The Emperor of All Maladies: A Biography of Cancer (71 page)

In 1982, Weinberg
, Barbacid, and Wigler independently published their discoveries and compared their results. It was a powerful, unexpected convergence: all three labs had isolated the same fragment of DNA, containing a gene called
ras
, from their respective cancer cells.
^†
Like
src, ras
was
also a gene present in all cells. But like
src
again, the
ras
gene in normal cells was functionally different from the
ras
present in cancer cells. In normal cells, the
ras
gene encoded a tightly regulated protein that turned “on” and “off” like a carefully modulated switch. In cancer cells, the gene was mutated, just as Varmus and Bishop had predicted. Mutated
ras
encoded a berserk, perpetually hyperactive protein permanently locked “on.” This mutant protein produced an unquenchable signal for a cell to divide—and to keep dividing. It was the long-sought “native” human oncogene, captured in flesh and blood out of a cancer cell. “
Once we had cloned
a cancer gene,” Weinberg wrote, “the world would be at our feet.” New insights into carcinogenesis, and new therapeutic inroads would instantly follow. “It was,” as Weinberg would later write, all “a wonderful pipe dream.”

In 1983, a few months after Weinberg had purified mutant
ras
out of cancer cells,
Ray Erikson traveled to Washington
to receive the prestigious General Motors prize for his research on
src
activity and function. The other awardee that evening was Tom Frei, being honored for his advancement of the cure for leukemia.

It was a resplendent evening. There was an elegant candlelit dinner in a Washington banquet hall, followed by congratulatory speeches and toasts. Scientists, physicians, and policymakers, including many of the former Laskerites,
^*
gathered around linen-covered tables. Talk turned frequently to the discovery of oncogenes and the invention of curative chemotherapy. But the two conversations seemed to be occurring in sealed and separate universes, much as they had at Temin’s conference in Houston more than a decade earlier. Frei’s award, for curing leukemia, and Erikson’s award, for identifying the function of a critical oncogene, might almost have been given to two unconnected pursuits. “I don’t remember any enthusiasm among the clinicians to reach out to the cancer biologists to synthesize the two poles of knowledge about cancer,” Erikson recalled. The two halves of cancer, cause and cure, having feasted and been feted together, sped off in separate taxis into the night.

The discovery of
ras
brought one challenge to a close for cancer geneticists: they had purified a mutated oncogene from a cancer cell. But it threw open another challenge. Knudson’s two-hit hypothesis had also generated a risky prediction: that retinoblastoma cancer cells contained two inactivated copies of the
Rb
gene. Weinberg, Wigler, and Barbacid had proved Varmus and Bishop right. Now someone had to prove Knudson’s prediction by isolating his fabled tumor suppressor gene and demonstrating that both its copies were inactivated in retinoblastoma.

This challenge, though, came with an odd conceptual twist. Tumor suppressor genes, by their very nature, are asserted in their
absence.
An oncogene, when mutated, provides an “on” signal for the cells to grow. A tumor suppressor gene when mutated, in contrast, removes an “off” signal for growth. Weinberg and Chiaho Shih’s transfection assay had worked because oncogenes can cause the normal cells to divide uncontrollably, thus forming a focus of cells. But an anti-oncogene, transfected into a cell, cannot be expected to create an “anti-focus.” “
How can one capture genes
that behave like ghosts,” Weinberg wrote, “influencing cells from behind some dark curtain?”

In the mid-1980s, cancer geneticists had begun to glimpse shadowy outlines behind retinoblastoma’s “dark curtain.” By analyzing chromosomes from retinoblastoma cancer cells using the technique pioneered by Janet Rowley, geneticists had demonstrated that the
Rb
gene “lived” on chromosome thirteen. But a chromosome contains thousands of genes. Isolating a single gene from that vast set—particularly one whose functional presence was revealed only when inactive—seemed like an impossible task. Large laboratories professionally equipped to hunt for cancer genes—Webster Cavenee’s lab in Cincinnati, Brenda Gallie’s in Toronto, and Weinberg’s in Boston—were frantically hunting for a strategy to isolate
Rb.
But these efforts had reached a standstill. “
We knew where
Rb
lived,” Weinberg recalled, “but we had no idea what
Rb
was.”

Across the Charles River from Weinberg’s lab, Thad Dryja, an ophthalmologist-turned-geneticist, had also joined the hunt for
Rb.
Dryja’s laboratory was perched on the sixth floor of the Massachusetts Eye and Ear Infirmary—the Eyeball, as it was known colloquially among the medical
residents. The ophthalmological infirmary was well-known for its clinical research on eye diseases, but was barely recognized for laboratory-based research. Weinberg’s Whitehead Institute boasted the power of the latest technologies, an army of machines that could sequence thousands of DNA samples and powerful fluorescent microscopes that could look down into the very heart of the cell. In contrast, the Eyeball, with its proud display of nineteenth-century eyeglasses and lenses in lacquered wooden vitrines, was almost self-indulgently anachronistic.

