College of American Pathologists
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  Bladder markers gunning for
  green light


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Genetic model of human urothelial carcinogenesis
Assembly of whole-organ histologic and genetic maps

March 2002
Cover Story

Karen Titus

Though it starts out as a joke, there’s nothing funny about the punch line.

"I practice in Peoria, Illinois, so I guess the question is, ’How does it play in Peoria?’" says urologist Thomas Stanisic, MD. "And none of them are playing here."

The "them" in question: bladder cancer tumor markers. Few, if any, are catching on in general clinical practice. As Dr. Stanisic puts it, "Academics write about them, but basically, no one uses them very much."

This despite the demand for them, despite the need for them, despite the whopping number of possible choices-thanks to strong commercial interest in this area, the literature is lousy with potential markers.

Some of them even work. David Bostwick, MD, medical director of Bostwick Laboratories and clinical professor of pathology, University of Virginia, says p53 immunohistochemical staining, for example, is of value for patients with carcinoma in situ of the bladder as well as those with papillary malignancy of the bladder. Changes in p53 expression have been shown to correlate with a number of outcome variables, including pathologic stage at cystectomy and, ultimately, patient survival. "Those have been retrospective studies. But there has been a consistency of the data from one center to another," Dr. Bostwick says.

Given that the current gold standards for detecting bladder cancer are less than golden, the interest in finding reliable markers is more than reasonable.

Cystoscopy is invasive, expensive, and, depending on who you talk to, either downright painful or no worse than a routine trip to the dentist-though some might argue these two poles are one and the same. And no urologist can lay claim to 100 percent visual accuracy.

The standard noninvasive marker, voided urine cytology, has problems of its own. "That’s because of the cytologic similarity of benign epithelium to the lowest grades of cancer," says Dr. Bostwick. "The overlap in the cytologic spectrum is such that without the papillary architecture, it may be difficult or impossible to make that determination."

That makes for a considerable gap. "At the Cleveland Clinic, we have excellent cytopathologists," says Craig D. Zippe, MD, on staff at the institution’s section of urologic oncology and co-director of its prostate center. "But we still miss 30 percent of significant tumors with cytology. And we miss probably 50 percent of low-grade, low-stage cytology."

Those misses can be costly. "Proponents of urinary cytology will say, ’So it misses the smaller, low-grade ones; we’ll eventually pick them up,’" Dr. Zippe says. "But what happens is, when you eventually pick them up, there’s no guarantee that they’re still low grade, low stage. Not only that, even if they are low grade, low stage, you have to do a much bigger resection—and the more likely you’ll leave residual cancer cells on the resected edge that come back as a recurrence. And people often will then need intravesical chemotherapy."

"It’s so much easier when someone just has a little 5-mm lesion to remove it," he contends. "Ask a colorectal surgeon or a gastroenterologist if he wouldn’t prefer to take out a 5-mm polyp versus a 2-cm polyp."

Bladder cancer also "has an awful tendency to recur," says Dario C. Altieri, MD, professor of pathology at the Boyer Center for Molecular Medicine, Yale University School of Medicine. "All malignant cancers do that, of course, but for bladder, that translates into about 80 percent of the time." Which, in turn, translates into a considerable need for good followup.

Urine cytology generally earns good marks for detecting high-grade bladder cancer tumors, though it’s not perfect in this sphere, either. "People have this idea that urine cytology never misses the bad guys. Well, that’s not true," says Dr. Zippe. "Urine cytology is only about 60, 70 percent accurate at most for grade 3 muscle invasive tumors."

No wonder experts want the equivalent of a PSA for bladder cancer. Getting from here to there is easy only on paper, however.

What does it take for a tumor marker to make it into practice? Promise, for one.

On that front, survivin has plenty to offer. The survivin gene, first cloned in Dr. Altieri’s laboratory, inhibits the apoptosis program built into all cells. It’s not the only gene to do that. "But for reasons that are beginning to emerge now in the literature, this particular mechanism has been tremendously exploited in cancer," says Dr. Altieri—virtually all aggressive and invasive human cancers have selected survivin for re-expression. "There is tremendously increased expression of the gene in cancer, and the absence in the vast majority of normal tissues."

Dr. Altieri and colleagues at Yale—including those from the urology section, headed by Robert Weiss, MD—reasoned that survivin might make a good diagnostic marker. The results of their research efforts were published in JAMA a little over a year ago (Smith SD, et al. 2001;285:324-328).

