Musings of a Cancer Doctor
Wide-ranging views and perspective from George W. Sledge, Jr., MD
Wednesday, December 03, 2014
Case 75, an infant, presented to the Clinical Genomics Center at UCLA with a history of multifocal complex partial epilepsy and regression of developmental milestones. The child, as well as the two parents, underwent clinical exome sequencing (so-called trio-CES). You can imagine what it must have been like to be the parents of such a child, their desire to understand what caused such misery, and the hope that something beneficial might emerge from genomic analysis. Sequencing revealed that the child carried a previously undescribed missense variant in KCTN1, which encodes the KCa4.1 protein, a member of the calcium-activated potassium channel protein family.
When I was in training, and for many years thereafter, the description of such a case, and the identification of its etiology, warranted a paper in the New England Journal of Medicine or The Lancet. When the Human Genome Project opened its doors there were only some 25 well-delineated inherited diseases of metabolism. Now, according to the authors of a stunning recent JAMA paper on clinical whole-exome sequencing, the Online Mendelian Inheritance in Man website lists some 4000 disease-gene relationships. What was once impossible has become commonplace, even trivial.
At the UCLA center, 814 patients underwent whole exome sequencing, and 26% had a molecular diagnosis. Similar results were seen in 2000 patients studied at Baylor, where 25.2% had a diagnostic mutation detected. The Houston authors state that 58% of the diagnostic mutations were previously unreported. New diseases are raining down on us faster than any of us can possibly comprehend.
The genomic revolution continues to dazzle. I cannot imagine any more exciting time to be alive. Can there be any time in history when we have learned so much about human biology in so short a time? This seems the equivalent of that period early in the twentieth century when physics exploded, with atomic theory, special and general relativity and quantum mechanics all unrolled over a decade or two, the world utterly changed.
Some of it is cancer biology, and some just (just!) general biology. Starting with the human genome project, and continuing with The Cancer Genome Atlas project and its many relatives, we've seen a technological juggernaut roll through every aspect of human biology, indeed every aspect of biology. We've gone both deeper and wider, as the two JAMA papers suggest.
Leaving aside the "cool" factor of such work, this new look at human biology has profound implications for physicians and their patients.
When whole exome sequencing becomes cheap and ubiquitous, basically something you get on a newborn like Tay-Sachs testing, what do you do with the results? Do you tell the child's parents, their pediatrician (or geriatrician--some of these things may take a lifetime to emerge), or their insurance company?
And when you go looking, you frequently find something else, something unexpected. In the 2000 patients tested at Baylor, 92 patients (4.6%) had a medically actionable incidental finding. Looking over the list reported in the paper, I see old friends such as BRCA 1 and 2, PALB2, RAD51D and RET, among many others. Remember, they were not being tested for this: just incidental findings with the promise of future misery, or perhaps medical salvation.
What to Do with Results of Panel Testing?
One of the current debates going on in guideline committees involves what to do with the results of panel testing, and whether even to order panel testing. If I order a panel test because I suspect my patient has a BRCA mutation, and I find a gene predicting an increased risk of colorectal cancer, or (further afield) hypertrophic cardiomyopathy, what is my ethical and practical responsibility as a physician? What do I owe my patient, or my patient's children, or society (health economics rearing its ugly head almost immediately)?
My personal bias, for what it’s worth, is that such broad panel testing is inevitable technologic imperialism, like we see with just about every potentially useful diagnostic technology. There is part of me that wants to shout, just like when viewing some not very bright teenagers in a slasher movie, “Don’t look behind that door. There are monsters there.” But we always open that door. Always.
And how do the tests affect a patient sitting in the room with the doctor? How does knowledge of a KCTN1 missense variant help a baby with partial complex epilepsy? And if it does not, do we even want to know? We all know Francis Bacon's dictum that knowledge is power, but what happens when knowledge leaves the doctor powerless in the face of a previously undescribed disease?
O brave new world, that has such testing in it.
Most oncologists aren't cancer geneticists, at least not yet, though we may be forced into the role sometime soon. But the broader, deeper aspects of the genomic revolution are clearly affecting how we think about the cancer patients we see. When, a couple of years ago, the first decently sized genomic evaluations of human cancer began to come out, we were all impressed with the large number of mutations, and in particular the ubiquity of rare driver mutations seen across common human cancers.
These studies, it now seems obvious, seriously underestimated the problem for many cancers. The original TCGA work, for instance, tended to focus only on mutations occurring in more than 5% of a particular cancer's cells. This meant, as was recognized at the time, that we were missing many low frequency mutations buried within a cancer's genome. Now we are beginning to see what deep, dark waters there are in the genome's abyss.
Wang and colleagues (Nature, 2014; 512(7513):155-60) performed single cell whole genome sequencing on tumors from two breast cancer patients, one ER-positive and one triple-negative. In the first cancer genome studies, one chose an area with high tumor cellularity, ground it up, and took what was essentially a family portrait. If the family had eight adult brunettes and a runty blond baby sitting behind them, you only saw the brunettes. With single cell whole exome sequencing, if you sequence enough cells (the Nature paper sequenced about 50 per tumor), the rare family members pop out. Single cell sequencing allows a collection of individual portraits to complement the family group picture.
