Ames could now test thousands of chemicals to create a catalog of chemicals that increased the mutation rate—mutagens. And as he populated his catalog, he made a seminal observation: chemicals that scored as mutagens in his test tended to be carcinogens as well. Dye derivatives, known to be potent human carcinogens, scored floridly, causing hundreds of colonies of bacteria. So did X-rays, benzene compounds, and nitrosoguanidine derivatives—all known to cause cancers in rats and mice. In the tradition of all good tests, Ames’s test transformed the unobservable and immeasurable into the observable and measurable. The invisible X-rays that had killed the Radium girls in the 1920s could now be “seen” as revertant colonies on a petri dish.
Ames’s test was far from perfect. Not every known carcinogen scored in the test: neither DES nor asbestos sprinkled on the disabled Salmonella caused significant numbers of mutant bacteria. (In contrast, chemical constituents of tobacco smoke did cause mutation in the bacteria, as noted by several cigarette manufacturers who ran the test and, finding it disconcertingly positive, quickly buried the results.) But despite its shortcomings, the Ames test provided an important link between a purely descriptive approach toward cancer prevention and a mechanistic approach. Carcinogens, Ames suggested, had a common, distinctive functional property: they altered genes. Ames could not fathom the deeper reason behind this observation: why was the capacity to cause mutations linked to the ability to induce cancer? But he had demonstrated that carcinogens could be found experimentally—not retrospectively (by investigating cases and controls in human subjects) but by prospectively identifying chemicals that could cause mutations in a rather simple and elegant biological assay.
Chemicals, it turned out, were not the only carcinogens; nor was Ames’s test the only method to find such agents. In the late 1960s, Baruch Blumberg, a biologist working in Philadelphia, discovered that a chronic, smoldering inflammation caused by a human hepatitis virus could also cause cancer.
A biochemistry student at Oxford in the 1950s, Blumberg had become interested in genetic anthropology, the study of genetic variations in human populations. Traditional biological anthropology in the 1950s mainly involved collecting, measuring, and categorizing human anatomical specimens. Blumberg wanted to collect, measure, and categorize human genes—and he wanted to link genetic variations in humans to the susceptibility for diseases.
The problem, as Blumberg soon discovered, was the lack of human genes to be measured or categorized. Bacterial genetics was still in its infancy in the 1950s—even the structure of DNA and the nature of the genes was still largely undiscovered—and human genes had not even been seen or analyzed. The only tangible hint of variations in human genetics came from an incidental observation. Proteins in the blood, called blood antigens, varied between individuals and were inherited in families, thus implying a genetic source for this variation. These blood proteins could be measured and compared across populations using relatively simple tests.
Blumberg began to scour far-flung places in the world for blood, drawing tubes of serum from Fulani tribesmen in Africa one month and Basque shepherds the next. In 1964, after a brief tenure at the NIH, he moved to the Institute for Cancer Research in Philadelphia (later renamed the Fox Chase Cancer Center) to systematically organize the variant blood antigens that he had cataloged, hoping to link them to human diseases. It was a curiously inverted approach, like scouring a dictionary for a word and then looking for a crossword puzzle into which that word might fit.
One blood antigen that intrigued him was present in several Australian aboriginal subjects and found frequently in Asian and African populations, but was typically absent in Europeans and Americans. Suspecting that this antigen was the fingerprint of an ancient genetic factor inherited in families, Blumberg called it the Australia antigen or Au for short.
In 1966, Blumberg’s lab set out to characterize the aboriginal antigen in greater detail. He soon noted an odd correlation: individuals carrying the Au antigen often suffered from chronic hepatitis, an inflammation of the liver. These inflamed livers, studied pathologically, showed signs of chronic cycles of injury and repair—death of cells in some pockets and compensatory attempts to repair and regenerate liver cells in others, resulting in scarred, shrunken, and burnt-out livers, a condition termed chronic cirrhosis.
