Like Rats Fleeing a Sinking Hypothesis

I claim to be an expert on rat mammary gland anatomy, a surprisingly complex topic that might seem to be of limited use in today’s world. And like many who claim expertise in a tiny niche, the truth is that it was my assistant in the lab (that is, a medical student) who did all the hard work in discovering nuances in rat anatomy that are not in the textbooks. Still, in accordance with the unwritten rules of academia, I claim all the credit.

You might think I’m kidding, since I often dabble in semi-comedic hyperbole. What could rat mammary gland anatomy possibly have to do with a clinical controversy? So, to avoid burying the lead, here’s where I’m headed – For over 30 years, researchers and clinicians have been quoting the studies of preventive mastectomies performed on female Sprague-Dawley rats in the DMBA (7,12-Dimethylbenz[a]anthracene) carcinogenesis model, wherein the number of cancers emerging after exposure to DMBA is – amazingly – the same whether or not mastectomy is performed. Of several articles on this topic, one of the more commonly referenced publications is from Wong JH et al in Surgery 1986; 99:67-71. Inexplicably, in this and similar studies, preventive mastectomies did not even make a dent in the number of cancers, roughly 5 breast cancers per rat, with or without “mastectomies.”

Then comes a non sequitur – as one prominent breast surgeon stated in the years leading up to the discovery, sequencing, and commercialization of BRCA-1 testing in humans: “Because the BRCA mutation will be present in every cell, it is possible that preventive mastectomy will have no effect whatsoever, as in the case of DMBA-induced tumors that occur with the same frequency whether or not the rat has undergone preventive mastectomy.”

Even at the time (circa 1992), that statement sounded like it contained a logical fallacy, though I couldn’t pinpoint the error from my short experience in Philosophy 101. Yet, there is an enormous difference between a “mutation in every cell,” versus “every cell will become cancer,” or even “every cell is equally at very high risk for cancer.” The human body is estimated to be composed of 32 trillion cells. If we assign 1% of those cells to breast epithelium, we’re talking about 300 billion cells. I attempt this rough calculation to avoid co-opting the phrase, “billions and billions,” attributed to Carl Sagan (but only adopted as mantra after Johnny Carson used the phrase on his TV show). Now, with 300 billion breast epithelial cells already carrying a BRCA-1 mutation, why is it that without preventive surgery, breast cancer will arise from only one or two or three clones over a lifetime? Alternatively stated, 99.999999999% of these cells never become cancer.

From the pre-BRCA era, we knew that patients with very strong hereditary risks were most likely to develop “only” one or two cancers during their lifetimes, the primary difference being earlier age of onset, far more impressive than the actual number of cancers over time. Even without a BRCA mutation, breast cells accumulate many somatic mutations over the course of one’s lifetime, sometimes generating a growth advantage to each clone, so it remains an oddity as to why only one or two clones of cancer cells emerge clinically. Perhaps the first cancer fires up the immune system to keep all the other “premalignant,” mutated cells in check. Perhaps it doesn’t even require a full-blown cancer to accomplish this. Maybe the body recognizes pre-malignant clones and acts accordingly for self-preservation. I don’t know. I only know that the mathematics don’t add up. 300 billion breast epithelial cells primed for cancer, yet only one tumor in most cases, two in some, and three or more in a few?

Many years ago (circa 1989), when I had the delusion that I was going to build a benign breast tissue research empire at the University of Oklahoma, I attended a basic science conference on the topic of early carcinogenesis in breast cancer. In my naiveté, when I checked in, I asked at the registration desk how I would get my CME. The registrar was caught off guard and seemed puzzled as I explained the meaning of CME. But she maintained composure as she broke the news to me – there wasn’t such a thing as CME at a basic science conference. I was the only physician attending.

Anyway, after I settled in, I realized that I was barely able to follow the presentations beyond the introductory 35mm slides. However, one talk was both understandable and memorable – a presentation on the somatic mutations in breast cells having normal morphology under light microscopy. Specifically, in breast cancer patients, when looking at adjacent normal tissue, many of the mutations were identical to those in the tumor, though not quite as many. But even more remarkable, some of these same mutations were present in tissue far away from the tumor, including the opposite breast. After all, what environmental insult (other than focal radiation) generates genetic “hits” in only one breast? The cells might look normal, but they are not. They accumulate somatic mutations long before changes occur under the microscope.

