SLIDES & TRANSCRIPTS
Friday, December 13, 2002

Huggins Lecture

Prostate Disease/Urologic Oncology: Personal Experience and Random Thoughts in Half a Century of Involvement

John Grayhack, M.D.

Dr. Huggins was a native of Halifax, Nova Scotia. There he is as a young man. He attended and graduated from Harvard Medical School in 1924.

Following this, he took the go west, young man, go west advice seriously. He went to the University of Michigan to expand his surgical experience under the tutelage of Hugh Cabot.

Do any of you know who his roommate was at the University of Michigan? Well, it was Reed Nesbitt. He and Reed Nesbitt were roommates for two years. It was remarkable that two men who contributed so much to advancing the care of patients with urologic disease were actually roommates in the infancy of this specialty.

After a two year internship he became an instructor in surgery in 1926, and in 1927, he accepted an appointment to the surgical faculty at the University of Chicago. That's where I went to medical school, and that's where I ran into Dr. Huggins.

Dr. Dallas Femister, the head of surgery at the University of Chicago was at that time, assembling a unique group of men to constitute the surgical faculty at the new on campus hospital that was being developed. Each of these men had a special interest. This was in the mid-1920s.

Dr. Huggins became a professor of surgery in 1936, and essentially remained at the University of Chicago until his death in 1997. He had a remarkable ability to develop well-conceived and thorough laboratory studies that enhanced his understanding of clinical phenomena he saw as a urologist. His decision to alter the hormonal environment by castration or suppression of the testes was fostered by his knowledge of the effect of these manipulations on the prostate of the dog.

Perusal of three of his early publications gives an insight into his depth of understanding of, and careful, thoughtful approach to evaluating the responses of the patient in his cancer to castration and hormone therapy.

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This publication was publication number one under the title of, "Studies on Prostate Cancer: The Effect of Castration of Estrogen and Androgen Injection on Serum Phosphatase in Metastatic Carcinoma of the Prostate." It was published in 1941.

The observations by Gutman and Gutman, and Berenger and Woodard that patients with disseminated prostate carcinoma often exhibited increased serum acid phosphatase activity, provided an unusual opportunity to possibly use a secretory product to reflect tumor activity. Castration or administration of estrogen or testosterone was employed in groups of patients.

Serum levels of acid phosphatase activity fell with castration and estrogen administration, and was increased in the small group of previously castrated individuals who received testosterone propionate. Based on these observations, Drs. Huggins and Hodges concluded that prostate cancer was influenced by the level of androgenic activity in the body.

Furthermore, the observations of serum phosphatase supported the hypothesis that disseminated carcinoma of the prostate is inhibited by eliminating androgens by castration or estrogen. They also concluded that cancer of the prostate is activated by androgen injection. Essentially these observations were very important in establishing the potential role for appropriate serum markers in human tumor biology.

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Paper number two entitled, "Studies of Prostate Cancer and the Effects of Castration on Advanced Cancer of the Prostate," reported by Dr. Huggins with Drs. Stevens and Hodges was also published in 1941. In it, the authors formulated their famous syllogism, namely that in many instances, malignant prostate tumors are an overgrowth of adult epithelial cells.

All known types of adult prostatic epithelium undergoes atrophy when androgenic hormones are greatly reduced in amount or in activated. Then the syllogism slips a little bit. Therefore, they expected and found that all but 3 of the 23 patients, 15 of whom had x-ray evidence of metastatic disease improved noticeably with castration.

All patients gained from 3 to 18 kilograms in weight. Their appetites and red cell counts increased. In 9 of the 11 patients with severe pain, complete or nearly complete relief of pain was achieved and maintained. You can imagine the effect this had on the patients at the time. Some of them were bedridden, and barely able to eat or move.

The prostate underwent regression in size in all but one case. Serum phosphatase levels decreased in all but two cases. The improvement was greater than the authors had observed in any case in which far advanced or metastatic carcinoma was treated in any other way.

Dr. Huggins used to receive letters from these patients through the years as they survived, thanking him, and reminding him of what he had done for them.