A link between an ancient antigen and cirrhosis suggested a genetic susceptibility for liver disease—a theory that would have sent Blumberg off on a long and largely fruitless tangent. But a chance incident overturned that theory and radically changed the course of Blumberg’s studies. The lab had been following a young patient at a mental-disability clinic in New Jersey. Initially, the man had tested negative for the Au antigen. But during one of the serial blood draws in the summer of 1966, his serum suddenly converted from “Au negative” to “Au positive.” When his liver function was measured, an acute, fulminant hepatitis was discovered.
But how could an “intrinsic” gene cause sudden seroconversion and hepatitis? Genes, after all, do not typically flicker on and off at will. Blumberg’s beautiful theory about genetic variation had been slain by an ugly fact. Au, he realized, could not mark an inherent variation in a human gene. In fact, Au was soon found to be neither a human protein nor a blood antigen. Au was a piece of a viral protein floating in the blood, the sign of an infection. The New Jersey man had been infected by this microbe and thus converted from Au negative to positive.
Blumberg now raced to isolate the organism responsible for the infection. By the early 1970s, working with a team of collaborators, his lab had purified particles of a new virus, which he called hepatitis B virus, or HBV. The virus was structurally simple—“roughly circular . . . about forty-two nanometers in diameter, one of the smallest DNA viruses that infect humans”—but the simple structure belied extraordinarily complex behavior. In humans, HBV infection caused a broad spectrum of diseases, ranging from asymptomatic infection to acute hepatitis to chronic cirrhosis in the liver.
The identification of a new human virus set off a storm of activity for epidemiologists. By 1969, Japanese researchers (and subsequently Blumberg’s group) had learned that the virus was transmitted from one individual to another through blood transfusions. By screening blood before transfusion—using the now familiar Au antigen as one of the early biomarkers in serum—the blood-borne infection could be blocked, thereby reducing the risk of hepatitis B.
But another illness soon stood out as linked to HBV: a fatal, insidious form of liver cancer endemic in parts of Asia and Africa that arose out of scarred, ashen livers often decades after chronic viral infection. When cases of hepatocellular cancer were compared to controls using classical statistical methods, chronic infection with HBV, and the associated cycle of injury and repair in liver cells, stood out as a clear risk factor—at about five- to tenfold the risk for uninfected controls. HBV, then, was a carcinogen—although a live carcinogen, capable of being transmitted from one host to another.
The discovery of HBV was an embarrassment to the NCI. The institute’s highly targeted and heavily funded Special Virus Cancer Program, having inoculated thousands of monkeys with human cancer extracts, had yet to find a single cancer-associated virus. Yet a genetic anthropologist exploring aboriginal antigens had found a highly prevalent virus associated with a highly prevalent human cancer. Blumberg was acutely aware of the NCI’s embarrassment, and of the serendipity in his work. His departure from the NIH in 1964, although cordial, had been driven by precisely such conflicts; his interdisciplinary curiosity had chafed against the “discipline-determined rigidity of the constituent institutes,” among which the NCI, with its goal-directed cancer virus hunt, was the worst culprit. Worse still for the strongest enthusiasts of the cancer virus theory, it appeared as if Blumberg’s virus itself was not the proximal cause of the cancer. The inflammation induced by the virus in liver cells, and the associated cycle of death and repair, appeared to be responsible for the cancer—a blow to the notion that viruses directly cause can
cer.
But Blumberg had little time to mull over these conflicts, and he certainly had no theoretical axes to grind about viruses and cancer. A pragmatist, he directed his team toward finding a vaccine for HBV. By 1979, his group had devised one. Like the blood-screening strategy, the vaccine did not, of course, alter the course of the cancer after its genesis, but it sharply reduced the susceptibility to HBV infection in uninfected men and women. Blumberg had thus made a critical link from cause to prevention. He had identified a viral carcinogen, found a method to detect it before transmission, then found a means to thwart transmission.
The strangest among the newly discovered “preventable” carcinogens, though, was not a virus or a chemical but a cellular organism—a bacterium. In 1979, the year that Blumberg’s hepatitis B vaccine was beginning its trial in America, a junior resident in medicine named Barry Marshall and a gastroenterologist, Robin Warren, both at the Royal Perth Hospital in Australia, set out to investigate the cause of stomach inflammation, gastritis, a condition known to predispose patients to peptic ulcers and to stomach cancer.