The first thing one discovers is the incredible variability of cancer cells. The authors state “No two single tumor cells are genetically identical,” which I find somewhat scary. The triple negative breast cancer they examined didn't even pretend to be a single cancer, having three distinct subtypes buried within the cancer and a myriad of private mutations.
For the past several years the emerging genomics have depressed me even as they have fascinated me. Hypervariation is obviously a bad thing if one is throwing kinase inhibitors at a cancer: the whack-a-mole problem of compensatory resistance mechanisms dooms monotherapy approaches to inevitable failure.
But the silver lining of genomic hypervariability is now beginning to be seen in the immune checkpoint field. Genomic hypervariability is associated with neoantigen diversity. A recent evaluation of melanoma patients treated with the checkpoint inhibitor ipilimumab demonstrates a link between genomic instability and tumor response: the more mutations per megabase, the better the response to Ipi.
Mutations per megabase is not a great way to select therapy (the same New England Journal of Medicine article presents a signature that is a better predictor of response), but I suspect the concept is sound. The current issue of Nature, as I write this blog, has five manuscripts devoted to cancer immunotherapy. Among them are some fascinating pieces of information: the emerging evidence that checkpoint inhibition immunotherapy will be useful in bladder cancer (a disease sorely in need of new therapies), and the importance of PD-L1 expression on tumor infiltrating lymphocytes as a marker of response.
But what really caught my eye was evidence, in two of the papers, that checkpoint inhibition would be particularly successful where there are specific tumor neoantigens. Much of the genome literature in recent years has distinguished “driver” and “passenger” mutations. One can imagine a driver mutation saying to a passenger “You’re just along for the ride. I’m the one that matters. I drive the cancer.” And this statement is no doubt true when one speaks of kinase inhibition for a cancer. It’s been the basis for drug discovery in the last decade: find the mutant growth factor, find a molecule to block it, treat.
But in the world of immunotherapy, the drivers are just chauffeurs; the important guys sit in back, like fat cat bankers in a black stretch limousine. The “passenger” mutations signal the immune system, unleashed by anti-PD1 antibody, to attack the cancer cell. So finding genomic hypervariabilty in a patient's cancer may lead, not to despair, but to a PD1 inhibitor.
Getting back to my earlier concern that knowledge is not always power, it is perhaps best to add a qualifier: "yet." Identifying the genomic disorder underlying a baby's seizures may not be actionable today, but if you believe that progress results from the progressive accumulation of facts, and our ability to weave those facts together into testable hypotheses and new therapeutic approaches, then some day that KCTN1 missense variant test result may be accompanied by a doctor telling the parents "but we've got a treatment for that" and a prescription. So what if there are 4000 gene-disease relationships in the online databases? Indeed, so what if it is 10,000 next year? So what if the cancer has a myriad of mutations? We have lots of assistant professors, and all the time in the world, to solve these problems. That's what we do.
Sunday, November 16, 2014
No birds sing on the island of Guam. Sometime in the 1940s the brown tree snake, native to Australia and New Zealand, arrived there, probably in the cargo hold of a passing ship. It had no natural predators, and was let loose on an island rich in wildlife. It proliferated madly, causing the extinction of numerous vertebrate species as it spread.
Something very similar is happening in Florida. The Burmese python, introduced by the pet trade, was first sighted in the Everglades in the 1980s. By 2000 or so there were well-established reproductive populations, and between 2000 and 2010 these grew exponentially. There are now estimated to be somewhere between 30,000 and 300,000 Burmese pythons in southern Florida, with effects on wildlife similar to those in Guam, writ large. These pythons are now genetically distinct from the pythons of Burma, but remarkable in their own lack of diversity: a clonal population run wild.
Or, if you appreciate unintended irony, take the case of Darwin’s frogs, the Chilean amphibian first described by the great evolutionary biologist during his voyage of discovery on HMS Beagle. Darwin’s frogs (Rhinoderma rufum is the proper scientific name) have likely gone extinct, the result of the introduction of a toxic fungal species, Batrachochytrium dendrobatidis.
Batrachochytrium dendrobatidis causes chytridiomycosis, a disease characterized by the inability to breathe, hydrate, osmoregualte, or thermoregulate correctly. It is sweeping around the globe, wiping out amphibian species wherever it lands: as many as 30 percent of amphibian species worldwide may be affected. Amphibians have been around for 365 million years, but their biodiversity is collapsing due to an invasive species.
The brown tree snake and the Burmese python and chytridiomycosis are part of a growing, and global problem: the spread, through human agency, of invasive species. These invasive species are an important part of what has been labeled “the sixth extinction.”
The previous five great extinctions (such as the meteor that wiped out the dinosaurs) were caused by cataclysmic natural events. The sixth extinction is on us: the species have spread sometimes through intent (the Burmese python) and sometimes by mistake (the brown tree snake), but always through human intervention.