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But the third paper, which was really my favorite, deserves your attention and is entitled, "The Prostatic Secretion," and was published by Dr. Huggins in the Harvey Lecture Series in 1947. It is a fascinating and informative document based primarily on the observations made in the prostatic fistula dogs. The dogs were subjected to hormone manipulation, including administration of gonadatropins, stilbestrol, testosterone, progesterone, and dihydrocorticosterone.

The effect of these manipulations on the pilocarpia induced quantity and composition of seminal fluid of these patients was assessed. The concentration of various anions and cations and prostatic fluid from the dog and man was reported. And remarkably, the proteolitic enzymes in the secreted dog fluid were studied extensively.

Dr. Huggins was obviously fascinated by the fact that the dog and man had differing seminal clotting and lysing mechanisms. But he chose to study the dog. After several studies he concluded that the principle proteolytic activity of the dog prostatic fluid, fibrinogenase resembled, but is not identical with trypsin.

Had he chosen to study human semen as thoroughly as he studied the dog prostatic fluid, he may well have identified PSA at that early point. Nevertheless, one cannot read this manuscript without being impressed by Dr. Huggins' powers of observation, and his determination to unravel the mechanisms involved in the phenomenon he had studied so carefully. If you want to get an insight into a Nobel Prize winner's work and investigative effort, this manuscript is worth reading.

Subsequently, Dr. Huggins utilized other therapeutic approaches to carcinoma of the prostate,

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and developed an interest in breast cancer and malignancy in general. He made contributions in these areas, but none to my knowledge, approaching his impact on prostate cancer itself, or the general field of cancer that these early efforts produced.

I knew Dr. Huggins, or Charlie as the students called him when he wasn't around. He was a challenging and interesting teacher, who had an actively participatory teaching technique that inspired some, and was not appreciated -- that means they were scared to death of him -- by others.

I found him provocative and stimulating, and believed him when he said that urology was the queen of sciences. It is my impression that he made a great change in the way at least most of us manage and think about cancer. He demonstrated the value of blood markers. He essentially provided significant evidence that the more you know about an organ and the cells that make it up, as well as those that control it, the better chance you have to manipulate the information to the advantage of your patient.

I remember well that he called me when I was a junior medical student in surgery and asked if I could come and help him come and do a circumcision. But I couldn't, because of the circumstances. However, the next time I saw him, he told me with his hand on my shoulder, young man, when opportunity knocks, grab it.

However, when I returned to Chicago, he was gracious and kind to me. He invited my wife and myself to his 85th birthday party. His memorial service following his death in 1997, was attended by scientists from all over the world. I'm certain that the picture of the field of urology that he painted played an unrecognized role in my request as a surgical assistant resident to be allowed to take my year in the laboratory with Bill Scott and Charles Teser.

There is one last thing about Dr. Huggins that deserves emphasis. He had a lasting impact on the practice of urology through his urologic offspring. He and Bill Scott were very different personalities, but both were committed to science and to scientific doctrine.

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If you look at this slide, which I put up with great trepidation, and acknowledge it immediately that many deserving people are omitted, I think you cannot help but be impressed by the contributions of the individuals listed that have made to the understanding and management of prostate cancer.

Some are like his grandchildren, and some are his great-grandchildren, if I figured it out right. But all of these, and I'm sure several others that he had trained have made the assessment and care of the patients with carcinoma of the prostate much better than it had been.

So, it is not enough to bake a good cake, unless you can get somebody to eat it. In all of those, and myself and many others have been eating the cake that they served for a long time. He and Bill Scott helped to set the table for many of us.

Now, for the remaining minutes that I have, I have decided --

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you'll be relieved by this -- not to talk about the topics listed on this slide, since you are going to talk about them in the rest of the meeting anyway, and I'll have a chance to learn from you.

So instead, I plan to discuss two projects that I am still pursuing. They both center on secretory proteins produced by male sex organs, and are in a sense, at least hypothesis-based. They are clustered around the identification, quantification, and selective physiological characterization of accessory secretory products of the testes and/or the prostate.

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I cannot overemphasize the value of the information of the information in this book by Thaddeus Mann. This is the second edition of the book. When the first edition came out when I was a resident, it was about three-eighths of an inch thick. And then ten years later, it is much thicker. But it is filled with useful information about the accessory sex glands, their secretions in all the animals that studies were known on. I know of no competitive book, even at this time. So I used it a great deal.