For centuries, gastritis had rather vaguely been attributed to stress and neuroses. (In popular use, the term dyspeptic still refers to an irritable and fragile psychological state.) By extension, then, cancer of the stomach was cancer unleashed by neurotic stress, in essence a modern variant of the theory of clogged melancholia proposed by Galen.
But Warren had convinced himself that the true cause of gastritis was a yet unknown species of bacteria, an organism that, according to dogma, could not even exist in the inhospitable acidic lumen of the stomach. “Since the early days of medical bacteriology, over one hundred years ago,” Warren wrote, “it was taught that bacteria do not grow in the stomach. When I was a student, this was taken as so obvious as to barely rate a mention. It was a ‘known fact,’ like ‘everyone knows that the earth is flat.’”
But the flat-earth theory of stomach inflammation made little sense to Warren. When he examined biopsies of men and women with gastritis or gastric ulcers, he found a hazy, blue layer overlying the craterlike depressions of the ulcers in the stomach. When he looked even harder at that bluish layer, he inevitably saw spiral organisms teeming within it.
Or had he imagined it? Warren was convinced that these organisms represented a new species of bacterium that caused gastritis and peptic ulcers. But he could not isolate the bacteria in any form on a plate, dish, or culture. Others could not see the organism; Warren could not grow it; the whole theory, with its blue haze of alien organisms growing above craters in the stomach, smacked of science fiction.
Barry Marshall, in contrast, had no pet theory to test or disprove. The son of a Kalgoorlie boilermaker and a nurse, he had trained in medicine in Perth and was an unwhetted junior investigator looking for a project. Intrigued by Warren’s data (although skeptical of the link with an unknown, phantasmic bacteria), he started to collect brushings from patients with ulcers and spread out the material on petri dishes, hoping to grow a bacterium. But as with Warren, no bacteria grew out. Week after week, Marshall’s dishes piled up in the incubator and were discarded in large stacks after a few days of examination.
But then serendipity intervened: over an unexpectedly busy Easter weekend in 1982, with the hospital overflowing with medical admissions, Marshall forgot to examine his plates and left them in the incubator. When he remembered and returned to examine them, he found tiny, translucent pearls of bacterial colonies growing on the agar. The long incubation period had been critical. Under the microscope, the bacterium growing on the plate was a minuscule, slow-growing, fragile organism with a helical tail, a species that had never been described by microbiologists. Warren and Marshall called it Helicobacter pylori—helicobacter for its appearance, and pylorus from the Latin for “gatekeeper,” for its location near the outlet valve of the stomach.
But the mere existence of the bacteria, or even its association with ulcers, was not proof enough that it caused gastritis. Koch’s third postulate stipulated that to be classified as a bona fide causal element for a disease, an organism needed to re-create the disease when introduced into a naive host. Marshall and Warren inoculated pigs with the bacteria and performed serial endoscopies. But the pigs—seventy pounds of porcine weight that did not take kindly to weekly endoscopies—did not sprout any ulcers. And testing the theory on humans was ethically impossible: how could one justify infecting a human with a new, uncharacterized species of bacteria to prove that it caused gastritis and predisposed to cancer?
In July 1984, with his experiments stalled and his grant applications in jeopardy, Marshall performed the ultimate experiment: “On the morning of the experiment, I omitted my breakfast. . . . Two hours later, Neil Noakes scraped a heavily inoculated 4 day culture plate of Helicobacter and dispersed the bacteria in alkaline peptone water (a kind of meat broth used to keep bacteria alive). I fasted until 10 am when Neil handed me a 200 ml beaker about one quarter full of the cloudy brown liquid. I drank it down in one gulp then fasted for the rest of the day. A few stomach gurgles occurred. Was it the bacteria or was I just hungry?”