The results have been devastating, with loss of biodiversity on a worldwide scale. They are also very expensive: one estimate puts the economic burden of the 6500 harmful invasive species in the United States at $100 billion per year. Not too different from the cost of cancer care in the U.S., I would guess, or at least the same order of magnitude.
Analogies are always dangerous, because biology is so particular and contextual, but the invasive species studied by wildlife ecologists seem, well, almost like cancer. They spread from their initial ecosystem, establish themselves at a distant site, proliferate madly, and push aside the normal hosts, in the process reconfiguring the microenvironment of the distant site. Uncontrolled, they eliminate their hosts.
This connection has not been lost on either ecologists or cancer researchers, both of whom now make use of each other’s scientific approaches. It is one of the delights of science that we can make important contributions through repurposing insights from one field to another, and the cancer-wildlife ecology connection is a good example of this phenomenon.
Take the Shannon index. Claude Shannon was one of the greatest, and least recognized, 20th century scientists. He is, for practical purposes, the father of information theory, and his work underlies much of the computer technology we take for granted. Shannon was interested in entropy (information loss) in strings of transmitted text, and his Shannon index was designed to measure the degree of that entropy.
Wildlife ecologists use the Shannon index to measure species biodiversity. A big issue surrounding the introduction of invasive species involves its effects on the overall biodiversity of an ecosystem. If the brown tree snake assassinates Guam’s birdlife, the biodiversity of the island diminishes, an impoverishment that ultimately affects us all. One can measure biodiversity using the Shannon index, the equation for which, for those who are interested, is as follows:
The Shannon index has been applied to cancer ecosystems as well. Intratumor diversity differs among cancers. For instance, among breast cancers, triple negative breast cancers are more genotypically diverse than luminal breast cancers, and their “biodiversity,” as measured by the Shannon Index, may predict patient survival. Distant metastatic deposits (the true “invasive species”) tend to have a greater degree of diversity than the primary tumor, perhaps a reflection of the treatments they have been exposed to. Vanessa Alemendro’s recent Cancer Research paper (Cancer Res; 74(5); 1338-48, 2014) is a nice starting point on this topic. Similar results have been seen in Barrett’s esophagus, where the Shannon index of a lesion predicts progression to frank esophageal cancer.
Evolutionary Imbalance Hypothesis
A colleague recently referred to Charles Darwin as “the first and best oncologist.” The Shannon index/biodiversity story is further evidence that this is true. Recently two evolutionary biologists, Dov Sax of Brown University and Jason Fridley of Syracuse University, have proposed what they call the Evolutionary Imbalance Hypothesis of invasive species.
The EIH goes something like this: species from regions with deep and diverse evolutionary histories are more likely to be successful invaders of regions with less deep, less diverse evolutionary histories.
Sax and Fridley have put EIH to the test through statistical analysis of multiple “host” and “recipient” ecosystems, looking both in the plant and animal kingdoms, and the hypothesis always appears to ring true. Humans have created several unintended experimental tests of EIH: digging the Suez and Panama canals exposed, in each case, a more evolutionarily diverse ecosystem to a less well-developed ecosystem. Guess who invaded whom?
EIH is nothing new, the authors point out: Darwin proposed it in 1859, saying that better tested species have "consequently been advanced through natural selection and competition to a higher stage of perfection or dominating power." When I think of a nice, well-behaved tubular breast cancer, each cell looking like its neighbor, each with a low mutational load, and compare it with a high-grade, genomically diverse basal breast cancer, and the subsequent fate of their hosts, I can only repeat, “Darwin was the first and best oncologist.”
Ken Pienta’s group at Hopkins has taken the connection even further, explicitly making the link between metastasizing cancers and invasive species. Invasive species, they point out in a recent paper (Journal of Cellular Biochemistry 115:1478–1485, 2014), are ecosystem engineers, reconfiguring their microenvironment “as they construct a niche that is favorable to their own survival.”
This niche construction results in ecologic inheritance, “the inheritance, via an external environment, of one or more natural selection pressures previously modified by niche-constructing organisms.” Pienta’s group has championed the use of mathematical approaches derived from the ecology literature (the Tilman equations for modeling the invasion of two species into a defined space) to describe bone marrow metastasis.
Tumors are great ecosystem engineers, through the secretion of cytokines and growth factors that permanently alter the neighborhood they live in, making life easier for their progeny. But whereas many invasive species eventually reach some sort of homeostasis with their new ecosystem, cancers rarely do so. Their ecosystem engineering, successful in the short term, ultimately results in environmental collapse, and the death of the host.
So are they “successful” invasive species, or not? It’s all a matter of perspective, and the duration of the perspective. They are successful invaders right up to the patient’s last breath.