One member of the team, lead author Shannon Smith, MD, developed what Dr. Altieri calls a "very simple, straightforward detection system for survivin." Urine specimens were filtered onto a nitrocellulose membrane, and the blots were analyzed for the presence of survivin using a polyclonal antibody.

None of the normal controls had urine survivin. "No surprise there," says Dr. Altieri. The second group consisted of patients with inflammatory infection of the urinary tract—only two of them expressed survivin. In the third group—patients with nonbladder genitourinary tract cancer—none were positive for urine survivin.

A fourth group consisted of 46 patients with newly diagnosed or recurrent bladder cancer. All of them were positive for survivin in the urine. The fifth group was made up of those who had had bladder cancer, been treated, and now had negative cystoscopy results. All but three of the 35 patients in this group were negative for survivin.

Of those three patients in the latter group, two turned out to have a recurrence. "Which suggests that perhaps the presence of survivin could be used as a screening tool for people who’ve had cancer and have to undergo routine followup," Dr. Altieri says.

Of the two survivin-positive patients in the second group, one developed bladder cancer. "So this could be application No. 2—a prospective screening tool, perhaps for categories of individuals who are at risk for bladder cancer," he suggests.

Since the publication of the JAMA paper, Dr. Altieri reports, others have closed in on a broad-based, highly sensitive urine screening assay. "Once that is in place, then we are ready for the harder experiments, the clinical trials."

Survivin is just one of the many stories awaiting a successful ending. It joins an already sizable (and evolving) collection of marker wanna-bes, perhaps to the pleasure only of ardent list-makers:

  • NMP22 (nuclear matrix protein)
  • BTA Stat (qualitative), BTA Trak (quantitative), for human complement factor H-related protein
  • Immunocyt (multiple protein antigens with fluorescence tags)
  • p53
  • p16
  • RB (retinoblastoma)
  • telomerase
  • fibrin-fibrinogen degradation products
  • hyaluronic acid
  • hyaluronidase
  • Quanticyt (Feulgen-stained automated image analysis by dual parameter morphometry)
  • HER-2/neu
  • CYFRA 21-1
  • tissue polypeptide antigen
  • bcl-2
  • Ki67, Ki67 nuclear antigen
  • microsatellite DNA
  • DNA ploidy
  • FNG actins
  • microvessel density
  • epidermal growth factor receptor, acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor
  • BLCA-4 (nuclear matrix protein)
  • oncogenes c-erb-B2, c-ras, c-myc, c-jun, mdm2
  • Lewis X antigen
The list hardly needs to be longer to invite the inevitable question: Which one to choose? Or, says Dr. Stanisic, "Which, if any?"

To begin with, says H. Barton Grossman, MD, "It’s important to clarify what you want to get out of the marker. There’s a lot of confusion right now, because people don’t have their endpoints in mind." Dr. Grossman, who holds the W.A. "Tex" and Deborah Moncrief, Jr. Distinguished Chair of Urology and is professor and deputy chairman of the Department of Urology, University of Texas M.D. Anderson Cancer Center, offers several scenarios.

"If you want to use a marker to perhaps substitute for cystoscopic followup, then you want to maximize your sensitivity, and you’re willing, perhaps, to trade off specificity," he says.

At M.D. Anderson, researchers are trying to develop microsatellite markers to predict which tumors are more likely to progress aggressively. "That’s a whole different use of markers," Dr. Grossman says.

Then there’s early detection. "It’s not so straightforward as one might think," he says.

One option is to look at markers that detect disease—either low grade or high grade—before its appearance cystoscopically. Another route would be to detect genes that become altered in the disease process, which could serve as prognostic markers for aggressive tumors. In either scenario, the markers would need to be highly specific. "Particularly if you’re picking up very early changes, you don’t want to act on it unless you believe it’s actually a true event," says Dr. Bostwick. Specific markers for early development could be useful in screening certain populations, such as smokers.

Dr. Zippe suggests markers might be useful in patients who’ve had bladder cancer and been treated with intravesical chemotherapy, since they often develop secondary tumors in the distal ureter. "The markers can help raise your suspicion that maybe something else is going on in the upper tracts," he says.