Then, the speaker drove the point home – “Halsted was right for the wrong reasons when he described his “field effect” as the justification for mastectomy. In fact, the field effect is real, but it’s a bilateral field effect at the molecular biologic level that only rarely translates to the clinic. And this is the case whether one is talking about accumulating somatic mutations in breast cells over a lifetime, or having germline mutations at birth. Having germline mutations in every cell certainly increases long-term risk of breast cancer, but the eventual crossover to malignancy occurs in surprisingly few cells, be it from either somatic or germline mutations…or both.

Coming at this from another angle, let’s apply the crystal ball to a 25 year-old BRCA-positive patient, and condense her future into the present. Yes, we know there are about 300 billion mutated cells, yet in the crystal ball, we see only 3 cancers emerging over 50 years, one in the RUOQ in the year 2026, one in the RLIQ in the year 2037, and one in the LLOQ in the year 2049. All 3 future cancers, however, are located within the tissue that is removed with bilateral preventive mastectomies. Thus, it does not matter that microscopic tissue containing mutated cells is left in this patient if she opts for preventive surgery, and this seems to be the case in 90-95% of patients who opt for preventive mastectomies.

Whether or not there is a direct correlation between percentage of tissue removed to relative risk reduction is unknown. I suspect those pesky residual cells in BRCA+ patients do, in fact, keep the correlation from being exact, but it’s close. My guess is something like this: If 99% of the breast epithelium is removed, there will be a 90-95% risk reduction. Since it’s a relative risk reduction, the BRCA+ patients will have a higher absolute risk of future cancer after preventive mastectomies than someone at lower risk (e.g., 90% risk reduction applied to 80% absolute risk leaves 8% lifetime risk post-mastectomies in a BRCA+ patient, whereas a 90% risk reduction applied to someone with a 30% risk leaves a 3% remaining risk).

Other variables prevent strict adherence to mathematical probabilities, however. Some patients have well-defined boundaries to their breast parenchyma while others are poorly defined. Then, there are varying degrees of precision from one surgeon to the next in carefully removing all grossly evident breast parenchyma. As for leaving the nipple-areola complex, this should be a moot point – not only are there fewer TDLUs in this area, but also if we resort to our crystal ball again, how many cancers are going to occur directly beneath the nipple? A few, but not many.

In 2017, the oncologic safety of nipple-sparing mastectomy in women with breast cancer was published in the Journal of the American College of Surgeons by Smith BL et al (Vol 225: 361-365). In 311 patients with established cancer, only 3.7% recurred locally, but there were ZERO recurrences near the retained nipple-areola complex. (We await their data on the 2,000 mastectomies performed for risk reduction in patients without cancer.) More pertinent to this blogatorial is the performance of risk-reducing nipple-sparing mastectomies in BRCA-positive patients before cancer occurs, addressed in “Oncologic Safety of Prophylactic Nipple-Sparing Mastectomy in a Population with BRCA Mutations” that appeared in JAMA Surg. 2018; 153:123-129 (and was my prompt to write this article).

In the short-term, 22 cancers were expected in BRCA1 and BRCA2 mutation carriers undergoing preventive surgery, yet no cancers actually developed. That’s ZERO cancers after 548 nipple-sparing mastectomies! In the invited commentary, there it was again, after all these years, just like in 1986: “Although it seems intuitive that reducing the volume of breast tissue would likely reduce the risk of developing breast cancer, BRCA carriers have germline mutations. Any residual breast tissue remains at the same inherent risk of developing breast cancer.”

So, we’re back to the making the distinction between “every cell has the mutation” as distinct from “every cell has an equally high potential to become cancer.” First of all, the only time each cell has perfectly equal propensity for cancer is at conception. After that, somatic mutations begin to accumulate in women (yes, even in utero) with or without germline mutations, and certain clones with a growth advantage will emerge in focal regions, not equally scattered throughout the breast tissue. Within those focal regions, further mutations will provide yet another growth advantage in even fewer focal areas, until there is eventually crossover to malignancy in only a tiny fraction of the original 300 billion cells. (Yes, the “two-hit” hypothesis for tumor suppressor genes is currently the rule, but my guess is that “clinical emergence of cancer” is more complicated than two-hits, as immune surveillance enters the picture.)