And, at the need of the day, so what? Both of the groups mentioned above have suggested that we might use ecologic principles as part of a therapeutic attack on metastatic lesions. One wonders whether the recent immunologic approaches using checkpoint inhibitors (anti-PD1 and PDL-1) are a partial answer to the ecologic observations regarding tumor biodiversity: the more genomically and antigenically diverse a cancer (think melanoma and smoking-induce lung cancer), the more sensitive to immunotherapy? Is the Shannon index as a predictor of immunotherapy benefit? It’s a thought.
Wildlife ecologists are just beginning to draw on the cancer experience, if my cursory review of their literature is correct. While they regularly refer to invasive species as a form of ecosystem cancer, they are just beginning to think about what it takes to wipe out these “cancerous” species. A recent press release by the US Geological Survey pointed out that the cancer model of “prevention, early detection, diagnosis, treatment options and rehabilitation” made perfect sense, and this combined approach is being used to combat invasive American bullfrogs in the Yellowstone River of the Northern Rockies. I just hope invasive American bullfrogs are easier targets than triple negative breast cancer.
Humans are the ultimate invasive species, and the one that allowed all the others to spread. We’re responsible for the extinction (outside of 1.5% of our own genome) of our closest relatives, the Neanderthals, as well as the many other large mammalian and avian species that have disappeared since we conquered the world. We’ve provided the conduits for all the other invasive species that are performing ecosystem engineering on a global scale.
It would be easy to say that we are the ultimate cancer, the one doing its level best to foul its global environment. Indeed, many have said something like this. But if so, we are surely the first cancer with a conscience, and perhaps the only one ultimately capable of reining itself in before it kills its host.
Let’s hope that the fields of wildlife ecology and cancer biology continue to cross-fertilize, to the benefit of both planetary and human ecosystems. Let’s pray that we continue to be a “successful” invasive species.
Friday, September 19, 2014
Recently I was dining with friends, enjoying a pleasant Palo Alto evening on their porch. We noticed a family of quail walking on the ledge of the wooden fence that enclosed the yard, a mother and her brood of children. They hopped down into the yard. A peregrine falcon was sitting atop a tall tree, off in the distance. To me it was a thin smudge, featureless and motionless, perhaps a football field away. I suspect the falcon saw every minor blemish on my face. A falcon's vision is considerably better than that of an aging medical oncologist: they can see prey three kilometers away.
At some intellectual level I was aware that falcons were predators, capable of swift, violent action. My hostess even mentioned that she hoped the falcon had not seen the covey of quail. But we gave the falcon little thought until it fell from the sky, talons out, into the gathering of quail: a large, close blur, shocking in its suddenness, striking just feet from where we sat. I could have sworn I had seen it on that distant tree just seconds before, and that was in fact probably the case: falcons have been clocked at speeds as high as 242 miles per hour during their hunting stoop. They are, as I now know, the fastest animals on the planet.
One of the memes running through oncology right now is that of "cancer as a chronic disease." If you enter it as a search term in Google you get 54,100 hits. The idea taking hold is that we are entering a time when the average patient's cancer will be held in check through the judicious use of systemic targeted agents. Years will pass, the patient in generally good health.
I must admit that the phrase "chronic disease" always gives me pause when I hear it in relation to cancer. Historically, physicians separated acute illnesses such as pneumonia from chronic illnesses such as rheumatoid arthritis or diabetes. Prior to the advent of antibiotic therapies in the 1940's it was the acute illnesses that tended to kill; afterwards there was a progressive shift to chronic disease as a cause of death.
From a definitional standpoint, the standard definition of a "chronic disease" is “a long-lasting condition that can be controlled but not cured.” Most human solid tumors meet this definition, and are recognized as such by the CDC.
But I think that when we talk about "cancer as a chronic disease" we are usually thinking of something else: basically, the cancer goes into the stasis field of modern medicine and does not emerge to kill you. My patients certainly think in those terms.
There are, of course, some real candidates for what I might consider true chronic disease status, the oncoequivalent of "take your insulin and it won't kill you": chronic myelogenous leukemia, controlled with imatinib, or an ER-positive breast cancer held in check with an aromatase inhibitor for prolonged periods. Or, to a somewhat lesser degree, metastatic HER2-positive breast cancer, or metastatic colorectal cancer, or renal carcinoma, all diseases where survival has significantly improved through the application of targeted therapies.
Scientifically this rapidly brings us to the question of treatment duration. Here we are trapped in empiricism. Mother Nature selected a year of HER2-targeted adjuvant therapy with trastuzumab as optimum. Not six months, not two years, but the time it takes the earth to circle the sun. Or maybe not: adding a year of adjuvant neratinib to your year of trastuzumab may improve disease-free survival. Three years of adjuvant imatinib is better than one year for GIST tumors post-surgery. How about five?
For early stage ER-positive breast cancer, ten years of tamoxifen is better than five years is better than three years is better than one year: every time we have studied duration, longer is better. Though, of interest, the disease-free survival curves never quite plateau: are we are just delaying the inevitable?