In Peoria, Dr. Stanisic proposes several ways to put bladder cancer markers into play. One would be as an adjunct to cystoscopy, to pick up tumors that are not visible either because they occur in the upper tract, or because they’re flat, in situ tumors. Urologists would also find markers useful when faced with equivocal cytologies, he says, or when "you look and can’t see something, but you’re pretty convinced there’s something there."

Another use would be as a general screen. "There are a whole bunch of people who come into our office every day with blood in the urine. What do you do with those?" he asks. "Ten percent of those people have bladder tumors, so maybe it would be nice if you didn’t have to subject 90 percent of those people to cystoscopy, if you could find something that would say, OK, this person’s probably got a bladder tumor. That would avoid an invasive procedure in a large segment of the population."

Just for the record, no one is talking about giving up cystoscopy anytime soon. "That’s the million-dollar question, but I don’t see it happening in the United States," says Dr. Zippe. Even if the markers achieve the sensitivities and specificities demanded by pathologists and clinicians, "I think in the U.S. most urologists are too nervous to eliminate routine cystoscopy as a screening tool."

"If markers got to be very accurate for very low-grade, low-stage tumors, then they could replace cystoscopy," says Dr. Stanisic. "It is possible. But I don’t see that happening in the next four or five years because of the lack of confidence in the current markers. Medico-legally and from an ethical point of view, if somebody has blood in the urine or symptoms of bladder cancer, it’s easy enough to look in their bladder in the office with cystoscopy, even if it does cost a couple hundred dollars."

If physicians are careful to hedge their bets when describing the possible use of bladder cancer markers, they’re just as cautious when asked to name the markers that look most promising.

Urologists and pathologists alike report being encouraged by the data they’re seeing, but the day when markers come into their own is generally acknowledged to be in the somewhat fuzzy distance. Certainly no marker is going anywhere without more trials. The word "comparative" has only recently made its way into the titles of articles on these markers; the word "prospective" remains scarce. Yet without prospective, randomized trials, it’s impossible to know whether markers can deliver on their promise. It probably also bears repeating that, as with other forms of cancer and other diseases, the ability to diagnose may outstrip the ability to treat.

The abundance of markers also creates a logistics quagmire. "I have experience with three markers: BTA Stat, BTA Trak, and NMP22," says Dr. Zippe. "Initially when I started researching these, I looked at all three of them—but you can’t do that forever. So for the last several years, I’ve been exclusively looking at NMP22."

Dr. Bostwick says he’s interested in exploring several of the commercially available microsatellite markers that have demonstrated, in comparative studies, a marked ability to identify early as well as high-grade and high-stage carcinomas. "We haven’t gotten to it yet," he says. "There are so many markers available."

Using fluorescence in situ hybridization to analyze cell-based markers is one of the newest and—based on the limited available data—one of the more promising approaches, says Dr. Grossman. "It may have very high sensitivity and specificity." One convincing study, published in the Journal of Urology (Halling KC, et al. 2000;164:1768-1775), compared cytology and FISH for detecting urothelial carcinoma. The researchers, who used a mixture of fluorescent labeled probes to the centromeres of chromosomes 3, 7, and 17 and band 9p21 (P16/CDKN2A gene), reported that FISH had statistically significant higher sensitivity for detecting pTis, pT1-pT4, grade 3, and all tumors, and that it clarified cases with equivocal or suspicious cystoscopy or cytology results.

Dr. Grossman admits to being optimistic about microsatellite markers as well, which should also offer high sensitivity and specificity. "There are, again, several small publications suggesting that it’s going to be very useful. One problem, of course, is converting this high technology into an easily applicable clinical test. But I think that’s where we may be moving in the next decade." Automating FISH, likewise, could also be difficult as well
as expensive.

He’s also intrigued by p53 and RB. Unfortunately, he says, the number of papers supporting their use are nearly balanced by those that don’t. He fingers the usual suspects: "It depends on who’s doing the assay, how it was run, what population of patients you’re looking at. The data are not absolutely convincing that these markers are going to be clinically useful, and making patient decisions on these nonvalidated markers is dangerous."

Yale’s Dr. Altieri, understandably, has a bit of a soft spot for survivin. "And telomerase has attracted a lot of attention—again, it’s an enzyme that’s selected only for cancer cells. But it’s expensive, because it’s not an antibody-based detection."