Now, back to the rats. It is helpful to note that the 2018 commentary (above) to the JAMA Surg article, reminding us of the “same inherent risk” in residual breast tissue, was written by the lead author of the DMBA article from 32 years ago wherein preventive surgery had zero impact on the rate of development of cancers. One can readily appreciate his skepticism about the short-term clinical data. Nevertheless, it takes working with the DMBA model to understand how it is analogous to the human situation, but more importantly – how the DMBA rats are different.

There are two reasons why the DMBA model is not a good way to study preventive surgery – 1) rat mammary anatomy, and 2) the DMBA carcinogen turns many cells malignant within a very short time frame.

Rat mammary anatomy – speaking as a bona fide expert now, there are indeed some analogies between humans and rats, e.g., breast tissue close to the skin predisposes to residual epithelium, very few TEBs beneath the nipple (TEBs are terminal end buds that are analogous to human TDLUs), etc., but it’s the differences that are important here. In spite of anatomic discussions describing the rat mammary “fat pad” where all the action is, there is no breast mound, or discrete parenchymal cone that one can call a “breast.” The rat has 12 nipples, each with a single duct opening to the outside, but the supporting breast parenchyma is a diffuse sheet that covers nearly the entire ventral surface of the animal, extending from lower jaw to anus and even wrapping around to the dorsal surface of the rat in some places. Diagrams of the extent of this parenchymal sheet indicate a formidable task for the surgeon who believes he or she can remove the diffuse breast tissue associated with 12 nipples. But in “our” experience, the extent of the parenchymal sheet is even more impressive, such that one is talking about removing the breast tissue from approximately one-third of the surface area of the entire animal. And in the words of my medical student assistant: “…a striking feature is the lack of boundaries in the mammary tissue.”

But that’s not where the problems end. As “we” discovered in “our” meticulous dissections, there are some mighty forces that keep the surgeon from a truly extirpative procedure. Again, in the words of James Banta, MS-2 on summer fellowship (who, after his experience with me, became an ophthalmologist): “Approaching the axilla, the most difficult part of the procedure, one encounters the cutaneous trunci muscle. It originates on the lesser tubercle of the humerus and inserts directly into the skin forming a broad, thin sheet that thickens as it approaches the axilla. This muscle penetrates the second and third mammary glands and separates them into superficial and deep layers. While the deep layer is easily removed, the remnant of breast tissue in the superficial layer cannot be properly excised without removing the thickened portion of the cutaneous trunci, which along with the breast tissue remnant, is tightly adherent to the dermis. Removing this muscle, with its breast tissue remnant, requires ligature of numerous tributaries of the dorsal branch of the lateral thoracic artery, resulting in necrosis of the skin flap.”

Yes, we have a difficult time getting rid of all the microscopic breast tissue in humans, too, but if you use Google Images for “DMBA tumors in rats,” you’ll see tumors under the animal’s jaw, or on its back, or near its tail, and it will give the term “residual breast tissue” a whole new meaning.

Add to this the second reason why this particular model is not a good one for surgical prevention — the DMBA carcinogen turns many cells fully malignant within a very short time frame.

 The DMBA model is sometimes used as a good example in distinguishing “initiation” from “promotion,” a basic principle behind carcinogenesis. DMBA does the initiating (mutations), while hormones (esp. estrogen and prolactin) do the promotion. But not so fast. As early as 1962, the prominent breast cancer researcher at Roswell Park, Dr. Thomas Dao, made the case that the hormonal milieu in the DMBA model is part of the initiation of tumor cells. So what? Well, it means that cancer cells are “created” in one lockstep, unlike human carcinogenesis. In addition, these malignant cells are widely scattered throughout all the (diffuse) breast tissue.

On the average, if you give DMBA to female, virgin Sprague-Dawley rats at the age of sexual maturity, you’ll see 100% of them develop 3-5 breast cancers after a short latency of 8 to 22 weeks. And to show how powerful the hormonal contribution is, if you perform oophorectomy on these animals 4 weeks prior to the DMBA, you’ll get zero cancers. As Dr. Dao pointed out, initiation with prompt carcinogenesis (without true promotion) is thus accomplished through the combination of DMBA and hormones together. Hormones might have some additional promoter aspects, but this is secondary. Cancer cells are created at the git-go, and they are widely scattered.