And if so, is it really a chronic disease? If you stop treating rheumatoid arthritis and your joints get hot again, you may be miserable but you probably will not die, and have a good chance of going back into remission with the same old drug: joints don't mutate. If you have metastatic breast cancer you are walking around under the cloud of a death sentence, any temporary stay of execution provided by fulvestrant or T-DM1 notwithstanding. Part of my problem with "cancer as a chronic disease" is exactly this: I know few individuals who would trade RA for metastatic cancer. Equating them seems somehow disingenuous.
I keep thinking of that peregrine falcon and the quail family. I have had a number of patients who I have sheparded along for years, occasionally decades, with either estrogen receptor-targeted or HER2-targeted treatments. It is easy to contract and propagate the "cancer as a chronic disease" meme with such patients. The clinic visits become celebrations of the doctor's therapeutic virtuosity and the patient's family anniversaries, with drugs being switched in and out every now and then when the CAT scan demonstrates modest progression.
You can almost fool yourself into believing that the patient doesn't have a lethal disease. And then the falcon swoops in. “Chronic disease” can turn into “acute and lethal” in a shockingly brief period. One clinic visit you are discussing an upcoming high school graduation, and then the next you are having an end of life discussion.
I do not know why it is--perhaps because the patients have transitioned from being patients to old friends--but the death of a patient whom I have treated for a decade usually hits me harder than one who dies quickly. There is no intellectual reason for this: the death of a long-term survivor suggests I was actually doing my job, and I should be delighted that I have added many years of high quality life rather than just a few. But it always leaves a bitter taste in my mouth. I doubt it is an experience a rheumatologist deals with very often.
Like some old soldier looking over the parapets at a distant enemy, I have learned to respect my foe's endless ingenuity, its treachery, its patient evil, and its almost maniacal drive to escape the barriers I try and throw up around it. We have, again and again, seen the ultimate failure of kinase-based approaches that have dominated the last decade, the consequence of smart tumor’s ability to mutate and evade.
The real "chronic disease" possibilities may lie within the realm of immune checkpoint inhibitors, where some (though certainly not all) melanomas appear to go into stasis for years and years, the body's immune system unleashed to keep the wolves at bay. The idea of metastatic melanoma as a chronic disease still boggles the imagination, but it certainly looks to be a real possibility for many patients. Whether I want my T regulatory system ramped up for the next decade or two remains to be seen. I suspect we will be keeping the immunologists and rheumatologists very busy: more chronic disease.
Are there any other approaches that might turn human cancer into a true chronic disease?
I present for your edification the naked mole rat. These are probably the ugliest mammals on the planet, almost disgustingly so. They are also the longest-lived rodents we have, clocking in at 30 years, a good life if living in total darkness underground in what one leading student of the species has described as a “dictatorship” is your cup of tea.
But here is the really interesting thing: they never die of cancer. I don't mean rarely, I mean never.
It doesn't matter how hard you try, either. Pump them full of carcinogens, and they just go on their merry, repulsive way, burrowing through the earth as if nothing had ever happened. And why is this? It turns out that the naked mole rat produces generous amounts of a high molecular weight form of hyaluronic acid, an evolutionary adaption that changes their skin to something quite stretchy and allows them to easily traverse underground passages. This form of hyaluronic acid has the pleasant side effect of walling off individual cancer cells before they can gain mass or metastasize. The naked mole rat buries the cancer cell in concrete. This is a spectacular example of a microenvironmental approach to cancer.
Cancer cells live in neighborhoods, and for the past decade or so we have been enlisting the neighbors (blood vessels, T cells) in the neighborhood watch. These microenvironmental approaches have started to take off, and I suspect (based partly, but not entirely, on the naked mole rat story) that we just at the beginning of such interventions. Hopefully we will not end up looking like naked mole rats.
Naked mole rats also have exceptional transcriptional fidelity. This error-free life also protects them from cancer. So taken are laboratory researchers with the naked mole rat that Science magazine declared it the “Vertebrate of the Year” in 2013, and there has been an explosion of scientific articles plumbing the rodent’s exceptional longevity and freedom from cancer. And, of course, a naked mole rat genome project.
But back to the falcon and the quail. For a brief second after the falcon struck, we were unsure of the outcome. We couldn't see whether the falcon had succeeded in snatching one of the young covey. Then there was an explosion of quail, noisy, vectored in multiple contradictory directions, their panic reigning supreme. And then, much more quietly than the quail and more slowly than he had arrived, the falcon took off, talons empty: not today. Not today. Not today!
Sunday, August 24, 2014
The crickets in my back yard have been particularly noisy these last few days, louder than they have been all summer, so loud they have kept me awake with their chirping. But I enjoy listening to them. Sometimes I just hear one, sometimes a veritable orchestra, or at least a robust string section. It is, for reasons I cannot explain, a deeply comforting sound.
I have had warm feelings towards them since my youth. I grew up as part of a generation raised on Walt Disney's Pinocchio, where a charming Jiminy Cricket served as (somewhat ineffective) conscience for the long-nosed wooden boy. Hollywood anthropomorphism favored crickets, and why not? Unlike mosquitoes and lice, these insects never mean us any harm.