Dryja, too, was an unlikely cancer geneticist. In the mid-1980s, having completed his clinical fellowship in ophthalmology at the infirmary in Boston, he had crossed town to the science laboratories at Children’s Hospital to study the genetics of eye diseases. As an ophthalmologist interested in cancer, Dryja had an obvious target: retinoblastoma. But even Dryja, an inveterate optimist, was hesitant about taking on the search for
Rb.
“Brenda [Gallie] and Web [Cavenee] had both stalled in their attempts [to clone
Rb
]. It was a slow, frustrating time.”

Dryja began his hunt for
Rb
with a few key assumptions. Normal human cells, he knew, have two copies of every chromosome (except the sex chromosomes), one from each parent, twenty-three pairs of chromosomes in all, a total of forty-six. Every normal cell thus has two copies of the
Rb
gene, one in each copy of chromosome thirteen.

Assuming Knudson was right in his two-hit hypothesis, every eye tumor should possess two independent inactivating mutations in the
Rb
gene, one in each chromosome. Mutations, Dryja knew, come in many forms. They can be small changes in DNA that can activate a gene. Or they can be large structural deletions in a gene, stretching over a large piece of the chromosome. Since the
Rb
gene had to be
inactivated
to unleash retinoblastoma, Dryja reasoned that the mutation responsible was likely a deletion of the gene. Deleting a sizable piece of a gene, after all, is perhaps the quickest, crudest way to paralyze and inactivate it.

In most retinoblastoma tumors, Dryja suspected, the two deletions in the two copies of the
Rb
gene would lie in different parts of the gene. Since mutations occur randomly, the chance of both mutations lying in precisely the same region of the gene is a little akin to rolling double sixes in dice that have one hundred faces. Typically, one of the deletions would “hit” the
front end of the gene, while the other deletion might hit the back end (in both cases, the functional consequences would be the same—inactivating
Rb
). The two “hits” in most tumors would thus be asymmetric—affecting two different parts of the gene on the two chromosomes.

But even hundred-headed dice, rolled many times, can yield double sixes. Rarely, Dryja knew, one might encounter a tumor in which both hits had deleted exactly the same part of the gene on the two sister chromosomes. In that case, that piece of chromosome would be completely missing from the cell. And if Dryja could find a method to identify a completely missing piece of chromosome thirteen in a retinoblastoma tumor cell, he would instantly land on the
Rb
gene. It was the simplest of strategies: to hunt the gene with absent function, Dryja would look for absence in structure.

To identify such a missing piece, Dryja needed structural mileposts along chromosome thirteen—small pieces of DNA called probes, which were aligned along the length of the chromosome. He could use these DNA probes in a variant of the same “sticking” reaction that Varmus and Bishop had used in the 1970s: if the piece of DNA existed in the tumor cell, it would stick; if the piece did not exist, the probe would not stick, identifying the missing piece in the cell. Dryja had assembled a series of such probes. But more than probes, he needed a resource that he uniquely possessed: an enormous bank of frozen tumors. The chances of finding a shared deletion in the
Rb
gene in both chromosomes were slim, so he would need to test a vast sample set to find one.

This, then, was his crucial advantage over the vast professional labs in Toronto and Houston. Laboratory scientists rarely venture outside the lab to find human samples. Dryja, a clinician, had a freezer full of them. “
I stored the tumors obsessively
,” he said with the childlike delight of a collector. “I put news out among patients and doctors that I was looking for retinoblastoma cases. Every time someone saw a case, they would say, ‘Get that guy Dryja.’ I would then drive or fly or even walk to pick up the samples and bring them here. I even got to know the patients by name. Since the disease ran in families, I would call them at home to see if there was a brother or sister or cousin with retinoblastoma. Sometimes, I would know [about a tumor] even before doctors knew.”

Week after week, Dryja extracted the chromosomes from tumors and ran his probe set against the chromosomes. If the probes bound, they usually made a signal on a gel; if a probe was fully missing, the signal was
blank. One morning, having run another dozen tumors, Dryja came to the lab and held up the blot against the window and ran his eyes left to right, lane after lane automatically, like a pianist reading a score. In one tumor, he saw a blank space. One of his probes—H3-8, he had called it—was deleted in both chromosomes in that tumor. He felt the brief hot rush of ecstasy, which then tipped into queasiness. “
It was at that moment
that I had the feeling that we had a gene in our hands. I had landed on retinoblastoma.”

Dryja had found a piece of DNA missing in tumor cells. Now he needed to find the corresponding piece present in normal cells, thus isolating the
Rb
gene. Perilously close to the end, Dryja was like an acrobat at the final stretch of his rope. His one-room lab was taut with tension, stretched to its limit. He had inadequate skills in isolating genes and limited resources. To isolate the gene, he would need help, so he took another lunge. He had heard that researchers in the Weinberg lab were also hunting for the retinoblastoma gene. Dryja’s choices were stark: he could either team up with Weinberg, or he could try to isolate the gene alone and lose the race altogether.