, lying in wait to strike when opportunity presents itself.
Among the detectives on Cote’s team were postdoctoral fellow Vincenzo Pagliarulo and biostatistician Susan Groshen, Ph.D., professor of preventive medicine, the point persons on a hunt through medical records and specimens of 556 patients who had been treated for prostate cancer at the Keck School and the USC/Norris Comprehensive Cancer Center. The records and specimens date back 23 years, to 1983. From this meticulous detective work, Pagliarulo, Groshen and others on the Cote team focused on 274 of these patients.
The reasons for the search—and this particular focus—require some background: First, prostate cancer is very common; one in six American men will be diagnosed with prostate cancer. Second, earlier studies by Cote and others identified occult cells as likely culprits in prostate cancer that recurs in many patients. Finally, there is how the prostate functions and how it is removed when cancer strikes.
The prostate gland and the adjacent seminal vesicles are tucked away in the lower abdomen between the urinary bladder and rectum. Snaking through the prostate is the urethra, which carries urine from the bladder during urination. It also carries semen from prostate and seminal vesicles during ejaculation.
Men over age 50 who listen to their doctors have their blood chemistry examined at least once a year. The labs check cholesterol levels, of course, to weigh the risk of coronary artery disease. If requested, the labs also measure PSA, or Prostate Specific Antigen, which is always present at low levels in men’s blood and indicates the rate of prostate cell growth. As PSA levels rise—or if they rise quickly—it is one significant sign the prostate is in trouble. Doctors examine the prostate at that time with a gloved finger inserted through the rectum, feeling for knobs or bulges on the surface of the dense tissue of the walnut-sized prostate. The next step is to insert a long, thick needle through the rectum into the prostate itself, snipping off tiny pieces that can be examined histologically, under microscopes.
If cancer is detected, patients consult with their doctors and decide to choose one or a combination of choices: watchful waiting, in which patients decide to keep track of the progress of the cancer before intervening; chemotherapy, injecting powerful anti-cancer drugs to try to kill the cancer cells; hormone therapy, injecting hormones to lower the patient’s testosterone levels, which are often linked to cancer production in the prostate. Then there is surgery, which many men opt for in hopes that all of the cancer cells will be excised from their bodies permanently.
The choice is difficult because all have adverse effects. But as one cancer patient said: “My cancer was diagnosed early because I had been diligent about checking my PSA levels. When the needle biopsy confirmed the cancer, I reasoned that if I had my prostate taken out, my chances were good for permanent recovery.” Many men echo those sentiments and choose a type of surgery called a radical retropubic prostatectomy.
During this surgery, doctors open the lower abdomen and carefully cut away the prostate and seminal vesicles, trying to spare sensitive nerves while searching for signs of the cancer’s spread to nearby tissues.
To be as sure as possible that all of the cancer is removed, they also remove some nearby tissues, including a number of lymph glands that line the area. Pathologists examine these tissues through microscopes. If they find cancer has spread, further therapeutic measures can be taken.
Samples of these tissues are carefully categorized and safely stored inside small blocks of paraffin or plastic for future reference. It is these samples, and the medical records that follow them, that are the fountainhead for the Cote team’s detective work.
The 274 patients studied were chosen from 556 patients whose prostatectomy surgeries had been preceded by extensive removal of nearby lymph tissue. These 274 patients had cancer that had spread slightly beyond the prostate, putting them at increased risk for developing a recurrence of their cancer. Importantly for the detectives, the medical histories of the 274 had been carefully annotated by the primary surgeons in this study: Donald Skinner, M.D., the Hanson-White Chair in Medical Research and chair of urology, and Gary Lieskovsky, M.D., the Donald G. Skinner Chair in Urology and professor of urology. John Stein, M.D., associate professor of urology, assisted in the maintenance of the database.
Cote and Groshen designed a study that called for searching the stored lymph tissues of all 274 patients for occult cells and examining the many thousands of data points to determine how the patients fared. Did prostate cancer recur? If so, how long after surgery? What was the age of the patient at surgery? How invasive was the tumor? Were other therapies used following surgery? The answers to these and many other questions were ferreted out by Pagliarulo, Groshen, Debra Hawes, M.D., assistant professor of clinical pathology and postdoctoral fellow Frank H. Brands, all under the oversight of Cote.
What Cote and his colleagues had going for them is a 20-year-old science—immunohistochemistry—they have refined to fish out occult cancer cells that escape the scrutiny of the microscope. It has become a crucial technique widely used in research laboratories and in clinical diagnostics: immuno for immune system; histo for histology, or microscope; chemistry for the underlying chemical processes used to identify targets.