As a child I was fascinated with their amazing ability to tell me the temperature. Maybe you learned this as I did. Count the chirps in 15 seconds, add 40, and you have the temperature in degrees Fahrenheit. This observation was formulated as Dolbear's Law in 1897, and it still works, more or less. More or less, because there are over 900 cricket species worldwide, and they do not all chirp, and do not chirp at the same rate for a given temperature. So Dolbear’s law is not exactly a law of nature.
I was taught that these living thermometers rubbed their hind legs together to create the chirps, and believed it for decades, but it just isn't so: the crickets have something called a stridulatory organ, a large vein running along the bottom of each wing. In fact, the scientific name for chirping is stridulation; I’ll stick with chirping. Crickets run the top of one wing along the bottom of the other to create the sound. And, in case you are wondering, crickets have a tympanic membrane to hear the chirps--though oddly enough it is located just below the knee.
Why crickets chirp is another matter: largely this is mating behavior, the male of the species announcing himself to potential mates. Entomologists distinguish four separate chirping behaviors, including a calling song that attract females and repels other males, a quiet courting song used when a female is near, an aggressive song triggered by the near presence of other males, and a brief copulatory song produced after a successful mating. You can’t make this stuff up.
The timekeeping aspect of the chirping has nothing to do with its underlying reproductive purposes. Crickets are, like all insects, cold-blooded, and their chirpings heat up along with their bodies. The thermometer is coincidence, an artifact of physicochemical design, albeit a happy one for a six-year-old boy on a warm summer’s night in a field in Wisconsin.
Nature is full of living thermometers. Measuring temperature must be something basic to all living organisms, for even lowly bacteria are capable of it: they contain temperature-sensing RNA sequences, known as RNA Thermometers, or RNATs, in their mRNAs. RNATs control virulence, heat shock, and cold shock genes in E. coli.
Their two structures are simple but clever: a zipper-like structure that gradually melts as temperature increases, and a switch mode, where two mutually exclusive structures depend on ambient temperature. RNATs are such a clever natural design that they are now being co-opted by biotechnologists.
Sometimes these internal thermometers can have seemingly bizarre purposes. Red-eared slider turtles (and many other reptiles) lack sex chromosomes. Turtle gender is determined by the temperature at which their eggs are incubated, with colder temperatures producing males and warmer temperatures females. The cut point is right around 29oC.
How this occurs has been partially elucidated in recent years. Aromatase (which, as all good medical oncologists know, converts androgens to estrogens) is under epigenetic control, so that, in the words of a recent PLOS paper, “female-producing temperature allows demethylation at the specific CpG sites of the promoter region which leads the temperature-specific expression of aromatase during gonad development.” And, in case the breast cancer docs are wondering, you can change the turtle’s gender by exposing its egg to letrozole. And you thought it was just a breast cancer drug.
But mammals are the ultimate living thermometers. If you are a warm-blooded animal whose edge over crocodiles and snakes involves continuous thermoregulation (and we live within a very narrow temperature range), then you need to have some means of measuring your degree with precision.
And we are quite good at it, we humans. The skin at the base of our thumbs can perceive temperature differences of 0.02-0.07o C. I find this little short of amazing: our fingers are thermometers. The explanation for this impressive ability is an evolutionary masterpiece. Temperature-sensitive transient receptor potential channels (or thermoTRPs) are a family of ion channels, activated at different temperature thresholds, each exquisitely sensitive to a particular temperature range. One of them, TRPV1, is also activated by capsaicin, which is why those red-hot chili peppers make your throat feel like it is burning up.
We rarely think of this internal thermometer, perhaps because it is hidden in the background, unlike the more showy, in-your-face senses of sight and hearing and taste and smell. It gets lumped in with "sense of touch" and promptly forgotten. And, since the invention of thermometers in the 17th century, we have rarely felt the need to rely on it, or even recognize its existence.
We are the only species on the planet to have an external thermometer--two if you count our cricket biothermometers. But the old thermometer is still there, sitting quietly in the back.
This temperature sense is hard-wired into our psyche, as our language (indeed, every human language) commemorates. We speak of "an icy stare" or say that a relationship is "heating up" or that someone has a "burning desire" or of a lawman being "in hot pursuit" of a criminal, only to discover that "the trail has gone cold." We live by metaphors, and temperature metaphors are exceptionally common.
It goes even deeper than language. Psychological studies have shown that the simple act of handing someone a warm cup increases interpersonal warmth, and that someone excluded from a conversation will judge a room to be cooler than one who is included. Our subconscious is deeply invested in temperature, and it is wired into our internal thermometer.
The oncologist in me always wonders whether such things affect cancers. Temperature dysregulation is, of course, common in cancers such as Hodgkin’s disease, and a rare complication of treatment for Hodgkin’s is prolonged hypothermia (lasting up to 10 days) following chemotherapy administration. I’m sure there is some interesting biology there, perhaps involving thermoTRP’s, but at the end of the day it probably doesn’t matter all that much.