Marshall was not “just hungry.” Within a few days of swallowing the turbid bacterial culture, he was violently ill, with nausea, vomiting, night sweats, and chills. He persuaded a colleague to perform serial biopsies to document the pathological changes, and he was diagnosed with highly active gastritis, with a dense overlay of bacteria in his stomach and ulcerating craters beneath—precisely what Warren had found in his patients. In late July, with Warren as coauthor, Marshall submitted his own case report to the Medical Journal of Australia for publication (“a normal volunteer [has] swallowed a pure culture of the organism,” he wrote). The critics had at last been silenced. Helicobacter pylori was indisputably the cause of gastric inflammation.
The link between Helicobacter and gastritis raised the possibility that bacterial infection and chronic inflammation caused stomach cancer.* Indeed, by the late 1980s, several epidemiological studies had linked H. pylori–induced gastritis with stomach cancer. Marshall and Warren had, meanwhile, tested antibiotic regimens (including the once-forsaken alchemical agent bismuth) to create a potent multidrug treatment for the H. pylori infection.* Randomized trials run on the western coast of Japan, where stomach and H. pylori infection are endemic, showed that antibiotic treatment reduced gastric ulcers and gastritis.
The effect of antibiotic therapy on cancer, though, was more complex. The eradication of H. pylori infection in young men and women reduced the incidence of gastric cancer. In older patients, in whom chronic gastritis had smoldered for several decades, eradication of the infection had little effect. In these elderly patients, presumably the chronic inflammation had already progressed to a point that its eradication made no difference. For cancer prevention to work, Auerbach’s march—the prodrome of cancer—had to be halted early.
Although unorthodox in the extreme, Barry Marshall’s “experiment”—swallowing a carcinogen to create a precancerous state in his own stomach—encapsulated a growing sense of impatience and frustration among cancer epidemiologists. Powerful strategies for cancer prevention arise, clearly, from a deep understanding of causes. The identification of a carcinogen is only the first step toward that understanding. To mount a successful strategy against cancer, one needs to know not only what the carcinogen is, but what the carcinogen does.
But the set of disparate observations—from Blumberg to Ames to Warren and Marshall—could not simply be stitched together into a coherent theory of carcinogenesis. How could DES, asbestos, radiation, hepatitis virus, and a stomach bacterium all converge on the same pathological state, although in different populations and in different organs? The list of cancer-causing agents seemed to get—as another swallower of unknown potions might have put it—“curiouser and curiouser.”
There was little precedent in other diseases for such an astonishing diversity of causes. Diabetes, a complex illness with complex manifestations, is still fundamentally a d
isease of abnormal insulin signaling. Coronary heart disease occurs when a clot, arising from a ruptured and inflamed atherosclerotic plaque, occludes a blood vessel of the heart. But the search for a unifying mechanistic description of cancer seemed to be sorely missing. What, beyond abnormal, dysregulated cell division, was the common pathophysiological mechanism underlying cancer?
To answer this question, cancer biologists would need to return to the birth of cancer, to the very first steps of a cell’s journey toward malignant transformation—to carcinogenesis.
*H. pylori infection is linked to several forms of cancer, including gastric adenocarcinoma and mucosa-associated lymphoma.
*Marshall later treated himself with the regimen and eradicated his infection.
“A spider’s web”
It is to earlier diagnosis that we must look for any material improvement in our cancer cures.
—John Lockhart-Mummery, 1926
The greatest need we have today in the human cancer problem, except for a universal cure, is a method of detecting the presence of cancer before there are any clinical signs of symptoms.
—Sidney Farber, letter to Etta Rosensohn,
November 1962
Lady, have you been “Paptized”?
—New York Amsterdam News,
on Pap smears, 1957
The long, slow march of carcinogenesis—the methodical, step-by-step progression of early-stage lesions of cancer into frankly malignant cells—inspired another strategy to prevent cancer. If cancer truly slouched to its birth, as Auerbach suspected, then perhaps one could still intervene on that progression in its earliest stages—by attacking precancer rather than cancer. Could one thwart the march of carcinogenesis in midstep?