With no one magic bullet in sight, several published papers have suggested that using a combination of several markers would be a safe, effective, and less costly route to go. "I expect that story is probably true, but there’s no prospective data to prove it," says Dr. Grossman.

Dr. Stanisic is just as circumspect.

"It would be nice if a panel could be done," he says. But given the demographics of this particular patient population—he estimates that 85 to 90 percent of bladder cancer cases occur in long-term smokers, the majority of whom are of or nearing Medicare age-such an approach might be too costly. "It’s pretty hard to say if that’s going to become reality."

Dr. Bostwick, however, is unequivocal.

"Reliance on multiple markers? Absolutely," says Dr. Bostwick. "That’s why we currently use the Immunocyt test—it actually incorporates more than one marker into a simple immunofluorescence test."

Dr. Bostwick’s laboratory offers p53 IHC staining as well as the Immunocyt test; the latter has been useful in confirming the presence of malignancy in cases with worrisome cystoscopic findings.

"I think it’s time for pathologists to incorporate some of these tests that have undergone tremendous evaluation already, in multiple different centers," he says. "Urine cytologies have a recognized failure rate, and we probably can improve on that by using some other studies."

Clinicians, however, aren’t ready to cast their lots with the markers.

Despite his lab’s success with p53, says Dr. Bostwick, "We find that there is remarkably little interest among the urologic community for using the stain. I don’t think urologists are convinced that the use of p53 in their practice is going to change what they’re going to do in terms of treatment."

You can almost hear the chorus from outside the lab: "Bingo!"

Simply put, clinicians lack confidence in the current markers. It’s not just the lack of prospective data, though no one’s ignoring that need. Nor are urologists clinging to current detection methods out of blind habit, though becoming comfortable with new methodologies always takes time. No one wants to throw away the old until they’re sure the new truly works.

"If you combine cytology with cystoscopy, you will probably pick up most tumors," Dr. Stanisic says. "The same thing would probably be true of some of these tumor markers. The difference is, most of us have become familiar with the individuals who look at the cytologies, and we have an idea as to their reliability or lack of same. But with NMP22 and some of the other markers, we don’t have a lot of experience, at least not to date."

Sometimes they do have experience. Sometimes it’s bad.

"We’ve tried the BTA test in the past, which had the advantage of being done in the office. We did that for a long time," Dr. Stanisic recalls. "Our office found that we had so many false positives, we almost felt ashamed to do it. We had to bill the patient for it, and it led to a lot of investigations down the wrong path. So we stopped doing that one."

Even when the experience is more affirming, urologists remain cautious. Noting p53’s usefulness as a marker for tumor aggressiveness, Dr. Stanisic says, "In a difficult situation, I might use it to give me another piece of information. But I don’t do it on everyone, because we’re not exactly sure what it means in everybody."

"I personally don’t have the confidence to use these markers every day," he continues. "Maybe in five years we’ll use them more, but right now I don’t think it’s something we can justify routinely."

With IHC stains, such as p53 or RB, standardization is lacking. "The methodology is not uniform, the cutoffs for a positive test are not uniform, and interpreting the data becomes very problematic," says Dr. Grossman. "So I would strongly caution clinicians and pathologists about relying too heavily on these assays and making clinical decisions based on IHC for a given protein."

Problems with false positives are the chief reason bladder cancer markers aren’t used in the United States. That hurdle may be difficult to clear, at least for certain markers. NMP22, for example, is basically based on a nuclear matrix protein and looks at nuclear markers from the cell. "But it’s not specific to the cancer nucleus; it’s a nucleus of any exfoliated cell," Dr. Zippe explains. Anything that causes rapid exfoliation of the transitional cell epithelium—cystitis, bladder stones, ureteral stents—elevates NMP. "So when you have any abnormal epithelium, then you carry the false positive rate." It’s also possible the high rate is linked with cellular proliferation, which can occur with inflammation from an in-dwelling stent, for example, or ileal conduits, as well as bladder neoplasms.

Given these complications, the marker that wins out, some say, will be one with an antigen-antibody specificity to a cancer nucleus.

Or maybe not.

Maybe, says Bogdan Czerniak, MD, PhD, the single-gene marker approach is no longer where it’s at. "Can it move this field forward for early cancer detection? I don’t think so," says Dr. Czerniak, associate professor of pathology, Department of Pathology, University of Texas M.D. Anderson Cancer Center. "We can make minor steps forward, but when we talk about incipient clinical occult phases of bladder neoplasia and detecting early occult bladder cancer, we have to develop different approaches."