Thermoregulation has not been a huge therapeutic player in the cancer field, despite half-hearted attempts at cryosurgery and hyperthermia. Are cancers essentially cold-hearted, or do they burn with the desire to harm? Or both at the same time? Metaphor only carries you so far, and attributing emotions to lethal Darwinian machines is pointless. But when I lay my hand on an inflammatory breast cancer I am always impressed, sometimes even shocked, by its malignant heat, its angry redness. Perhaps ion channels are irrelevant to their darker purpose, or perhaps like so many other things we haven't looked closely enough.
As it turns out, there is a growing literature on capsaicin as an anti-cancer agent: the active agent in chili peppers induces necrotic cell death in cancer cells, and does so in direct relation to TRPV1 expression levels. Is that cool, or what? Or a hot research area? Whichever.
So I sit on my back porch pondering all this as the evening proceeds, the temperature gradually falling, the symphony of chirps slowing bit by bit, and eventually dying out. Perhaps the crickets have found their special ones tonight. Is that a celebratory chirp I hear?
Thursday, July 31, 2014
Recently, in preparation for a lecture I gave on writing scientific papers, I had the opportunity to re-read Watson and Crick’s original 1953 Nature paper, “Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid.” It is not just the most important scientific paper in biology written in the last century, it is also one of the most beautifully written: concise, clear, jargon-free, and with the greatest punch line for any paper in the scientific literature: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Try and top that for a discussion section.
I downloaded the paper from the Nature website, which required me to go to the actual April 25, 1953 issue. My eyes wandered for a few seconds over the table of contents for the issue, and suddenly I had a thought: I wonder what it was like to publish a paper in that issue of Nature if your name wasn’t Watson or Crick? To be remembered, forever in the annals of science, as one of the also-rans.
There are many equivalents, both inside and outside science: giving a talk at the Linnaean Society in 1858 in the same session where Darwin and Wallace’s first papers on evolution were presented. Or, perhaps, being the back-up act on the 1964 Ed Sullivan Show for the Beatles. A wonderful trivia question, by the way: Las Vegas entertainers Allen & Rossi, and the impressionist Frank Gorshin, in case you want to wow someone at a dinner party.
6 Articles, 23 Letters
So who were the also-rans? There were six articles and 23 letters in the issue. Of the six articles, three were about the structure of DNA. In addition to the Watson and Crick article, there was Maurice Wilkins’ “Molecular Structure of Nucleic Acids: Molecular Structure of Deoxypentose Nucleic Acids” and Rosalind Franklin’s “Molecular Configuration in Sodium Thymonucleate.”
Franklin’s paper includes the beautiful x-ray crystallography that has become a commonplace for biology texts ever since, a ghostly X of salmon DNA.
The Wilkins and Franklin papers represented important support to the Watson-Crick modeling paper, which is why the two Cambridge researchers had encouraged their King’s College colleagues to submit papers at the same time.
The interaction of Watson and Crick with Wilkins and Franklin (both of King’s College. London), and for that matter the fraught relationship of Wilkins and Franklin, are part of the lore of molecular biology. Watson and Crick ran no experiments, relying instead on the work of others, most prominently Rosalind Franklin’s X-ray diffraction studies of DNA, which they correctly interpreted as evidence in favor of a double helix. It was a crucial piece of experimental data, and one that both Wilkins and Franklin had misinterpreted. Watson’s classic memoir, The Double Helix, describes the interactions in picturesque detail, and his description of Franklin in particular struck many then and later as both petty and sexist.
The Nobel Prize can be given out to a maximum of three investigators, and in 1962 Wilkins was investigator number three. Franklin had, in the intervening years, developed ovarian cancer, and died at the age of 37, some four years before the prize was awarded. She spent some of her final months with Crick and his wife, receiving chemotherapy. The oncologist in me has always wondered whether the Jewish Franklin carried an Ashkenazi BRCA mutation.
Would she have been awardee number three had she lived? This is an unanswerable question, but her premature death doomed her to permanent also-ran status, making her a feminist icon in the process. Could she have come up with the double helix structure on her own, given sufficient time? Perhaps. But her Nature article concludes with a sad, might-have-been-but-wasn’t, admission: “Thus our general ideas are not inconsistent with the model proposed by Watson and Crick in the preceding communication.” Not inconsistent with: a grudging admission that she had failed to see what her own data supported.
Editors of scientific journals, and contributors to those journals, scrutinize the impact factor. The impact factor is quite simple: add up the total number of citations for a journal in the first two years after publication, and divide by the number of citable items (usually articles and reviews). The best journals (Science, Nature, Lancet, New England Journal of Medicine) usually have the highest impact factors. The vast remainder are decidedly mediocre: a sea of journals with an impact factor of 2 or thereabouts. Most published science is unmemorable, rapidly forgotten even by the authors. Citations do not have a Gaussian distribution; instead they follow a power law distribution, with a few 900-pound gorillas followed by a long tail. And seldom has the power law been so severe as that issue of Nature.