Through immunohistochemistry the researchers used colored stains to tag antibodies that react with antigens in cancer cells. Cote’s system uses antibodies known to react to prostate cancer antigens as a way of finding the cancer—sort of how detectives construct a “sting” operation, utilizing the propensities of criminals to reveal themselves when tempted.
The tags pinpoint the location of the occult cancer cells in the preserved samples that had escaped detection during surgery.
What team members set out to do with their sensitive system was to determine how often—and where—occult prostate cancer cells metastasized into nearby lymph nodes undetected by analysis under the microscope. Further, they wanted to know whether these hidden cancer cells were significant in terms of the patient’s health. And, they wanted to know whether the technique combined with adjuvant therapy would extend lives.
They started with a base of 274 men whose cancerous prostate glands had been completely removed through radical prostatectomy. Of these, 94 had cancers that had metastasized to the lymph nodes, as determined by visual examination under the microscope.
But the lymph nodes of the other 180 had been judged cancer free by the same methods.
It was on these 180 that Cote focused the attention of his laboratory researchers, testing tiny slices of the stored lymph node samples for the presence of tumor cells.
Their immunohistochemical approach probed with a cocktail of two commercially available biological tools called monoclonal antibodies. If the cocktail detected a protein called a cytokeratin, a soft-tissue structural protein, they examined tissue from the same sample with another antibody designed to detect antigens from prostate cancer.
The researchers found that 24 of the 180 patients judged cancer free had tumor cells detectable in pelvic lymph tissue—13.3 percent. They analyzed the progression of the disease over an average of nearly 12 years in these patients as well as the other patients in the study. Statisticians Groshen and Jie Cai oversaw the extremely detailed statistical analysis.
The team divided the 274 patients into three groups: those on whom the cytokeratin immunochemistry did not detect occult metastases; those in which immunochemistry detected occult cancer cells, and those in which metastasis had been detected by routine microscopic examination.
Of the 274 total, 141 experienced a recurrence of cancer, either observed in the clinic or by PSA examination. Bottom line: this study demonstrates that immunohistochemical assessment of the lymph nodes can detect tumors not recognized at initial histologic examination.
Occult tumor cells found in the lymph nodes of patients with prostate cancer identified those at increased risk for recurrence and at increased risk for dying of their disease. Cote and his colleagues showed, surprisingly, that the risk associated with occult metastases was equal to the risk associated with metastases identified by routine histologic methods.
Although the current study did not examine whether early adjuvant therapy has better results than deferred therapy, Cote points to studies by others that “suggest that patients with occult lymph node metastases from prostate cancer may benefit from immediate adjuvant systemic treatment,” because chemotherapy and hormonal therapy will have its greatest impact in patients with the lowest amount of cancer in their bodies, such as occult metastases. Cote and his colleagues will pursue this idea.
Cote’s findings are also widely used to evaluate patients with breast cancer. But searching for occult metastases in bone marrow, blood and lymph nodes is not routinely done for most cancers. Cote believes technical difficulties and high costs curtail routine screening of bone marrow, and the procedure is uncomfortable for the patient.
Taking a blood sample is much gentler and patients will be more likely to accept this method, Cote says, but current methods for capturing circulating tumor cells are problematic, costly and unwieldy. So Cote’s lab, in collaboration with engineers at Caltech, have devised a sort of high-tech net to fish out tumor cells from bone marrow or blood. He hopes to develop the net, a micropore membrane, into a microchip device for processing blood, bone marrow and eventually other bodily fluids. The project, part of USC Provost C.L. Max Nikias’s Nanotechnology Initiative, could be applicable for identifying other cancers very early, when they are most susceptible to successful therapy.
“This microdevice has the potential to revolutionize tumor cell capture and identification,” Cote says.
Cote has demonstrated another promising approach using certain characteristics of stem cells to define the aggressiveness of occult tumor cells in bone marrow, blood and lymph nodes. He is the first to demonstrate that the earliest metastases have stem cell characteristics, which could lead to a radically different view of early metastases and therapeutic approaches to early cancer spread.
For Cote, simple histologic examination has never been enough. He has devoted his professional career to the improvement of cancer diagnostics. As far back as 1990, when Cote came to USC from Memorial Sloan Kettering Cancer Center, he felt sure there were other ways to find the hidden metastases lurking in the body but invisible to microscopic examination. “Some of the first papers I published were on finding regional (lymph) and systemic (bone marrow and blood) metastases, how it might be done, why it might be important,” he recalls. “I felt it was important to create a system that could look for cells on an even smaller scale.”
It appears that now he and his fellow detectives are doing just that—and improving the means to save the lives of cancer patients.
Alfred Kildow is a freelance writer relaxing in Monterey, California.
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