His approach has been to identify early, predisposing genetic "hits," blending the concept of cancer as a genomic disorder with informatics and data from the Human Genome Project. He and his colleagues are developing a genetic map of human bladder cancer, which is based on hypervariable DNA markers; they are linking it to a physical map, which contains fragments of human genome organized in the form of YAC, BAC, or PAC clones, and finally to the ultimate map, the genome sequence itself (Czerniak B, et al. Genes, Chromosomes and Cancer. 2000;27:392-402; Kram A, et al. Laboratory Investigation. 2001;81:1039-1048).

The beauty of the genetic map, says Dr. Czerniak, is that the hypervariable markers can be tested by simple PCR reactions. "Just envision that you will extract DNA from small areas of mucosa and have it in individual samples—typically 50 to 60 of them—covering the entire organ, in this case the bladder. They are specifically related to various intraurothelial premalignant conditions, and you test multiple hypervariable markers on a total genomic scale and look for distribution of genomic imbalances in those samples (Genetic model of human urothelial carcinogenesis).

"If you look at the histological map of premalignant conditions and invasive cancer, then superimpose those on the genetic map, you will be able to trace the sequence of events," he continues. The result is a map of the early phases of bladder cancer (Assembly of whole-organ histologic and genetic maps).

"This is how we’re trying to target the early events of human carcinogenesis," Dr. Czerniak says. "This way we can take the markers, which are mapping to these regions, and use them as markers for early detection. At the same time, we can develop strategies to target the genes that are mapping to the same regions."

This is a giant leap forward, says Steve Scherer, PhD, who’s helped move specific bladder cancer markers from the genetic map to the physical and sequence-based maps. Though the first, classic steps of gene hunting—identifying families and tracing markers to those families—remain, once a region has been identified, it’s relatively easy to go into that region, grab sequences, and identify the genes within the region. "You can start to get very specific about genes and their protein products that may be a central part of cancer development," says Dr. Scherer, assistant professor, Department of Molecular and Human Genetics, Baylor College of Medicine.

"Obviously, this opens all kinds of doors in terms of diagnostics," he adds.

Earlier detection is one possibility. "It’s especially true if it looks like there might be a heritable component to this, where we could screen people who are at risk based on environmental factors," Dr. Scherer says. "We could look to see whether there’s some way to tell, at the molecular level, that this person is beginning to develop the foci within the bladder that are the very, very earliest signs of cancer."

The entire genetic map and integration with the sequence map are nearly complete and will be submitted for publication within a month or so, says Dr. Czerniak. "We’re in the last phases of correcting the data and updating certain chromosomes."

The mapmakers still have plenty of work ahead of them. "The next step is to narrow the regions on the map," Dr. Scherer says. "Right now they’re too big, and there’s a heck of a lot going on in there."

Once that’s accomplished, they hope to characterize the genes within those regions, identifying those that might be involved with bladder cancer progression. "Then we’d like to see what’s going on with those genes in tumor samples versus nontumor samples, begin to build those kinds of correlations," says Dr. Scherer. "And we’d like to explore using diagnostics while we are working out exactly which genes are involved." They’re also starting to explore SNPs—single nucleotide polymorphisms—in the mapped regions. "If we can take our data and tie it into the genesis of a human haplotype map"—and work is already underway on such a map, Dr. Scherer reports—"we’ve got one more tool that will potentially allow us to do major correlations between specific mutations, a particular haplotype, and
tumor progression."

"We’ve got a lot to do," he admits.

So does everyone, for that matter.

While bladder cancer tumor markers appear to be of some benefit, they’re far from being an overall solution.

But no one’s turning out the lights on them, either, because bladder cancer has yet to see a significant drop in mortality rates. "We haven’t changed it a dent," says Dr. Zippe.

"What else do we have on the horizon that can change it?" he asks. "Cytology alone, at least the way we read it now, is unacceptable. Can we change it by doing better surgery? No. Have we developed better chemotherapeutic drugs to change the mortality? No."

Screening with early markers is where it’s at, he says. "This is where the action is if we’re going to make a change."

Karen Titus is CAP TODAY contributing editor and co-managing editor.