Impact factor is a way of keeping score, though not without its own issues. One problem with it is that it cannot measure the long-term impact of a paper. With 60-plus years of follow-up, however, we can look at the long-term impact of the work published in that April 25, 1953 issue of Nature. Watson and Crick are the huge winners, of course: Google Scholar says their paper has been cited 9866 times, and we hardly need the citation numbers to realize the revolutionary importance of that paper. The Wilkins and Franklin papers clock in at 618 and 833 citations, marking them as important contributions. But what of the others?
Let’s begin with the other three articles. They are, in order of presentation, “Refractometry of Living Cells”; “Microsomal Particles of Normal Cow's Milk”; and “Measurement of Wind Currents in the Sea by the Method of Towed Electrodes.” None is particularly remembered today, I think it is safe to say. Google Scholar reports the three articles as having had, respectively, 172, 41, and 15 citations. OK citation numbers, even today, but definitely in the also-ran category. Interestingly, the refractometry paper was cited by a 1991 Science paper on optical adherence tomography that itself has been cited 8400 times.
The letters range from 0 to 235 citations, according to Google Scholar. The most recognized is W.R. Davis’s “An Unusual Form of Carbon”, which has been cited 235 times, most recently in 2010. The story here is interesting. Davis and his colleagues worked at the British Ceramic Research Association in Stoke on Trent in England, where they studied the carbon deposited on the brickwork of blast furnaces. They identified what they described as tiny “carbon vermicules,” some as small as 100 angstroms. In retrospect (hence the 235 citations) they were early discoverers of carbon nanotubes, members of the fullerene structural family, now actively studied for their fascinating physicochemical properties. Three researchers received Nobel prizes in the 1996 for fullerene studies, so one can feel for Davis and his fellow Stoke on Trent ceramicists. They were dual also-rans, publishing in the same issue as Watson and Crick, and coming along a few decades too early in the as-yet-unnamed fullerene field.
Of the other papers published as letters, what is there to say? I love the title of K.A. Alim’s “Longevity and Production in the Buffalo,” though R. Hadek’s “Mucin Secretion in the Ewe's Oviduct” runs a close second for my affections.
But is easy to understand why such articles are poorly cited and long forgotten, given the relative obscurity of the topics. One of the keys to success in science is choosing the right problem to work on, and mucin secretion in the ewe’s oviduct probably cannot compete with decoding the key to life on earth.
Least Cited, But...
The least cited paper (I could not find any citations using Google Scholar) is my favorite: Monica Taylor’s “Murex From the Red Sea”. Taylor wrote from Notre Dame College in Glasgow, where she curated a natural history collection. Sir John Graham Kerr had collected some 300 Murex tribulus shells from the Great Bitter Lake in Egypt. Murex was what would now be called an alien invasive species, introduced following the completion of the Suez Canal, and Sir John (would a 2014 letter to Nature ever refer to a Sir John?) and Monica wondered whether the species had altered its presentation, “relieved of the shackles of environment.” The letter was a plea for specimens from the Red Sea so that the comparison might be made. Wikipedia informs me that Murex tribulus is a large predatory sea snail, with a distribution from the Central Indian Ocean to the Western Pacific Ocean.
Did Monica Taylor ever get her Red Sea specimens? Life is full of small mysteries. Taylor herself is a fascinating soul. Born in 1877, the daughter of a science teacher and the cousin of Sir Hugh Taylor, one-time Dean of the Princeton Graduate School, she trained as a teacher prior to becoming a nun. So Monica Taylor was actually Sister Monica, and Sister Monica dearly wanted to become a scientist. But the road was not smooth for a woman, let alone a nun, wishing to be a scientist in the early twentieth century. She was refused permission to attend the University of Glasgow, and was unable to complete an external degree from the University of London due to Notre Dame’s inadequate laboratory facilities.
After several thwarted attempts, according to the University of Glasgow website, “She was eventually granted permission to do laboratory work in the Zoology Department of the University of Glasgow, provided she did not attend lectures and was chaperoned by another Sister at all times.” There she impressed Professor Graham Kerr, who encouraged her to pursue an advanced degree, and obtained permission for her to attend classes. After receiving a DSc from the University of Glasgow in 1917, she headed the Science Department at Notre Dame College until her retirement in 1946, all the while conducting significant research in amoebic zoology.
In 1953, the year of her Murex letter, she was awarded an honorary doctorate from the University of Glasgow for being "a protozoologist of international distinction." She died in 1968; six years after Watson and Crick got their Nobel prizes. No citations, and no Nobel, but perhaps you will remember this also-ran, a woman of courage and fortitude.
Most of us are also-rans, if judged against those carved into science’s Mount Rushmore. Glory would not be glorious if it was common. But maybe we have it wrong if we think the also-rans felt demeaned by their also-ranness. Maybe Dr. Alim or Dr. Hadek or Sister Dr. Taylor enjoyed their brush with greatness. And maybe, just maybe, they were satisfied with lives well lived in service to science and mankind.