In Memoriam
Myron Melamed, 1927–2013 Zbigniew Darzynkiewicz,1* Louis Kamentsky,2 Elena Holden3
WITH deep sadness we learned that Myron Roy Melamed, known to his friends and colleagues as Mike, died on September 18 after a six and a half year battle against pancreatic cancer. His name is well-known among researchers in the fields of pathology, flow cytometry, and imaging cytometry and his contributions to these fields are many, varied, and impactful. Mike earned his MD from the University of Cincinnati in 1950 and followed it with residency and fellowship training that included internal medicine, hematology, pathology, and histochemistry at the University of Cincinnati, Duke University Hospital, Mount Sinai Hospital in New York, and Hammersmith Hospital in London. During the Korean War he served as a Captain in the Army Medical Corps. From 1979 to 1989 Mike was the Chairman of the Pathology Department at Memorial Sloan-Kettering Cancer Center and Professor of Pathology and Biology at Cornell University Medical College. From1991 to 2007 he was Chairman of the Department of Pathology at Westchester Medical Center in Valhalla, NY. His contribution to the field of cytometry is enormous. In 1965, Mike and his friend and colleague Louis (Lou) Kamentsky, who was then at the IBM Watson Laboratory at Columbia University published the seminal paper “Spectrophotometer: New Instrument for Ultra-rapid Cell Analysis” (18). The extremely fruitful collaboration between Mike and Lou, which continued into the current century, led to the development of the several generations of flow cytometers and subsequently to the laser scanning slide-based cytometer (LSC). Their work together is described in the continuing segment of this In Memoriam article by Lou himself. Mike’s interest in cytometry stems from his recognition of the importance of quantitative analysis of cellular attributes in pathology. He pioneered the development of numerous 1
In the Pantheon of Fathers of Cytometry: Mort Mendelsohn, Mike Melamed and Lou Kamentsky (ISAC Congress 1998)
applications of flow- and laser scanning- cytometry for clinical diagnosis and prognosis, particularly for many types of cancer. A perusal of his publications reveals numerous original and review articles, extensively cited, utilizing cytometry for analyses of clinical material as well as in basic research in cell biology (1–28). From historical perspective it is likely that Mike will be recognized as the world most renowned pathologist contributing towards developments in cytometry. Mike’s contribution comes also from his work in organizing and coordinating developments in the field of cytometry worldwide. He was the originator, Founding Member and one of the first Presidents of the International Society for Analytical Cytology, currently known as the International Society for Advancement of Cytometry (ISAC). Under the auspices of the Engineering Foundation Mike organized one of the first
Professor of Pathology, Medicine and Immunology/Microbiology, New York Medical College, Valhalla, NY Inventor of Cytofluorograf V (Biophysics Systems/Ortho Instruments), and of Laser Scanning Cytometer (CompuCyte Corporation), currently retired 3 Former President and CEO of CompuCyte Corporation, currently Chief of Strategic Scientific Marketing Life Sciences, Thorlabs Imaging Systems, Sterling, VA 2
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*Correspondence to: Z. Darzynkiewicz. E-mail:
[email protected] Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.22413 C 2013 International Society for Advancement of Cytometry V
Cytometry Part A 83A: 1047 1050, 2013
In Memoriam international cytometry congresses, in Portsmouth, New Hampshire. In 1979, together with Mortimer Mendelsohn and Paul Mullaney, he coedited the seminal monograph “Flow Cytometry and Sorting” (17), which became a “bible” for all researchers engaged in flow cytometry. The monograph rapidly sold-out and the second, enlarged edition (13) also became one of the most sought-after books on cytometry at that time. Apart from cytometry, Mike’s contributions in field of pathology were also widely recognized. He served as President of the American Society of Cytology, and the New York Pathology Society, and sat on the board of directors of the New