In another departure from human counterparts, these histologically malignant DMBA tumors metastasize only rarely. If it were not for euthanasia, the tumors would kill through their bulk and local effects, draining the animal of all resources for life. So, most studies end by counting the initial wave of cancers, then putting the animal to sleep. So, what happens if you remove these cancers as quickly as they appear? They just keep coming. So many malignant cells are created by DMBA that there’s seemingly no end to the number of cancers.

And this is the scenario that some conceptualize for the BRCA-positive patient when they say “cells are just one step away from being cancer, so the risk is not lowered if any cells remain.” But this ignores our crystal ball for humans where we can see 3 or 4 cancers (max.) spread out over a 50-year time frame. (Yes, I know, we’ve all seen 5 or more simultaneous separate cancers in one breast, but this is a rare exception and raises questions if these are truly 5 different clones or, more likely, a pre-existing widespread DCIS, or intramammary lymphatic spread, or….but I digress.)

Pathology in the DMBA model is usually drawn from palpable tumors. But if you sample anywhere in the diffuse sheet of breast tissue, you’ll find wildly atypical cells that are lying in wait to emerge later. In contrast, when a BRCA-positive patient undergoes preventive mastectomy, most of the tissue is completely normal, maybe with too many lymphocytes in the lobules (perhaps keeping those billions of pre-malignant cells in check).

Yes, there is a fairly high rate of focal high-risk lesions in preventive mastectomy specimens, as well as 2-4% with invasive cancer in BRCA+ patients. But compare the reported pathology findings in BRCA-positive mastectomy specimens to the much-lower-risk preventive mastectomy patients, and you won’t find a great deal of difference in the incidence of ADH, ALH/LCIS, borderline lesions, or even occult DCIS (highly dependent on sampling technique, of course). A few articles even describe the BRCA patients with a lower incidence of high-risk lesions. But nothing compares to the DMBA model where it’s hard to find normal breast tissue anywhere. The point is that, in humans, there is a mismatch between the “molecular biologic field effect” that is present with either somatic mutations or germline mutations versus what emerges clinically. Therefore, we should rest our cases on clinical observations and not the DMBA model.

The primary benefit of the DMBA model is to test various hormonal strategies in the prevention and treatment of breast cancer, given that the malignant cells created are usually hormonally responsive. And this was why I had my team at the University of Oklahoma adopt the DMBA model, which is no easy feat when you’re starting from scratch (lab space, funding, animal care and protection regulations, handling of the highly carcinogenic DMBA, etc.). From this, we published on the prevention of rat mammary carcinoma with leuprolide compared to oophorectomy, then again with leuprolide compared to tamoxifen, plus we dabbled in experimental GnRH agonists and melatonin prevention as well.

The summer after the first year of medical school used to be wide open, and I made the decision to offer a (competitive) summer fellowship that was one-half clinical and one-half research. Funded by a small army of women that helped me at the time, the fellowship became very popular and was always filled with top students, such that we tried to take on more every year (we peaked at 4 students one summer). My purpose was not altogether altruistic, as I hoped to make an early impression on the impressionable, developing the future personnel who would fill the multidisciplinary clinical spots as well as the multidisciplinary research spots at OU. It worked well. In fact, our first student in the fellowship, Elizabeth Jett, subsequently became a breast radiologist and is currently the Director of the OU Breast Institute, where I had served as the founding medical director in 1993. One of my favorite photos from that era is Betsy holding a Sprague-Dawley rat while attempting a smile through her disgust.

But of all my prior students, it was poor James Banta, future ophthalmologist, who got caught in a hair-brained scheme I had at the time – laser photodynamic therapy (could we tag indocyanine green to cytokeratin?) to eradicate the residual breast tissue after mastectomy in the Sprague-Dawley rat (with human applications, of course). It seemed like the natural thing to do, that is, remove all the breast tissue you can surgically, then obliterate the remaining epithelium non-surgically. We were not total hacks when it came to the laser approach. We had the expertise and advice from Wei Chen, PhD who, 20 years later, would be awarded an R-01 grant from the NCI for his “laser photodynamic therapy with combined immunologic boost” approach in a variety of cancers. But in the poor Sprague-Dawley rats, it was just too much, even though we applied the laser to just one side of the animal (see photo below for back-of-the-envelope planning as we designed our study groups).