The first project was based on the hypothesis that secretary products of the prostate will have characteristic quantitative or qualitative changes preceding or associated with the development of malignancy in this gland.

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The assumption that biochemical changes characteristic of malignancy will be present in recoverable prostatic fluid is based on the substantial evidence that carcinoma of the prostate has a multifocal origin.

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This was an editorial that I wrote for Tom Stamey. On reviewing the information, I became convinced that carcinoma of the prostate was a field change, and that it was highly probably that the cells that have undergone this change, at least some of them, would have maintained their normal relationship to the prostatic ductal system, and that the secretions obtained from their prostate might well manifest some significant abnormalities.

In this effort, we concentrated on LDH isoenzyme ratios and the concentration of transferin and complement C3 and C4. Now, this was in the late 1970s and 1980s, and it produced some -- and there is the title.

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And there are the results with the C3.

So the sophistication of the determinations was not what it would be today. But the mean of the observed C3 concentration of prostatic fluid was 10 milligrams percent or over, in 80 percent of the patients with cancer; 4 percent of the patients with BPH, without evidence of inflammation; and 13 percent of the patients with BPH and inflammation.

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With regard to transferin, the mean concentration of patients with documented cancer exceeded the mean concentration of all other groups by two and a half times. The lowest observed transferin level was 30 milligrams or higher, in 66 percent of the patients; 2 percent of the patients with BPH without evidence of inflammation, and 4 percent of BPH patients with evidence of inflammation.

These were significant findings at that time, and tended to confirm the hypothesis that one could learn what was going on with the cancer or the organ by sampling prostatic fluid. We judged these observations to be of considerable interest, but not a practical clinical tool at that time.

Now, in this day and age, this needs to be revisited in the likelihood that we will find proteins we were unaware of with modern tools is high, and Dr. Wright, who is with Paul Schellhammer, or Paul Schellhammer is with him, wants some of our fluid, and is going to use it in his cell studies. And some others are interested in it, and we are again. We are struggling to make the promised specimens available, but I have limited help, and working in the cold room is not one of my great joys these days.

Lastly, I'm going to tell you about something we are doing at this time, and it's essentially where we stand in the efforts to identify a non-androgenic testes secretory protein that may impact on pathologic prostate growth.

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We are pursuing this because of the biological evidence that supports its presence. That's what we found, although you have seen the end already.

This is an old slide that combines two hormone-regulated characteristics of the seminal vesicle. One is seminal vesicle fructose, and the other is seminal vesicle weight. The study was done in about 200 seminal vesicles obtained at autopsy. Proper controls were instituted to make certain there was no significant change in the fluid content of the fructose.

Well, what it showed was that with time the fructose level went down, just as you would predict, with decreasing androgen levels. But the weight of the vesicle stayed the same. The weight of the vesicles is maintained by a spectrum of hormones and factors. As far as I know, the fructose level is maintained only by androgen.

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And then this is a study of prostatic fluid in men as the age. And again, one would expect that acid phosphatase would go down as the male hormone goes down, and it does. We didn't know what LDH would do, but since we could measure it, we measured it; it went down. But we were aware from animal studies that the citric acid concentration in the secretions is controlled by multiple factors, hormonal.

And sure enough, in the middle age group instead of this constant rate of decrease, we saw an increase, again, suggesting that something was stimulating the -- and most likely a hormonal factor --

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stimulating the production of the secretory product by the prostate, mainly citric acid.

So if you put all that together, and here is the seminal vesicle fluid, and the fructose concentration is down, the citric acid concentration is up. And if you look at the prostate, it is growing. So that tended to confirm one of our hypotheses.

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And then following this, a whole series of individuals did studies in animals, in addition to those that were just reported in man, that indicated that the testes produced a non-androgenic factor that would stimulate the male accessory sex organ growth. The first was Gene Wilson. He was bound and determined to produce BPH with androgen, but he couldn't do it unless they had testes, essentially. So in his statement he is the first one who had targeted the testes as a source of the secretion that was male prostate stimulating, but not androgen.