York Transplant program. He coauthored a study, published in the New England Journal of Medicine in 1990 which confirmed for the first time the association of second-hand smoke and lung cancer (20). One of Mike’s recent contributions, coauthored with Leo Koss was the 5th edition of “Koss’ Diagnostic Cytology and Its Histopathologic Bases” is widely regarded as the most comprehensive and authoritative text in the field of pathology and clinical cytology (2). Mike Melamed received numerous awards and honors for his scientific research and teaching accomplishments including: the Papanicolaou Award of the American Society of Cytopathology, the Maurice Goldblatt Award of the International Society of Cytology, the Daniel Drake Award of the University of Cincinnati Medical School, and the Fred Stewart Award of the Memorial Sloan-Kettering Cancer Center for contributions in tumor pathology. He served on the Nobel Prize nominating committee for Medicine. I had the privilege and pleasure to work and collaborate with Mike from 1974 until 1990 at the Memorial SloanKettering Cancer Center, initially working in his laboratory, and then more independently as member of his Pathology Department. I will remember him as a very kind and helpful person. He was friendly and compassionate to all members of his laboratory, regardless of their position, from the technical personnel to graduate students, postdoctoral fellows and assistants. His knowledge of pathology and fascination in cytometry provided a fertile ground for creativity and productivity in research linking these disciplines. In his very busy work-day as an attending pathologist immersed in the daily chores of examining pathology specimens he was able to find time to drop into the research laboratory and inquire about the recent findings from the cytometric data. Frequently he would bring freshly examined tumor specimens for further analysis for DNA ploidy and other markers, by flow cytometry. Among the graduate students, and postdoctoral fellows working in his research laboratory were scientists who went to become renowned in their respective fields of interest: Frank Traganos in the area of cell cycle and development of new cytometry methods, Thomas (Tom) Sharpless in computer software development and Don Evenson in advancing male fertility assays. Don’s interests and his subsequent advances in the field of male fertility was an outcome of the seminal paper published when Don was still in Mike’s laboratory, describing the altered susceptibility of DNA to denaturation in abnormal sperm cells, as detected by acridine orange methodology (5). Lisa Staiano-Coico was also Mike’s graduate student. In the 1048
field of cytometry she is recognized as the past President of ISAC organizing the highly successful XXth ISAC Congress in Montpellier, France. Lisa’s distinguished academic career included positions of senior associate dean at Cornel University, provost of the Temple University and President of the City College of New York. Michael (Mike) Andreeff coming as the post-doctoral fellow from Germany, for a short period of time was also in Mike’s laboratory discovering importance of multiparametric analyses by flow cytometry for clinical diagnosis of hematological diseases (21). Mike Andreeff has a very distinguished career, and is currently holding a position of Professor of Medicine and Haas Chair in Genetics, Departments of Leukemia and Stem Cell Transplantation, and also as the Head of Flow Cytometry Core Facility, at the MD Anderson Cancer Center, Houston TX. Also passing through Mike’s laboratory was Marek Kimmel, a mathematician from Poland who just immigrated to the US. Their collaboration was extremely fruitful, leading to numerous publications in which Marek’s expertise in mathematics contributed to more comprehensive analysis of the data coming mostly from pathological samples (e.g., 12,26–28). Marek has also a distinguished career and is now holding a position of Professor of Statistics and Bioengineering at Rice University in Houston, TX. Wojciech (Voytek) Gorczyca was a resident in Mike’s Pathology Department at the New York Medical College. Voytek’c research accomplishments at the time of his residency included the development of one of the most widely used methods to identify apoptotic cells (the TUNEL assay) and application of this assay for pathological specimens (15,29,30). Subsequently Voytek became the Director of the Flow Cytometry Departments at Genzyme and then at the CSI Cancer Diagnosis Laboratories (Alpharetta, GA), and authored several extensive monographs including “Flow Cytometry in Neoplastic Hematology” (Informa), “Prognostic Markers in Hematologic Oncology” (Taylor & Francis) and “Atlas of Differential Diagnosis in Neoplastic Hematopathology (Informa). These books represent the dawn of modern pathology bridging classical pathology with flow and imaging cytometry. I am certain that it is the enthusiasm and devotion of Mike to science, in particular in applying cytometry in pathology and cell biology, as well as his leadership qualities that inspired his students to achieve such successes. I do consider Mike to have been my key mentor, the man who directed me towards cytometry and pathology. I owe most of my accomplishments in these fields to the support and the possibilities he granted to me. I will never forget my first steps in his laboratory in 1974 and my first experiments R , the state-of-the-art and analyses using the CytofluorographV cytofluometer developed by Lou Kamentsky and manufactured at his Bio/Physics System. With wonder, I was seeing for the first time the multiparametric measurements of thousands of cells per sample, completed in a matter of a few minutes. Frank Traganos was then an experienced user of this instrument. Together with Mike, Lawrence (Dick) Adams and Lou Kamentsky they already had published the first papers detailing the use of this flow cytometer (31–34). With Frank and Tom Sharpless we started to publish a series of papers expanding application of flow cytometry in different areas of In Memoriam
In Memoriam pathology and cell biology. The methodology for differential staining of DNA and RNA with the use of acridine orange was presented in the papers that received high citation rates (8,9). Another important study was conceived by Tom to measure electronic pulse height and width of cells passing in flow through the laser beam of the flow cytometer (35,36). The invention described in these papers, essential for identification and size measurement of single cells, is now widely used in nearly all types of flow cytometers. Mike was also my friend who helped me not only in my research but also was interested and strongly supportive in my personal matters. With much sentiment I can recall the warmhearted moments with him and his wife Barbara when we had time to be together outside the laboratory. Mike was always willing to share his wisdom advising me on different issues often stemmed from my lack of experience in the US as a fresh immigrant from Poland. Richard Dawkins, the well- known British evolutionary biologist and author in his book “The Selfish Gene” introduces the term “meme” defining a product of creativeness of the brain such as a new idea, a solution to a problem or a discovery. The memes propagate in the meme pool by leaping from brain to brain through generations; their inter-generational dissemination resembles that of promulgation of genes. The “immortality” of scientists is measured by the frequency and duration of the memes they create. As a mentor of so many accomplished scientists Mike was able to transmit a lot of his memes to other brains. His memes are also alive today transmitted worldwide through numerous highly regarded publications. His 25 most cited publications, all of them cited more than 200 times each, provide evidence of the extent of his memes (1–25). Although Mike will be sorely missed his loss is counterbalanced by the memes he leaves, which makes his passing less sorrowful. Zbigniew Darzynkiewicz
I would like to add some of my own thoughts to Zbigniew’s in remembrance of my close friend and mentor, Mike Melamed. This August Mike and I celebrated our 50th year of friendship and collaboration. I was at IBM’s Research Laboratory in August 1963 looking for something new to work on after completing a project to develop a computerized reading machine when I was introduced to Herbert Derman and asked to look at the possibility of computer-automating Pap smear slide screening. Herb in turn introduced me to Mike who convinced me that cancer screening was what I should indeed be working on. We began a conversation on cytometry that lasted 50 years. Mike continually made me aware of what is important to science and health care. He immediately understood that I needed to be educated and arranged for me to go to Memorial Sloan Kettering to attend cytology screening classes for a couple of weeks. Mike as always was positive, he never said or did anything to discourage achieving any scientific or business goal we might attempt. During my initial attempts to find a way to automatically distinguish cancer from normal cells Mike was always available with advice and whatever samples I might need to continue my Cytometry Part A 83A: 1047 1050, 2013
experiments. We tried first to scan stained cells to apply techniques I had used on image recognition, and quickly realized that technology was not up to the task. I became aware of the work of Torbj€ orn Caspersson at the Karolinska Institute and scanned photos of cells taken at ultraviolet wavelengths thinking a single low-data scan with an ultraviolet light source might make normal and cancer cells differentiable because of Caspersson’s finding of higher RNA content in cancer cells. We scanned some UV photos of cells, got an interesting result and Mike, Herb, and I submitted our first paper as coauthors to Science “Ultraviolet Absorption in Epidermal Cells” in December 1963 (37). Aware of the Coulter Counter, I realized that we did not need to scan cells if we could get the cells suspended in liquid to flow through focused light with one wavelength sensitive to RNA and normalize the first measurement with a second longer wavelength measuring total protein. I quickly built a two-wavelength flow cytometer using a microscope and various electronics. We began to study preparative techniques to maximize the resolution of the instrument in distinguishing different suspended cells from Mike’s as well as Herb Derman’s Kingston, NY labs, resulting in the Science paper by Mike, Herb and me, “Spectrophotometer: New Instrument for Ultrarapid Cell Analysis”, submitted in July 1965 (18). Although we knew about Richard Sweets ink-drop recorder invention, Mike and I decided to pursue fluidic sorting building on an existing IBM fluidic switching typewriter technology that could capture cells based on their optical properties and place them on a microscope slide. We were able to show that we could sort cells to generate slides containing highly enriched cancer cells using cervical cytology specimens and submitted our third Science paper, “Spectrophotometric Cell Sorter” in March 1967 (38). As we improved the instrument, adding more measurements, fluorescence, and a computer we coauthored more publications until I left IBM in 1968. Mike and I understood the future importance of flow cytometry to biomedical research and clinical medicine and I felt it best that I leave IBM and seek an academic appointment or find funding to start a company to commercialize the technology. Mike immediately agreed to become a Director of the new BioPhysics Systems. In order to obtain funding to continue, BioPhysics Systems was required to develop an instrument and perform a clinical test in a hospital within six months. We decided that the simplest application of the technology was to count, using two angles of scatter, live-versusdead leucocytes for cross-matching and with Mike’s help we were able to install an instrument in Ted Boyse’s lab at Memorial Sloan-Kettering and demonstrate that the instrument could match manual live/dead cell counts. The first scatter and fluorescence measuring instruments produced by BioPhysics Systems and subsequent versions as well as an El Bit microcomputer all went into Mike’s lab at Memorial Hospital to understand and improve the instruments capabilities, find applications, and provide publications. We were fortunate to have both Frank Traganos as the initial user and Zbigniew Darzynkiewicz later. Throughout the eight years until BioPhysics Systems became part of Johnson and Johnson’s Ortho Instruments Mike’s advice and encouragement was essential to determining the direction of the company’s development. 1049
In Memoriam When I left Johnson and Johnson in 1988, thinking that our original attempts to scan cells that was not feasible in 1963 was now possible, I started CompuCyte to develop a laser scanning cytometer (LSC). Of course, Mike agreed to be a Director of the new company and agreed to collaborate on studies using the LSC along with Frank and Zbigniew. I welcomed the renewed opportunity to have an excuse to be with Mike more frequently again and to benefit from his knowledge and judgment. Mike Melamed was a most extraordinary man. What was unusual about Mike was that he combined a warm, gentle, and caring nature with an extraordinarily brilliant understanding of and dedication to science and medicine. He used his gifts every day to help others. He never focused attention on himself or ever complained. I didn’t know the extent of his illness these past years because he never spoke of it. People speak of humility and lack of hubris but Mike embodied these characteristics as few others can. Louis Kamentsky
I first met Dr. Melamed upon joining CompuCyte in 1999. As a member of the Board of Directors he contributed greatly to the development of laser scanning cytometry technology providing his insight into molecular pathology and guiding us toward building the analysis platform used in research and clinical applications in cellular and tissue-based analysis. Through all the years that I knew Dr. Melamed he never wavered from the principles that he believed were right. He was a man of character, honor, courage, kindness, and with all his achievements a man of modesty. He talked to me often about the love for his family and how much joy they brought to his life. In his last days he remained involved in Compucyte affairs up until his last day. Mike, my dear friend, will be missed by many. I will always be proud and grateful to have known Dr. Melamed who will remain in my heart forever. God bless you Mike. Elena Holden
LITERATURE CITED 1. Cordon-Cardo C, O’Brien JP, Casals D, Rittman-Grauer L, Biedler JL, Melamed MR, Bertino JR. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites, Proc Natl Acad Sci USA 86; 895–698. (cited 1,506 times). 2. Koss’ Diagnostic Cytology and Its Histopathologic Bases, 2 Volumes, edited by LG Koss and MR Melamed, Lippincott Williams & Wilkins 2006. (cited 1,306 times). 3. Cordon-Cardo C, O’Brien JP, Boccia J, Casals D, Bertino JR, Melamed MR. Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 1990; 38:1277–1287. (cited 890 times). 4. Martini N, Melamed MR. Multiple primary lung cancers. J Thorac Cardiovasc Surg 1975;70:606–612. (cited 686 times). 5. Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science1980:1131–1133. (cited 573 times). 6. Houghton AN, Mintzer D, Cardon-Cardo C, Welt S, Fliegel B, Vadhan S, Carswell E, Melamed MR, Oettgen MR, Old LJ. Mouse monoclonal IgG3 antibody detecting GD3 ganglioside: A phase I trial in patients with malignant melanoma. Proc Natl Acad Sci USA 1985;82:1242–1248. (cited 566 times). 7. Melamed MR, Flehinger BJ, Zaman BM, Heelan RT, Perchick WA, MartiniN. Screening for early lung cancer. Results of the Memorial Sloan-Kettering study in New York. Chest 1984;86:44–53. (cited 538 times). 8. Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR. Lymphocyte stimulation: A rapid multiparameter analysis. Proc Natl Acad Sci USA 1976;73:2881–2884. (cited 451 times).
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9. Traganos F, Darzynkiewicz Z, Sharpless T, Melamed MR. Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. J Histochem Cytochem 1977;25:46–56. (cited 430 times). 10. Darzynkiewicz Z, Bedner E, Li X, Gorczyca W, Melamed MR. Laser scanning cytometry. A new instrumentation with many applications. Exp Cell Res 1999;249:1–12. (cited 378 times). 11. Darzynkiewicz Z, Traganos F, Kapuscinski J, Staiano-Coico L, Melamed MR. Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytometry 1984;5:355–363. (cited 362 times). 12. Flehinger BJ, Kimmel M, Melamed MR The effect of surgical treatment on survival from early lung cancer. Implications for screening. Chest 1992;102:1013–1018. (cited 348 times). 13. Melamed MR, Lindmo T, Mendelsohn ML. Flow Cytometry and Sorting. New York: Wiley; 1991. (cited 339 times). 14. Flehinger BJ, Melamed MR, Zaman MB, Heelan RT, Perchick WB, Martini N. Early lung cancer detection: Results of the initial (prevalence) radiologic and cytologic screening in the Memorial Sloan-Kettering study. Am Rev Respiratory Dis 1984;130: 555–560. (cited 311 times). 15. Gorczyca W, Bigman K, Mittelman A, Ahmed T, Gong J, Melamed MR, Darzynkiewicz Z. Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 1993,7:659–670. (cited 304 times). 16. Darzynkiewicz Z, Traganos F, Melamed MR. New cell cycle compartments identified by multiparameter flow cytometry. Cytometry 1980;1:98–108. (cited 301 times). 17. Melamed MR, Mullaney P, Mendelsohn ML. Flow Cytometry and Sorting. New York: Wiley; 1979. (cited 297 times). 18. Kamentsky LA, Melamed MR, Derman H. Spectrophotometer: New instrument for ultrarapid cell analysis. Science 1965;50:630–631. (cited 295 times). 19. Kim JH, Chu F. Woodard HQ, Melamed MR, Huvos A, Cantin J. Radiation-induced soft-tissue and bone sarcoma. Radiology 1978;129:501–508. (cited 294 times). 20. Janerich DT, Thompson WD, Varela LR, Geenwald P, Chorost S, Tucci C, Zaman MB, Melamed MR, Kiely M, MvKneally MF. Lung cancer and exposure to tobacco smoke in the household. N Engl J Med 1990;323:632–636. (cited 281 times). 21. Andreeff M, Darzynkiewicz Z, Sharpless TK, Clarkson BD, Melamed MR. Discrimination of human leukemia subtypes by flow cytometric analysis of cellular DNA and RNA. Blood 1980;55:282–293. (cited 270 times). 22. Fradet Y, Cordon-Cardo C, Thomson T, Daly ME, Whitmore WF Jr, Lloyd KO, Melamed MR, Old LJ. Cell surface antigens of human bladder cancer defined by mouse monoclonal antibodies. Proc Natl Acad Sci USA 1984;81:224–228. (cited 254 times). 23. Darzynkiewicz Z, Sharpless T, Staiano-Coico L, Melamed MR. Subcompartments of the G1 phase of cell cycle detected by flow cytometry. Proc Natl Acad Sci USA 1980: 77:6696–6700. (cited 253 times). 24. Bedner E, Li X, Gorczyca W, Melamed MR, Darzynkiewicz Z. Analysis of apoptosis by laser scanning cytometry. Cytometry 1999;35:181–195. (cited 248 times). 25. Herr HW. Laudone VP, Badalament RA, Oettgen HF, Sogani PC, Freedman BD, Melamed MR, Whitmore WF Jr. Bacillus Calmette-Guerin therapy alters the progression of superficial bladder cancer. J Clin Oncol 1988;6:1450–1455. (cited 247 times). 26. Flehinger BJ, Kimmel M, Melamed MR. Natural history of adenocarcinoma-large cell carcinoma in the lung: Conclusions from screening programs in New York and Baltimore. J Natl Cancer Inst 1988;80:337–344. 27. Myc A, DeAngelis P, Kimmel M, Melamed MR, Darzynkiewicz Z. Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle. Exp Cell Res 1990;192:198–202. 28. Enker WE, Kimmel M, Cibas ES, Cranor ML, Melamed MR. DNA/RNA content and proliferative fractions of colorectal carcinomas: A five-year prospective study relating flow cytometry to survival. J Natl Cancer Inst 1991 May 15;83:701–707. 29. Gorczyca W, Gong J, Darzynkiewicz. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 1993;53:1945–1951. (cited 887 times). 30. Gorczyca W, Gong JP, Ardelt B, Traganos F, Darzynkiewicz Z. The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res 1993;53:3186–3192. (cited 452 times). 31. Melamed MR, Adams LR, Traganos F, Zimring A, Kamentsky LA. Acridine orange metachromasia for characterization of leukocytes in leukemia, lymphoma and other neoplasms. Cancer 1972;29:1361–1368. 32. Traganos F, Adams LR, Kamentsky LA and Melamed MR. Critical effect of dye concentration on acridine orange fluorescence of fixed thymocytes. Acta Cytol 1972;16: 281–283. 33. Melamed MR, Adams LA, Traganos F, Kamentsky LA. Initial observations on instrumental differential blood leukocyte counts during chemotherapy of patients with leukemia. Eur J Cancer 1973;9:181–184. 34. Melamed MR, Adams LR, Traganos F, Kamentsky LA. Blood granulocyte staining with acridine orange. Change with infection. J. Histochem Cytochem 1974;22:526–530. 35. Sharpless T, Traganos F, Darzynkiewicz Z, Melamed MR. Flow cytometry: Discrimination between single cells and cell aggregates by direct size measurement. Acta Cytol 1075;19:577–581. 36. Sharpless TK, Melamed MR. Estimation of cell size from pulse shape in flow cytometry. J Histochem Cytochem 1976;24:257–264. 37. Kamentsky LA, Derman H, Melamed MR. Ultraviolet absorption in epidermoid cancer cells. Science 1963;142:1580–1583. 38. Kamentsky LA, Melamed MR. Spectrometric cell sorter. Science 1967;156:1364–1365.
In Memoriam