 

In spite of our best intentions, we had an unacceptable mortality rate, and our never-published paper that followed was a treatise on surgical technique used for preventive mastectomies in rats, coupled with an extensive discussion of aggressive post-op care of the animal to avoid mortality. Like rats fleeing a sinking hypothesis, we ended the laser project. And from that experience, an ophthalmologist was born.

Returning to the question at hand – what is the future breast cancer risk for patients who undergo bilateral preventive mastectomies for BRCA-positivity, or for that matter, any of the strong genetic predispositions? We already know short-term risk is dramatically reduced in several studies. And, longer-term risk is also apparently reduced, as evidenced by the Mayo Clinic data where the mastectomies were done many years ago, then BRCA tested later. Numbers are small (26 with BRCA mutations), but zero cancers occurred after a median follow-up of 13.4 years. Importantly, 90% of the women in the Mayo Clinic series had the old-fashioned “subcutaneous mastectomy” wherein more tissue was left behind than today’s iteration of “nipple-sparing mastectomy.” Perhaps, modern results will be even better. The meta-analysis of De Felice F, et al (Ann Surg Oncol 2015; 22:2876-2880) suggests a 93% relative risk reduction, although a number of caveats exist here (starting with the admitted possibility of some patients from different studies being counted twice).

Since we aren’t certain about lifetime risks (or even 20-year risks) after preventive mastectomies, how do we counsel patients as to future risk? While some are confident that no cancers will occur, and their patients are told “no need for imaging follow-up,” I am biased by a small group of patients in my care who underwent subcutaneous mastectomies (old school technique) many years prior to BRCA testing, then were found later to harbor the mutation (similar to the Mayo Clinic sub-group). Their preventive surgeries were performed on the basis of family history, but then later developed breast cancer in their skin flaps (with the longest interval between surgery and cancer being 37 years in a BRCA1 positive patient – surgery at 40, then triple-negative cancer arising beneath the skin flap at 77). I consider these patients to remain at lifelong risk (probably a linear risk), prompting my policy of ongoing imaging. Interestingly, none of the cancers in my patients arose beneath the salvaged nipple-areolar complex.

As we await better long-term data, I offer another example as to how I handle counseling: If we’re talking about a 40 year-old BRCA+ patient who has 40 more years of life expectancy, and whose strong family history places her at the high end of the wide range of risk (80%), and then she undergoes bilateral salpingo-oophorectomy, her risk will be reduced to an estimated 40% lifetime (50% relative reduction from an absolute 80% to 40%). This remaining risk can also be stated as “1% per year.” Then, for further risk reduction, she undergoes bilateral preventive mastectomies, which takes her from 40% to 4% (lifetime). In this case, the 90% relative risk reduction is applied to the absolute 40%, leaving 4% (or 0.1% per year).

If, however, someone is diagnosed with a BRCA mutation later in life, age 50, after the benefit of early-age BSO is lost with regard to breast cancer, then she has higher risk (though not the “full” 80% that she had at age 25). If she has a strong family history to support the mutation, then she has an approximate 60% remaining lifetime risk spread out over 30 years, or 2% per year. A 90% relative reduction of this 60% leaves her with a 6% lifetime risk of breast cancer after bilateral preventive mastectomies. This 6% is only slightly less than the average risk patient at the same age who has never undergone breast surgery. This is my mathematical justification for continued monitoring and screening in most gene-positive patients who have undergone preventive surgery. In reality, it’s a matter of logic pending data, using lower school math.

For a quick tutorial on the controversies surrounding our current attempts to project risk and counsel patients, let me recommend the invited editorial by David Euhus, MD (Ann Surg Oncol 2015; 22:2807-2809), written in response to the 2015 meta-analysis mentioned above.

And please, no more extracting DMBA data to apply to human preventive surgical approaches. Let the rats off the ship. We have enough clinical evidence to know that preventive surgery in humans lowers risk substantially. Although our data is largely short-term when compared to “lifetime,” there is no reason to believe that these dramatic reductions in risk are only temporary and that the genetically-primed cells are going to “catch up” later on, rendering surgery a waste of time. No, the most pressing data is going to be the impact of surgical prevention on survival. That data is, in fact, starting to trickle in, and things are looking good so far.