And then we radiated the testes. The Hopkins group took the testes away, and restored normal hormone levels. Every one of these studies in the dog support the fact that the testes was secreting something besides male hormone that affected the prostate. And then a series of studies in the rat -- Dalton and Darris are from our group, and Cook is from the University of Illinois -- also suggested that this was the case.

So the question is, how were we going to determine whether this hypothesis was correct, or had any merit or not? And it was very difficult. We tried many things. We aspirated the blood from the spermatic vein. We ground up testes and looked, and we had no chance.

And then we recognized that nature had provided us with a source of testicular secretions that is unparalleled really as far as we know in the animal world. And that is the spermatocele. So it has both testicular and epididimal secretions. And we chose it as a source of sampling secretory products of the testes.

And then because Jim Kozlawski spent time here, and could grow human prostate here, and because he was interested in the project, we tried both epithelial cells and stromal cells, and decided to concentrate our efforts as an assay tool on the human prostate stromal cell, because we were really targeting BPH, and it is our impression that the main controller of prostate size and so on is the stroma.

And so as a result of that -- and then we grew these cells in a media that was a basal as we could get. It had nothing in it essentially, but it was RPMI plus ITS plus, which is essentially just enough to keep a cell alive. And we gathered the spermatocele fluid and found that it stimulated stromal growth on every occasion. We never had a spermatocele fluid in which stimulation was not achieved.

Then we began to study the spermatocele fluid, and fractionated it by ultra filtration and stepwise ammonium sulphate precipitation. I didn't do this, so that's why I have to read it. I know about it. And Cepharosecolumn exchange chromatography.

And as we did that, each active fractions persisted. Eventually, we had a 2-D gel of the active fraction that contained eight commassie blue spots. And five of them could be sequenced. We had enough protein to sequence. Three of those were sequenced by normal sequencing mechanisms, and they were not of any interest. Two, 47 KD and 17 KD, protein or peptide, could not be sequenced.

Eventually, we contacted the people at the City of Hope a second time, and they at that point, carried out the MALDI-TOF mass spectrometry a second time, and we learned that the 47 KD protein was homologous with pigment epithelial derived factor.
In addition, it has a second very closely related serum protease that is called clad F.

Now, the first time we saw the report of pigment epithelial derived factor, we didn't know what it was. But then we looked into it, and it was discovered in the eye, and was sequenced at this institution. And it has neurotropic, anti-angiogenic, and a host of other activities that seem to make it almost chameleon-like in its abilities to alter tissue. So we got very interested in it.

I'm going to conclude here with what we found.

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We then used purified PEDF, and it did the same thing, except that it showed no definite relationship to dose.

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We did a Western blot, and could demonstrate on the spermatocele fluid, and it had a positive reaction with the PEDF antibody.

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Then we used two antibodies, one a polyclonal, and one a monoclonal, and added the spermatocele fluid. And the stromal stimulating effect of the fluid was abated.

IgG antibody did not reproduce this result.

So we concluded that PEDF is a prostate stromal stimulating protein that is secreted by the testes. We do not know whether it is the factor that we have been looking for as a non-androgenic prostate stimulating protein from the testes, but it certainly is a candidate.

And at this point, we are pursuing efforts to characterize its activity. And we also are pursuing efforts to identify other agents in the testicular secretions that might give us a clue about other agents that could play this role.

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So it's worthwhile knowing something about biology. And it's worthwhile struggling to get tissue specimens, and secretory specimens, because they add information you can't get any other way. And if there is any message that I have to give to this group, it's that you live in a very exciting world day after day. You are very busy, busier than anybody has ever been, because of all those forms you have to fill out.

And once in a year, or once in five years you will something you know shouldn't happen. And the thing is to say that's an unusual phenomenon, and put it aside, and think you'll do something about it. But don't put it aside. When you see something that shouldn't be, from what you know, then you should share it with other people, and you should pursue it until you understand it, because it may be that an experiment in nature has just been carried out that you could never even conceive, and never be allowed to do if you did conceive it.

So I envy you all as you are still young and pursuing your careers, and I wish you the best of luck, and thank you for having me today.

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