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1.  Reliability of an e-PRO Tool of EORTC QLQ-C30 for Measurement of Health-Related Quality of Life in Patients With Breast Cancer: Prospective Randomized Trial 
Background
Breast cancer represents the most common malignant disease in women worldwide. As currently systematic palliative treatment only has a limited effect on survival rates, the concept of health-related quality of life (HRQoL) is gaining more and more importance in the therapy setting of metastatic breast cancer. One of the major patient-reported outcomes (PROs) for measuring HRQoL in patients with breast cancer is provided by the European Organization for Research and Treatment of Cancer (EORTC). Currently, paper-based surveys still predominate, as only a few reliable and validated electronic-based questionnaires are available. Facing the possibilities associated with evolving digitalization in medicine, validation of electronic versions of well-established PRO is essential in order to contribute to comprehensive and holistic oncological care and to ensure high quality in cancer research.
Objective
The aim of this study was to analyze the reliability of a tablet-based measuring application for EORTC QLQ-C30 in German language in patients with adjuvant and (curative) metastatic breast cancer.
Methods
Paper- and tablet-based questionnaires were completed by a total of 106 female patients with adjuvant and metastatic breast cancer recruited as part of the e-PROCOM study. All patients were required to complete the electronic- (e-PRO) and paper-based versions of the HRQoL EORTC QLQ-C30 questionnaire. A frequency analysis was performed to determine descriptive sociodemographic characteristics. Both dimensions of reliability (parallel forms reliability [Wilcoxon test] and test of internal consistency [Spearman rho and agreement rates for single items, Pearson correlation and Kendall tau for each scale]) were analyzed.
Results
High correlations were shown for both dimensions of reliability (parallel forms reliability and internal consistency) in the patient’s response behavior between paper- and electronic-based questionnaires. Regarding the test of parallel forms reliability, no significant differences were found in 27 of 30 single items and in 14 of 15 scales, whereas a statistically significant correlation in the test of consistency was found in all 30 single items and all 15 scales.
Conclusions
The evaluated e-PRO version of the EORTC QLQ-C30 is reliable for patients with both adjuvant and metastatic breast cancer, showing a high correlation in almost all questions (and in many scales). Thus, we conclude that the validated paper-based PRO assessment and the e-PRO tool are equally valid. However, the reliability should also be analyzed in other prospective trials to ensure that usability is reliable in all patient groups.
Trial Registration
ClinicalTrials.gov NCT03132506; https://clinicaltrials.gov/ct2/show/NCT03132506 (Archived by WebCite at http://www.webcitation.org/6tRcgQuou).
doi:10.2196/jmir.8210
PMCID: PMC5620457  PMID: 28912116
breast cancer; patient-reported outcomes; HRQoL; EORTC QLQ-C30; reliability
2.  Pertuzumab, Trastuzumab, and Docetaxel in HER2-Positive Metastatic Breast Cancer 
The New England journal of medicine  2015;372(8):724-734.
BACKGROUND
In patients with metastatic breast cancer that is positive for human epidermal growth factor receptor 2 (HER2), progression-free survival was significantly improved after first-line therapy with pertuzumab, trastuzumab, and docetaxel, as compared with placebo, trastuzumab, and docetaxel. Overall survival was significantly improved with pertuzumab in an interim analysis without the median being reached. We report final prespecified overall survival results with a median follow-up of 50 months.
METHODS
We randomly assigned patients with metastatic breast cancer who had not received previous chemotherapy or anti-HER2 therapy for their metastatic disease to receive the pertuzumab combination or the placebo combination. The secondary end points of overall survival, investigator-assessed progression-free survival, independently assessed duration of response, and safety are reported. Sensitivity analyses were adjusted for patients who crossed over from placebo to pertuzumab after the interim analysis.
RESULTS
The median overall survival was 56.5 months (95% confidence interval [CI], 49.3 to not reached) in the group receiving the pertuzumab combination, as compared with 40.8 months (95% CI, 35.8 to 48.3) in the group receiving the placebo combination (hazard ratio favoring the pertuzumab group, 0.68; 95% CI, 0.56 to 0.84; P<0.001), a difference of 15.7 months. This analysis was not adjusted for crossover to the pertuzumab group and is therefore conservative. Results of sensitivity analyses after adjustment for crossover were consistent. Median progression-free survival as assessed by investigators improved by 6.3 months in the pertuzumab group (hazard ratio, 0.68; 95% CI, 0.58 to 0.80). Pertuzumab extended the median duration of response by 7.7 months, as independently assessed. Most adverse events occurred during the administration of docetaxel in the two groups, with long-term cardiac safety maintained.
CONCLUSIONS
In patients with HER2-positive metastatic breast cancer, the addition of per tuzumab to trastuzumab and docetaxel, as compared with the addition of placebo, significantly improved the median overall survival to 56.5 months and extended the results of previous analyses showing the efficacy of this drug combination. (Funded by F. Hoffmann–La Roche and Genentech; CLEOPATRA ClinicalTrials.gov number, NCT00567190.)
doi:10.1056/NEJMoa1413513
PMCID: PMC5584549  PMID: 25693012
3.  Metabolic shifts in residual breast cancer drive tumor recurrence 
The Journal of Clinical Investigation  null;127(6):2091-2105.
Tumor recurrence is the leading cause of breast cancer–related death. Recurrences are largely driven by cancer cells that survive therapeutic intervention. This poorly understood population is referred to as minimal residual disease. Here, using mouse models that faithfully recapitulate human disease together with organoid cultures, we have demonstrated that residual cells acquire a transcriptionally distinct state from normal epithelium and primary tumors. Gene expression changes and functional characterization revealed altered lipid metabolism and elevated ROS as hallmarks of the cells that survive tumor regression. These residual cells exhibited increased oxidative DNA damage, potentiating the acquisition of somatic mutations during hormonal-induced expansion of the mammary cell population. Inhibition of either cellular fatty acid synthesis or fatty acid transport into mitochondria reduced cellular ROS levels and DNA damage, linking these features to lipid metabolism. Direct perturbation of these hallmarks in vivo, either by scavenging ROS or by halting the cyclic mammary cell population expansion, attenuated tumor recurrence. Finally, these observations were mirrored in transcriptomic and histological signatures of residual cancer cells from neoadjuvant-treated breast cancer patients. These results highlight the potential of lipid metabolism and ROS as therapeutic targets for reducing tumor recurrence in breast cancer patients.
doi:10.1172/JCI89914
PMCID: PMC5451224  PMID: 28504653
4.  Cell-free circulating DNA integrity is an independent predictor of impending breast cancer recurrence 
Oncotarget  2017;8(33):54537-54547.
Non-invasive blood-based molecule markers are evaluated as promising biomarkers these days. Here we investigated the potential of cell-free circulating DNA Integrity (cfDI) as blood-based marker for the prediction of recurrence during the follow-up of breast cancer patients within a prospective study cohort. cfDI was determined in plasma of 212 individuals, by measuring ALU and LINE1 repetitive DNA elements using quantitative PCR. A significant decrease of cfDI in recurrent breast cancer patients was observed. The group of patients who had impending recurrence during the follow-up had significant lower cfDI compared to the group of non-recurrent patients (P < 0.001 for ALU and LINE1 cfDI). cfDI could differentiate recurrent breast cancer patients from non-recurrent breast cancer subjects (area under the curve, AUC = 0.710 for ALU and 0.704 for LINE1). Univariate and multivariate analysis confirmed a significant association of recurrence and cfDI. Breast cancer patients with a lower cfDI had a much higher risk to develop recurrence than the patients with a higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE1 cfDI, respectively). Further we show that cfDI is an independent predictor of breast cancer recurrence. In combination with other molecular markers, cfDI might be a useful biomarker for the prediction for breast cancer recurrence in clinic utility. We propose that cfDI might also be useful for the prediction of recurrence during the follow-up of other cancers.
doi:10.18632/oncotarget.17384
PMCID: PMC5589601  PMID: 28903362
breast cancer; recurrence; circulating DNA integrity; biomarker
5.  Electronic-Based Patient-Reported Outcomes: Willingness, Needs, and Barriers in Adjuvant and Metastatic Breast Cancer Patients 
JMIR cancer  2017;3(2):e11.
Background
Patient-reported outcomes (PROs) play an increasingly important role as an adjunct to clinical outcome parameters in measuring health-related quality of life (HRQoL). In fact, PROs are already the accepted gold standard for collecting data about patients’ subjective perception of their own state of health. Currently, paper-based surveys of PRO still predominate; however, knowledge regarding the feasibility of and barriers to electronic-based PRO (ePRO) acceptance remains limited.
Objective
The objective of this trial was to analyze the willingness, specific needs, and barriers of adjuvant breast cancer (aBC) and metastatic breast cancer (mBC) patients in nonexposed (no exposure to electronic assessment) and exposed (after exposure to electronic assessment decision, whether a tablet-based questionnaire is favored) settings before implementing digital ePRO assessment in relation to health status. We also investigated whether providing support can increase the patients’ willingness to participate in such programs.
Methods
The nonexposed patients only answered a paper-based questionnaire, whereas the exposed patients filled out both paper- and tablet-based questionnaires. The assessment comprised socioeconomic variables, HRQoL, preexisting technical skills, general attitude toward electronic-based surveys, and potential barriers in relation to health status. Furthermore, nonexposed patients were asked about the existing need for technological support structures. In the course of data evaluation, we performed a frequency analysis as well as chi-square tests and Wilcoxon signed-rank tests. Subsequently, relative risks analysis, univariate categorical regression (CATREG), and mediation analyses (Hayes’ bias-corrected bootstrap) were performed.
Results
A total of 202 female breast cancer patients completed the PRO assessment (nonexposed group: n=96 patients; exposed group: n=106 patients). Self-reported technical skills were higher in exposed patients (2.79 vs 2.33, P ≤.001). Significant differences were found in relation to willingness to use ePRO (92.3% in the exposed group vs 59% in the nonexposed group; P=.001). Multiple barriers were identified, and most of them showed statistically significant differences in favor of the exposed patients (ie, data security [13% in the exposed patients vs 30% in the nonexposed patients; P=.003] and no prior technology usage [5% in the exposed group vs 15% in the nonexposed group; P=.02]), whereas the differences in disease burden (somatic dimension: 4% in the exposed group vs 9% in the nonexposed group; P=.13) showed no significance. In nonexposed patients, requests for support services were identified, which could increase their ePRO willingness.
Conclusions
Significant barriers in relation to HRQoL, cancer-related restrictions, and especially the setting of the survey were identified in this trial. Thus, it is necessary to address and eliminate these barriers to ensure data accuracy and reliability for future ePRO assessments. Exposure seems to be a potential option to increase willingness to use ePRO and to reduce barriers.
doi:10.2196/cancer.6996
PMCID: PMC5565790  PMID: 28784595
breast cancer; patient-reported outcome measures; electronic patient- reported outcome; technical skills; willingness to use; needs and barriers
6.  Tumor biomarker conversion between primary and metastatic breast cancer: mRNA assessment and its concordance with immunohistochemistry 
Oncotarget  2017;8(31):51416-51428.
Biomarker changes between primary (PT) and metastatic tumor (MT) site may be significant in individualizing treatment strategies and can result from actual clonal evolution, biomarker conversion, or technical limitations of diagnostic tests.
This study explored biomarker conversion during breast cancer (BC) progression in 67 patients with different tumor subtypes and metastatic sites via mRNA quantification and subsequently analyzed the concordance between real-time qPCR and immunohistochemistry (IHC). Immunostaining for estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki-67 was performed on formalin-fixed, paraffin-embedded PT and MT tissue sections. RT-qPCR was performed using a multiplex RT-qPCR kit for ESR1, PGR, ERBB2, and MKI67 and the reference genes B2M and CALM2.
Subsequent measurement of tumor biomarker mRNA expression to detect conversion revealed significant decreases in ESR1 and PGR mRNA and MKI67 upregulation (all p < 0.001) in MT compared to PT of all tumor subtypes and ERBB2 upregulation in MT from triple-negative PT patients (p = 0.023). Furthermore, ERBB2 mRNA was upregulated in MT brain biopsies, particularly those from triple-negative PTs (p = 0.023). High concordance between RT-qPCR and IHC was observed for ER/ESR1 (81%(κ 0.51) in PT and 84%(κ 0.34) in MT, PR/PGR (70%(κ 0.10) in PT and 78% (κ −0.32) in MT), and for HER2/ERBB2 (100% in PT and 89% in MT). Discordance between mRNA biomarker assessments of PT and MT resulting from receptor conversion calls for dynamic monitoring of BC tumor biomarkers. Overall, RT-qPCR assessment of BC target genes and their mRNA expression is highly concordant with IHC protein analysis in both primary and metastatic tumor.
doi:10.18632/oncotarget.18006
PMCID: PMC5584258
breast cancer; tumor biomarkers; receptor conversion; immunohistochemistry; real-time quantitative polymerase chain reaction
7.  World Congress Integrative Medicine & Health 2017: Part one 
Brinkhaus, Benno | Falkenberg, Torkel | Haramati, Aviad | Willich, Stefan N. | Briggs, Josephine P. | Willcox, Merlin | Linde, Klaus | Theorell, Töres | Wong, Lisa M. | Dusek, Jeffrey | Wu, Darong | Eisenberg, David | Haramati, Aviad | Berger, Bettina | Kemper, Kathi | Stock-Schröer, Beate | Sützl-Klein, Hedda | Ferreri, Rosaria | Kaplan, Gary | Matthes, Harald | Rotter, Gabriele | Schiff, Elad | Arnon, Zahi | Hahn, Eckhard | Luberto, Christina M. | Martin, David | Schwarz, Silke | Tauschel, Diethard | Flower, Andrew | Gramminger, Harsha | Gupta, Hedwig H. | Gupta, S. N. | Kerckhoff, Annette | Kessler, Christian S. | Michalsen, Andreas | Kessler, Christian S. | Kim, Eun S. | Jang, Eun H. | Kim, Rana | Jan, Sae B. | Mittwede, Martin | Mohme, Wiebke | Ben-Arye, Eran | Bonucci, Massimo | Saad, Bashar | Breitkreuz, Thomas | Rossi, Elio | Kebudi, Rejin | Daher, Michel | Razaq, Samaher | Gafer, Nahla | Nimri, Omar | Hablas, Mohamed | Kienle, Gunver Sophia | Samuels, Noah | Silbermann, Michael | Bandelin, Lena | Lang, Anna-Lena | Wartner, Eva | Holtermann, Christoph | Binstock, Maxwell | Riebau, Robert | Mujkanovic, Edin | Cramer, Holger | Lauche, Romy | Michalsen, Andres | Ward, Lesley | Cramer, Holger | Irnich, Dominik | Stör, Wolfram | Burnstock, Geoffrey | Schaible, Hans-Georg | Ots, Thomas | Langhorst, Jost | Lauche, Romy | Sundberg, Tobias | Falkenberg, Torkel | Amarell, Catherina | Amarell, Catherina | Anheyer, Melanie | Eckert, Marion | Eckert, Marion | Ogal, Mercedes | Eckert, Marion | Amarell, Catherina | Schönauer, Annette | Reisenberger, Birgit | Brand, Bernhard | Anheyer, Dennis | Dobos, Gustav | Kroez, Matthias | Martin, David | Matthes, Harald | Ammendola, Aldo | Mao, Jun J. | Witt, Claudia | Yang, Yufei | Dobos, Gustav | Oritz, Miriam | Horneber, Markus | Voiß, Petra | Reisenberger, Birgit | von Rosenstiel, Alexandra | Eckert, Marion | Ogal, Mercedes | Amarell, Catharina | Anheyer, Melanie | Schad, Friedemann | Schläppi, Marc | Kröz, Matthias | Büssing, Arndt | Bar-Sela, Gil | Matthes, Harald | Schiff, Elad | Ben-Arye, Eran | Arnon, Zahi | Avshalomov, David | Attias, Samuel | Schönauer, Annette | Haramati, Aviad | Witt, Claudia | Brinkhaus, Benno | Cotton, Sian | Jong, Miek | Jong, Mats | Scheffer, Christian | Haramati, Aviad | Tauschel, Diethard | Edelhäuser, Friedrich | AlBedah, Abdullah | Lee, Myeong Soo | Khalil, Mohamed | Ogawa, Keiko | Motoo, Yoshiharu | Arimitsu, Junsuke | Ogawa, Masao | Shimizu, Genki | Stange, Rainer | Kraft, Karin | Kuchta, Kenny | Watanabe, Kenji | Bonin, D | Büssing, Arndt | Gruber, Harald | Koch, Sabine | Gruber, Harald | Pohlmann, Urs | Caldwell, Christine | Krantz, Barbara | Kortum, Ria | Martin, Lily | Wieland, Lisa S. | Kligler, Ben | Gould-Fogerite, Susan | Zhang, Yuqing | Wieland, Lisa S. | Riva, John J. | Lumpkin, Michael | Ratner, Emily | Ping, Liu | Jian, Pei | Hamme, Gesa-Meyer | Mao, Xiaosong | Chouping, Han | Schröder, Sven | Hummelsberger, Josef | Wullinger, Michael | Brodzky, Marc | Zalpour, Christoff | Langley, Julia | Weber, Wendy | Mudd, Lanay M. | Wayne, Peter | Witt, Clauda | Weidenhammer, Wolfgang | Fønnebø, Vinjar | Boon, Heather | Steel, Amie | Bugarcic, Andrea | Rangitakatu, Melisa | Steel, Amie | Adams, Jon | Sibbritt, David | Wardle, Jon | Leach, Matthew | Schloss, Janet | Dieze, Helene | Boon, Heather | Ijaz, Nadine | Willcox, Merlin | Heinrich, Michael | Lewith, George | Flower, Andrew | Graz, Bertrand | Adam, Daniela | Grabenhenrich, Linus | Ortiz, Miriam | Binting, Sylvia | Reinhold, Thomas | Brinkhaus, Benno | Andermo, Susanne | Sundberg, Tobias | Falkenberg, Torkel | Nordberg, Johanna Hök | Arman, Maria | Bhasin, Manoj | Fan, Xueyi | Libermann, Towia | Fricchione, Gregory | Denninger, John | Benson, Herbert | Berger, Bettina | Stange, Rainer | Michalsen, Andreas | Martin, David D. | Boers, Inge | Vlieger, Arine | Jong, Miek | Brinkhaus, Benno | Teut, Michael | Ullmann, Alexander | Ortiz, Miriam | Rotter, Gabriele | Binting, Sylvia | Lotz, Fabian | Roll, Stephanie | Canella, Claudia | Mikolasek, Michael | Rostock, Matthias | Beyer, Jörg | Guckenberger, Matthias | Jenewein, Josef | Linka, Esther | Six, Claudia | Stoll, Sarah | Stupp, Roger | Witt, Claudia M. | Chuang, Elisabeth | Kligler, Ben | McKee, Melissa D. | Cramer, Holger | Lauche, Romy | Klose, Petra | Lange, Silke | Langhorst, Jost | Dobos, Gustav | Chung, Vincent C. H. | Wong, Hoi L. C. | Wu, Xin Y. | Wen, Grace Y. G. | Ho, Robin S. T. | Ching, Jessica Y. L. | Wu, Justin C. Y. | Coakley, Amanda | Flanagan, Jane | Annese, Christine | Empoliti, Joanne | Gao, Zishan | Liu, Xugang | Yu, Shuguang | Yan, Xianzhong | Liang, Fanrong | Hohmann, Christoph D. | Steckhan, Nico | Ostermann, Thomas | Paetow, Arion | Hoff, Evelyn | Michalsen, Andreas | Hu, Xiao-Yang | Wu, Ruo-Han | Logue, Martin | Blonde, Clara | Lai, Lily Y. | Stuart, Beth | Flower, Andrew | Fei, Yu-Tong | Moore, Michael | Liu, Jian-Ping | Lewith, George | Hu, Xiao-Yang | Wu, Ruo-Han | Logue, Martin | Blonde, Clara | Lai, Lily Y. | Stuart, Beth | Flower, Andrew | Fei, Yu-Tong | Moore, Michael | Liu, Jian-Ping | Lewith, George | Jeitler, Michael | Zillgen, Hannah | Högl, Manuel | Steckhan, Nico | Stöckigt, Barbara | Seifert, Georg | Michalsen, Andreas | Kessler, Christian | Khadivzadeh, Talat | Bashtian, Maryam Hassanzadeh | Aval, Shapour Badiee | Esmaily, Habibollah | Kim, Jihye | Kim, Keun H. | Klocke, Carina | Joos, Stefanie | Koshak, Abdulrahman | Wie, Li | Koshak, Emad | Wali, Siraj | Alamoudi, Omer | Demerdash, Abdulrahman | Qutub, Majdy | Pushparaj, Peter | Heinrich, Michael | Kruse, Sigrid | Fischer, Isabell | Tremel, Nadine | Rosenecker, Joseph | Leung, Brenda | Takeda, Wendy | Liang, Ning | Feng, Xue | Liu, Jian-ping | Cao, Hui-juan | Luberto, Christina M. | Shinday, Nina | Philpotts, Lisa | Park, Elyse | Fricchione, Gregory L. | Yeh, Gloria | Munk, Niki | Zakeresfahani, Arash | Foote, Trevor R. | Ralston, Rick | Boulanger, Karen | Özbe, Dominik | Gräßel, Elmar | Luttenberger, Katharina | Pendergrass, Anna | Pach, Daniel | Bellmann-Strobl, Judit | Chang, Yinhui | Pasura, Laura | Liu, Bin | Jäger, Sven F. | Loerch, Ronny | Jin, Li | Brinkhaus, Benno | Ortiz, Miriam | Reinhold, Thomas | Roll, Stephanie | Binting, Sylvia | Icke, Katja | Shi, Xuemin | Paul, Friedemann | Witt, Claudia M. | Rütz, Michaela | Lynen, Andreas | Schömitz, Meike | Vahle, Maik | Salomon, Nir | Lang, Alon | Lahat, Adi | Kopylov, Uri | Ben-Horin, Shomron | Har-Noi, Ofir | Avidan, Benjamin | Elyakim, Rami | Gamus, Dorit | NG, Siew | Chang, Jessica | Wu, Justin | Kaimiklotis, John | Schumann, Dania | Buttó, Ludovica | Langhorst, Jost | Dobos, Gustav | Haller, Dirk | Cramer, Holger | Smith, Caroline | de Lacey, Sheryl | Chapman, Michael | Ratcliffe, Julie | Johnson, Neil | Lyttleton, Jane | Boothroyd, Clare | Fahey, Paul | Tjaden, Bram | van Vliet, Marja | van Wietmarschen, Herman | Jong, Miek | Tröger, Wilfried | Vuolanto, Pia | Aarva, Paulina | Sorsa, Minna | Helin, Kaija | Wenzel, Claudia | Zoderer, Iris | Pammer, Patricia | Simon, Patrick | Tucek, Gerhard | Wode, Kathrin | Henriksson, Roger | Sharp, Lena | Stoltenberg, Anna | Nordberg, Johanna Hök | Xiao-ying, Yang | Wang, Li-qiong | Li, Jin-gen | Liang, Ning | Wang, Ying | Liu, Jian-ping | Balneaves, Lynda | Capler, Rielle | Bocci, Chiara | Guffi, Marta | Paolini, Marina | Meaglia, Ilaria | Porcu, Patrizia | Ivaldi, Giovanni B. | Dragan, Simona | Bucuras, Petru | Pah, Ana M. | Badalica-Petrescu, Marius | Buleu, Florina | Hogea-Stoichescu, Gheorghe | Christodorescu, Ruxandra | Kao, Lan | Cho, Yumin | Klafke, Nadja | Mahler, Cornelia | von Hagens, Cornelia | Uhlmann, Lorenz | Bentner, Martina | Schneeweiss, Andreas | Mueller, Andreas | Szecsenyi, Joachim | Joos, Stefanie | Neri, Isabella | Ortiz, Miriam | Schnabel, Katharina | Teut, Michael | Rotter, Gabriele | Binting, Sylvia | Cree, Margit | Lotz, Fabian | Suhr, Ralf | Brinkhaus, Benno | Rossi, Elio | Baccetti, Sonia | Firenzuoli, Fabio | Monechi, Maria V. | Di Stefano, Mariella | Amunni, Gianni | Wong, Wendy | Chen, Bingzhong | Wu, Justin | Amri, Hakima | Haramati, Aviad | Kotlyanskaya, Lucy | Anderson, Belinda | Evans, Roni | Kligler, Ben | Marantz, Paul | Bradley, Ryan | Booth-LaForce, Cathryn | Zwickey, Heather | Kligler, Benjamin | Brooks, Audrey | Kreitzer, Mary J. | Lebensohn, Patricia | Goldblatt, Elisabeth | Esmel-Esmel, Neus | Jiménez-Herrera, Maria | Ijaz, Nadine | Boon, Heather | Jocham, Alexandra | Stock-Schröer, Beate | Berberat, Pascal O. | Schneider, Antonius | Linde, Klaus | Masetti, Morgana | Murakozy, Henriette | Van Vliet, Marja | Jong, Mats | Jong, Miek | Agdal, Rita | Atarzadeh, Fatemeh | Jaladat, Amir M. | Hoseini, Leila | Amini, Fatemeh | Bai, Chen | Liu, Tiegang | Zheng, Zian | Wan, Yuxiang | Xu, Jingnan | Wang, Xuan | Yu, He | Gu, Xiaohong | Daneshfard, Babak | Nimrouzi, Majid | Tafazoli, Vahid | Alorizi, Seyed M. Emami | Saghebi, Seyed A. | Fattahi, Mohammad R. | Salehi, Alireza | Rezaeizadeh, Hossein | Zarshenas, Mohammad M. | Nimrouzi, Majid | Fox, Kealoha | Hughes, John | Kostanjsek, Nenad | Espinosa, Stéphane | Lewith, George | Fisher, Peter | Latif, Abdul | Lefeber, Donald | Paske, William | Öztürk, Ali Ö. | Öztürk, Gizemnur | Boers, Inge | Tissing, Wim | Naafs, Marianne | Busch, Martine | Jong, Miek | Daneshfard, Babak | Sanaye, Mohammad R. | Dräger, Kilian | Fisher, Peter | Kreitzer, Mary J. | Evans, Roni | Leininger, Brent | Shafto, Kate | Breen, Jenny | Sanaye, Mohammad R. | Daneshfard, Babak | Simões-Wüst, Ana P. | Moltó-Puigmartí, Carolina | van Dongen, Martien | Dagnelie, Pieter | Thijs, Carel | White, Shelley | Wiesener, Solveig | Salamonsen, Anita | Stub, Trine | Fønnebø, Vinjar | Abanades, Sergio | Blanco, Mar | Masllorens, Laia | Sala, Roser | Al-Ahnoumy, Shafekah | Han, Dongwoon | He, Luzhu | Kim, Ha Yun | In Choi, Da | Alræk, Terje | Stub, Trine | Kristoffersen, Agnete | von Sceidt, Christel | Michalsen, Andreas | Bruset, Stig | Musial, Frauke | Anheyer, Dennis | Cramer, Holger | Lauche, Romy | Saha, Felix J. | Dobos, Gustav | Anheyer, Dennis | Haller, Heidemarie | Lauche, Romy | Dobos, Gustav | Cramer, Holger | Azizi, Hoda | Khadem, Nayereh | Hassanzadeh, Malihe | Estiri, Nazanin | Azizi, Hamideh | Tavassoli, Fatemeh | Lotfalizadeh, Marzieh | Zabihi, Reza | Esmaily, Habibollah | Azizi, Hoda | Shabestari, Mahmoud Mohammadzadeh | Paeizi, Reza | Azari, Masoumeh Alvandi | Bahrami-Taghanaki, Hamidreza | Zabihi, Reza | Azizi, Hamideh | Esmaily, Habibollah | Baars, Erik | De Bruin, Anja | Ponstein, Anne | Baccetti, Sonia | Di Stefano, Mariella | Rossi, Elio | Firenzuoli, Fabio | Segantini, Sergio | Monechi, Maria Valeria | Voller, Fabio | Barth, Jürgen | Kern, Alexandra | Lüthi, Sebastian | Witt, Claudia | Barth, Jürgen | Zieger, Anja | Otto, Fabius | Witt, Claudia | Beccia, Ariel | Dunlap, Corina | Courneene, Brendan | Bedregal, Paula | Passi, Alvaro | Rodríguez, Alfredo | Chang, Mayling | Gutiérrez, Soledad | Beissner, Florian | Beissner, Florian | Preibisch, Christine | Schweizer-Arau, Annemarie | Popovici, Roxana | Meissner, Karin | Beljanski, Sylvie | Belland, Laura | Rivera-Reyes, Laura | Hwang, Ula | Berger, Bettina | Sethe, Dominik | Hilgard, Dörte | Heusser, Peter | Bishop, Felicity | Al-Abbadey, Miznah | Bradbury, Katherine | Carnes, Dawn | Dimitrov, Borislav | Fawkes, Carol | Foster, Jo | MacPherson, Hugh | Roberts, Lisa | Yardley, Lucy | Lewith, George | Bishop, Felicity | Al-Abbadey, Miznah | Bradbury, Katherine | Carnes, Dawn | Dimitrov, Borislav | Fawkes, Carol | Foster, Jo | MacPherson, Hugh | Roberts, Lisa | Yardley, Lucy | Lewith, George | Bishop, Felicity | Holmes, Michelle | Lewith, George | Yardley, Lucy | Little, Paul | Cooper, Cyrus | Bogani, Patrizia | Maggini, Valentina | Gallo, Eugenia | Miceli, Elisangela | Biffi, Sauro | Mengoni, Alessio | Fani, Renato | Firenzuoli, Fabio | Brands-Guendling, Nadine | Guendling, Peter W. | Bronfort, Gert | Evans, Roni | Haas, Mitch | Leininger, Brent | Schulz, Craig | Bu, Xiangwei | Wang, J. | Fang, T. | Shen, Z. | He, Y. | Zhang, X. | Zhang, Zhengju | Wang, Dali | Meng, Fengxian | Büssing, Arndt | Baumann, Klaus | Frick, Eckhard | Jacobs, Christoph | Büssing, Arndt | Grünther, Ralph-Achim | Lötzke, Désirée | Büssing, Arndt | Jung, Sonny | Lötzke, Désirée | Recchia, Daniela R. | Robens, Sibylle | Ostermann, Thomas | Berger, Bettina | Stankewitz, Josephin | Kröz, Matthias | Jeitler, Mika | Kessler, Christian | Michalsen, Andreas | Cheon, Chunhoo | Jang, Bo H. | Ko, Seong G. | Huang, Ching W. | Sasaki, Yui | Ko, Youme | Cheshire, Anna | Ridge, Damien | