Τhe Official Journal of the Hellenic Senologic Society

MastologiaJuly - December 2015 Isuue 2, Volume 1 ISSN 1790 - 3963

Review An old pub, a brewery, some seafood, Noble prize for Physiology and Medicine and the treatment of breast cancer. An evolving tale  Breast Tomosynthesis - 3D Mammography  Psychosocial support for women with breast cancer Hereditary Breast and Ovarian Cancer BRCA 1&2 clinical article An overview of in situ carcinoma of the breast. The role of ultrasound with presentation of multiple cases A case from the Department of Mammography Screening Paderborn, Germany review Breast Cancer Treatment

An old pub, a brewery, some seafood, Noble prize for Physiology and Medicine and the treatment of breast cancer An evolving tale

Konstantinos T. Papazisis and Loukas F. Kontovinis “Euromedica” General Clinic of Thessaloniki, 54645, Thessaloniki, Greece www.oncomedicare.com

ABSTRACTabstract

Years of research in cell cycle, connected yeast scientists and cancer research. The elucidation of cell cycle control machinery led to the discovery of a new pharmacological category, cyclin-dependent kinase inhibitors (CDK inhibitors). This review is discussing the process that led yeast researchers to Nobel Prize winning and introduced a breast cancer treatment breakthrough.

Scientists, beer and seafood their discoveries concerning the molecular structure of Just some yards away from King’s College, Cambridge, nucleic acids and its significance for information transfer on the north side of Bene’s street there is an old pub, in living material”2. with the familiar name “The Eagle”, running since 1667. Some year later, in 1970, Lee Hartwell discovered the The pub was always a popular destination for a lunch cell division cycle (CDC) genes in baker’s (or brewer’s) break for scientists and workers nearby, as well as for a yeast (Saccharomyces cerevisiae)3, a system often used refreshing pint of beer after work. It was on such an oc- for studying cell division processes and for making beer. casion when on the 28th of February 1953 Francis Crick in- He cloned several genes, amongst them cell-division cy- terrupted lunch to announce that he and James Watson cle gene 28 (CDC28) and he help in the introduction of had discovered “the secret of life”, after they had come the concept of “cell cycle”, a carefully structured pro- up with the proposed structure of DNA1. The instance is cess that follows one direction, driving cells first to du- now commemorated with a blue plaque next to the en- plicate their genetic information (what we know as S trance and the pub serves ”Eagle’s DNA” ale… Some phase) and then divide (mitosis or M phase). He later in- years later, on 1962, both scientists shared with Maurice troduced the concept of cell cycle “checkpoints”4, where Wilkins the Nobel Prize in Physiology or Medicine “for DNA and the other cellular biochemistry is inspected, to

Corresponding author Konstantinos T. Papazisis E - mail: [email protected]

12 July - December 2015 | Issue 2 | Volume 1 Figure 1. Cell cycle is controlled by cyclins (c-D, c-E, c-A and c-B) and cyclin- dependent kinases (CDKs) that are asso- ciated in different phases of the cycle to orchestrate the biochemical processes for cellular proliferation

make sure everything is in order before going onwards pressed in cycling cells, but it was disappearing every time to the next step. the cells divided9. He went on to clone it and he found In those times of long hair, flared trousers and disco that the same molecule could work across species in the music, a young scientist from northern London, Paul frog eggs (Xenopus spp)10 where Marc Kirschner had al- Nurse, working at Murdoch Mitchinson’s laboratory at ready shown that the activity of MPF (maturation or mi- the University of Edinburgh, identified the cdc2 gene in tosis-promoting factor) was oscillating in a similar way11. It the fission yeast (Schizosaccharomyces pombe), a spe- was finally shown that MPF is a cyclin-CDK complex where cies used in traditional brewing5. As he had already spent the activity and the specificity of the kinase is regulated a year working at a local Guinness brewery6, one cannot (amongst other biological processes) by the presence of help but notice the influence his previous work may have the escorting cyclin12. As cyclins are destroyed before mi- had in his later career. He subsequently found that cdc2 tosis, the complex is no longer active and cyclins need to was the same as CDC28 and he discovered that it was be synthesized again for the whole process to re-initiate. transcribing for a serine/threonine kinase7 and cloned The Nobel Prize in Physiology or Medicine 2001 was a human homologue8, later known as CDK-1 (Cyclin-De- awarded jointly to Leland H. Hartwell, Tim Hunt and Sir pendent Kinase-1). The discovery that elements of cell cy- Paul M. Nurse “for their discoveries of key regulators of cle machinery were highly conserved between species as the cell cycle”13. Cell cycle research was largely expand- yeast and humans, with evolutionary distance of millions ed in the 1990s (publications with the tag “cell cycle” in- of years, helped scientists to further explore this com- creased by a factor of x60 between 1980s and 1990s) and plex phenomenon and boomed the research that led to the two British scientists Paul Nurse and Tim Hunt were our understanding of cell cycle. knighted in 1999 in 2006. Meanwhile, in 1982 Tim Hunt was teaching summer courses at Woods Hole Marine Biological Laboratory Cell cycle regulation (close to Massachusetts cost) and was working on the and G1 checkpoint control of protein synthesis in sea urchin eggs (a delicacy In a simplified version, mammalian cell cycle is the or- in many Mediterranean cuisines), when he noticed some- dered sequence of two important events: DNA synthe- thing very unusual: a protein, that he named cyclin, was sis and mitosis. A cell should not enter mitosis before barely if at all detectable in the unfertilized egg, was ex- DNA is correctly duplicated and should not be allowed

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Figure 2. G1 checkpoint. Positive and negative proliferative signals converge for the cyclin-D and CDK4/6 activation

to duplicate its DNA before it has successfully divid- ochemical pathways that interact to provide the sig- ed. Cyclins and Cyclin-Dependent Kinases help in con- nal for proliferation17. Growth factors, oncogenes, nu- trolling this process. In each phase of cell cycle a dif- tritional sufficiency, nearby cells (what we call “contact ferent cyclin escortes the specific CDK to promote the inhibition”) and inhibitory signals, all converge into the phosphorylation of the targets that needed to be acti- G1 checkpoint and specifically into the activation of Cy- vated in order of cell cycle to progress (Figure 1). The clin D-CDK4/6 complex. Once activated the complex picture is much more complicated as there are negative phosphorylates the tumor suppressor retinoblastoma regulators of the cyclin-dependent kinases (the INK4 protein (RB) that was inhibiting a crucial transcription family p16, p15, p18 and p1914 as well as p21, p27, etc.15) factor E2F. E2F is then released and increases transcrip- and enzymes (mostly kinases and phosphatases) that tion of several gens involved in cDNA synthesis, cell cy- activate or inhibit the cyclin/CDK complexes. Finally, in cle progress etc (Figure 2). By all means, this is a one-way metaphase, anaphase-promoting-complex (APC), an E3 decision. Once a cell passes the G1 checkpoint is destined ubiquitin ligase, that targets S and M-phase cyclins for to complete a whole cycle and divide or die. This is why degradation in the proteasome16, thus resetting the cell G1 checkpoint is such an attractive target for cancer cycle clock to start again in the G1 phase.Once enter- treatment. All cancers have lost the control of cellular ing the G1 phase cell faces a crucial dilemma: should it proliferation (e.g. all cancers have a deficiency targeting go on and prepare for another cycle (duplicate DNA - the G1 checkpoint)18 and restoring G1 checkpoint could divide, etc.) or should it rest, in what we call G0 phase lead to long-term control of cancer growth19. Thus, the (biochemically a G1 phase but with no “will to divide”, CDK4/6 inhibitors became the Holy Grail for drug devel- a status that many of our mature differentiated cells opment any many pharmaceutical companies entered have adopted)? What we call G1 checkpoint is all the bi- the race that started almost 20 years ago.

14 July - December 2015 | Issue 2 | Volume 1 An evolving tale: Breast Cancer Treatment

G1 checkpoint, cyclin D placebo and fulvestrant (pre- and peri-menopausal pa- and breast cancer treatment tients received goserelin as well). Almost 60% of the pa- Proliferation is an indication of aggressiveness in hor- tients enrolled had visceral metastases and only 25% re- mone receptor-positive breast cancer. In fact, 5 out of ceived treatment as first line. The results showed again the 16 genes analyzed by the Oncotype DX assay are a statistically significant prolongation of disease-free proliferation genes (Ki67, Cyclin B1, Survivin, MYBL2 survival from 3.8 months for the fulvestrant/placebo and STK15) are proliferation genes20. Cyclin D is overex- group to 9.2 months for the palbociclib plus fulvestrant pressed in breast cancer21,22, correlates with poor pro- group (HR 0.42, 95% CI 0.32-0.56, p<0.001). Palbociclib gression in hormone receptor-positive disease23 and its did not increase objective responses (from 6.3% with ful- expression has been associated to shorter relapse-free vestrant/placebo to 10.4% with the combination, p=0.16) and overall survival in women treated with tamoxifen and overall survival data have not yet been reported. in the adjuvant setting24,25. Cyclin D is therefore an at- The combination had an excellent tolerability profile, tractive therapeutic target for hormone receptor-pos- with neutropenia and leukopenia as the most common itive breast cancer26,27. Targeting protein-protein inter- grade 3 or 4 adverse events (62.0% and 25.2%, respec- actions (as cyclins interact with CDKs) is a very difficult tively). Febrile neutropenia was extremely rare (0.6%). task for drug development. Research has focused in- On February 3, 2015, the U. S. Food and Drug Administra- stead on the development of small molecules that mim- tion (FDA) granted accelerated approval to palbociclib ic the ATP-binding pocket of kinases and thus inhibit (Ibrance, Pfizer) for use in combination with letrozole the enzymatic activity of the kinase. However, CDK4/6 for the treatment of postmenopausal women with es- (the CDKs that partner cyclin D at the G1 checkpoint) trogen receptor (ER)-positive, human epidermal growth has been proven a difficult target and only recently factor receptor 2 (HER2)-negative advanced breast can- small molecules that inhibit CDK4/6 were developed. cer as initial endocrine-based therapy for their metastat- Palbociclib (formerly PD-0332991) is an orally ac- ic disease35. The drug is currently being validated by EMA tive potent and highly selective inhibitor of CDK4 and (European Medicines Agency). CDK6 kinases28,29. Palbociclib preferentially inhibits hor- Ribociclib (LEE011) is another CDK4/6 inhibitor devel- mone receptor-positive human breast cancer cells and oped by Novartis and tested in several malignancies36. has a synergistic effect with tamoxifen in resistant cell A phase I study showed an acceptable toxicity profile lines30,31. Inhibition of CDK4/6 with palbociclib leads to and preliminary signs of clinical activity37 and a phase suppression of a number of downstream genes required Ib/II study showed similar signs in combination with for proliferation including cyclin A and CDK1, a stable letrozole for estrogen receptor-positive breast cancer cell cycle arrest and induction of cellular senescence32. patients38. A phase 3 study in postmenopausal women A randomized phase 2 study (PALOMA-1/TRIO-18), as- with hormone receptor-positive and HER2-negative dis- sessing the safety and efficacy of palbociclib in combi- ease and no prior therapy for advanced breast cancer nation with letrozole as first-line treatment in women has recently completed accrual. Several other studies with advanced breast cancer, was recently reported33. (in the neoadjuvant setting, in premenopausal women, The study showed a statistically and clinically significant or in combination with PI3K or mTor inhibitors, etc.) are prolongation of disease-free survival from 10.2 months underway39. for the letrozole alone group to 20.2 months for the pal- Finally, abemaciclib (LY2835219) another selective bociclib plus letrozole group (HR 0.488, 95% CI 0.319- CDK4/6 inhibitor developed by Eli Lilly showed again 0.748), but no difference in overall survival (from 33.3 acceptable safety and evidence of clinical activity in pre- to 37.5 months, HR 0.813, 95% CI 0.492-1.345). The suc- treated patients with multiple tumor types40. A Phase cessful phase 2 study was followed by a large phase 3 2 trial, MONARCH 1, is studying abemaciclib’s impact as randomized study (PALOMA-3) in patients that had re- a monotherapy in women with hormone receptor-posi- lapsed or progressed during prior endocrine therapy34. tive, HER2-negative metastatic breast cancer. Abemac- PALOMA-3 randomized 521 patients with advanced hor- iclib is been tested in large phase 3 studies in combi- mone receptor-positive (and HER2-negative) disease, in nation with fulvestrant (MONARCH-2) or aromatase a 2:1 ratio, to receive either palbociclib and fulvestrant or inhibitors (MONARCH-3)41. Recently FDA handed Lilly

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the coveted “breakthrough therapy” designation for form of cancer in women. As we succeed in prolonging abemaciclib. life span and quality, we cannot avoid thinking Sir Paul Nurse’s comment in his biography, when he moved his Conclusions research to ICRF (Imperial Cancer Research Fund) at A fine line connecting the process of beer fermenta- Lincoln’s Inn Fields, London: “Perhaps, not unreason- tion, the tasteful sea urchins and very hard experimen- ably, some wondered what a yeast researcher was do- tal work, has elucidated the cell cycle control machin- ing in a cancer research institute”6… ery and identified several of its important components as potential anticancer targets. Though it seems a long Conflict of interests time, 20 years later we are able to bring the first of The authors declare that they have no commercial compet- these drugs to fight breast cancer, the most common ing interests or financial support for this study.

references

1. Molecular structure of nucleic acids; a structure for deox- Kirschner M. J Cell Biol. 1984 Apr;98(4): 1247-55. yribose nucleic acid. Watson JD, Crick FH. Nature. 1953 Apr 12. The role of cyclin synthesis and degradation in the control of 25; 171(4356): 737-8. maturation promoting factor activity. Murray AW, Solomon 2. http://www.nobelprize.org/nobel_prizes/medicine/ MJ, Kirschner MW. Nature. 1989 May 25;339(6222):280-6 laureates/1962/ 13. http://www.nobelprize.org/nobel_prizes/medicine/ 3. Genetic control of the cell-division cycle in yeast. I. Detec- laureates/2001/ tion of mutants. Hartwell LH, Culotti J, Reid B. Proc Natl 14. INK4 proteins, a family of mammalian CDK inhibitors with Acad Sci U S A. 1970 Jun;66(2):352-9. novel biological functions. Cánepa ET, Scassa ME, Ceruti JM, 4. Cell cycle arrest of cdc mutants and specificity of the Marazita MC, Carcagno AL, Sirkin PF, Ogara MF. IUBMB. RAD9 checkpoint. Weinert TA, Hartwell LH. Genetics. 1993 Life. 2007 Jul;59(7):419-26. May;134(1):63-80. 15. 15 Control of the cell cycle and apoptosis. Lundberg AS, 5. Genetic control of the cell division cycle in the fission Weinberg RA. Eur J Cancer. 1999.Dec;35(14):1886-94 yeast Schizosaccharomyces pombe. Nurse P, Thuriaux P, 16. Identification of subunits of the anaphase-promoting com- Nasmyth K. Mol Gen Genet. 1976 Jul 23;146(2): 167-78 plex of Saccharomyces cerevisiae. Zachariae W, Shin TH, 6. http://www.nobelprize.org/nobel_prizes/medicine/laure- Galova M, Obermaier B, Nasmyth K. Science. 1996 Nov ates/2001/nurse-bio.html 15;274(5290):1201-4 7. Cell. 1986 Apr 25;45(2):261-8. The cell cycle control gene 17. The G1/S cell-cycle checkpoint in eukaryotic cells. Peeper DS, cdc2+ of fission yeast encodes a protein kinase potentially van der Eb AJ, Zantema A. Biochim Biophys Acta. 1994 Dec regulated by phosphorylation. Simanis V, Nurse P. 30;1198(2-3):215-30. 8. Complementation used to clone a human homologue of the 18. Cell cycle checkpoints and their inactivation in human can- fission yeast cell cycle control gene cdc2. Lee MG, Nurse P. cer. Molinari M. Cell Prolif. 2000 Oct;33(5):261-74 Nature. 1987 May 7-13;327(6117):31-5 19. Cell cycle regulation in the G1 phase: a promising target for 9. Cyclin: a protein specified by maternal mRNA in sea urchin the development of new chemotherapeutic anticancer eggs that is destroyed at each cleavage division. Evans agents. Owa T, Yoshino H, Yoshimatsu K, Nagasu T. Curr T, Rosenthal ET, Youngblom J, Distel D, Hunt T. Cell. 1983 Med Chem. 2001 Oct;8(12):1487-503. Jun;33(2):389-96. 20. A population-based study of tumor gene expression and 10. Molecular cloning and characterization of the mRNA for risk of breast cancer death among lymph node-negative pa- cyclin from sea urchin eggs. Pines J, Hunt T. EMBO J. 1987 tients. Habel LA, Shak S, Jacobs MK, Capra A, Alexander C, Oct;6(10):2987-95. Pho M, Baker J, Walker M, Watson D, Hackett J, Blick NT, 11. Cell cycle dynamics of an M-phase-specific cytoplasmic fac- Greenberg D, Fehrenbacher L, Langholz B, Quesenberry CP. tor in Xenopus laevis oocytes and eggs. Gerhart J, Wu M, Breast Cancer Res. 2006;8(3):R25.

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21. Expression and amplification of cyclin genes in hu- 31. CDK inhibitors as potential breast cancer therapeutics: new man breast cancer. Buckley MF, Sweeney KJ, Hamil- evidence for enhanced efficacy in ER+ disease. Sutherland ton JA, Sini RL, Manning DL, Nicholson RI, deFazio A, RL, Musgrove EA. Breast Cancer Res. 2009;11(6):112 Watts CK, Musgrove EA, Sutherland RL. Oncogene. 1993 32. Therapeutically activating RB: reestablishing cell cycle con- Aug;8(8):2127-33. trol in endocrine therapy-resistant breast cancer. Thangav- 22. Amplification and overexpression of cyclin D1 in breast el C, Dean JL, Ertel A, Knudsen KE, Aldaz CM, Witkiewicz cancer detected by immunohistochemical staining. Gillett AK, Clarke R, Knudsen ES. Endocr Relat Cancer. 2011 Apr C, Fantl V, Smith R, Fisher C, Bartek J, Dickson C, Barnes D, 28;18(3):333-45. Peters G. Cancer Res. 1994 Apr 1;54(7):1812-7 33. The cyclin-dependent kinase 4/6 inhibitor palbociclib in com- 23. Overexpression of cyclin D1 messenger RNA predicts for bination with letrozole versus letrozole alone as first-line poor prognosis in estrogen receptor-positive breast can- treatment of oestrogen receptor-positive, HER2-negative, cer. Kenny FS, Hui R, Musgrove EA, Gee JM, Blamey RW, advanced breast cancer (PALOMA-1/TRIO-18): a randomised Nicholson RI, Sutherland RL, Robertson JF. Clin Cancer phase 2 study. Finn RS, Crown JP, Lang I, Boer K, Bondaren- Res. 1999 Aug;5(8):2069-76. ko IM, Kulyk SO, Ettl J, Patel R, Pinter T, Schmidt M, Shparyk 24. Cyclin D1 expression in breast cancer patients receiving Y, Thummala AR, Voytko NL, Fowst C, Huang X, Kim ST, Ran- adjuvant tamoxifen-based therapy. Rudas M, Lehnert dolph S, Slamon DJ. Lancet Oncol. 2015 Jan;16(1):25-35. M, Huynh A, Jakesz R, Singer C, Lax S, Schippinger W, 34. Palbociclib in Hormone-Receptor-Positive Advanced Breast Dietze O, Greil R, Stiglbauer W, Kwasny W, Grill R, Stier- Cancer. Turner NC, Ro J, André F, Loi S, Verma S, Iwata H, er M, Gnant MF, Filipits M; Austrian Breast and Colorec- Harbeck N, Loibl S, Huang Bartlett C, Zhang K, Giorgetti C, tal Cancer Study Group. Clin Cancer Res. 2008 Mar Randolph S, Koehler M, Cristofanilli M; PALOMA3 Study 15;14(6):1767-74 Group. N Engl J Med. 2015 Jul 16;373(3):209-19. 25. Adverse effect of adjuvant tamoxifen in premenopausal 35. http://www.fda.gov/Drugs/InformationOnDrugs/Approved- breast cancer with cyclin D1 gene amplification. Jirström Drugs/ucm432886.htm K, Stendahl M, Rydén L, Kronblad A, Bendahl PO, Stål O, 36. Dual CDK4/CDK6 inhibition induces cell-cycle arrest and se- Landberg G. Cancer Res. 2005 Sep 1;65(17):8009-16. nescence in neuroblastoma. Rader J, Russell MR, Hart LS, 26. Cyclin D as a therapeutic target in cancer. Musgrove EA, Nakazawa MS, Belcastro LT, Martinez D, Li Y, Carpenter EL, Caldon CE, Barraclough J, Stone A, Sutherland RL. Nat Rev Attiyeh EF, Diskin SJ, Kim S, Parasuraman S, Caponigro G, Cancer. 2011 Jul 7;11(8):558-72 Schnepp RW, Wood AC, Pawel B, Cole KA, Maris JM. Clin Can- 27. Cyclin D1 in breast cancer pathogenesis. Arnold A, Papan- cer Res. 2013 Nov 15;19(22):6173-82. ikolaou A. J Clin Oncol. 2005 Jun 20;23(18):4215-24. 37. A phase I study of the single-agent CDK4/6 inhibitor LEE011 28. Specific inhibition of cyclin-dependent kinase 4/6 by PD in pts with advanced solid tumors and lymphomas. Infante 0332991 and associated antitumor activity in human tu- JR, Shapiro G, Witteveen P, Gerecitano JF, Ribrag V,Chugh mor xenografts. Fry DW, Harvey PJ, Keller PR, Elliott R, Issa I, Chakraborty A, Matanao A, Zhao X, Parasuraman S, WL, Meade M, Trachet E, Albassam M, Zheng X, Leop- Cassier P. J Clin Oncol 32:5s, 2014 (suppl; abstr 2528^). old WR, Pryer NK, Toogood PL. Mol Cancer Ther. 2004 38. Phase Ib/II study of LEE011 and BYL719 and letrozole in ER+, Nov;3(11):1427-38. HER2– breast cancer: Safety, preliminary efficacy and -mo 29. Discovery of a potent and selective inhibitor of cyclin-de- lecular analysis. Juric D, Hamilton E, Estevez LG, De Boer RH, pendent kinase 4/6. Toogood PL, Harvey PJ, Repine JT, Mayer I, Campone M, Asano S, Bhansali S, Zhang V, Hewes Sheehan DJ, VanderWel SN, Zhou H, Keller PR, McNama- B, Munster P. SABCS 2014; abstr P5-19-24 ra DJ, Sherry D, Zhu T, Brodfuehrer J, Choi C, Barvian MR, 39. www.clinicaltrials.gov (NCT01958021, NCT02422615, Fry DW. J Med Chem. 2005 Apr 7;48(7):2388-406. NCT02278120) 30. PD 0332991, a selective cyclin D kinase 4/6 inhibitor, pref- 40. Shapiro G, Rosen LS, Tolcher AW, et al. A first-in-human erentially inhibits proliferation of luminal estrogen re- phase 1 study of the CDK4/6 inhibitor, LY2835219, for pa- ceptor-positive human breast cancer cell lines in vitro. tients with advanced cancer (abstract 2500). J Clin Oncol Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai 2013; 31(suppl). AJ, Ginther C, Atefi M, Chen I, Fowst C, Los G, Slamon DJ. 41. www.clinicaltrials.gov (NCT02102490, NCT02107703, Breast Cancer Res. 2009;11(5):R77. NCT02246621).

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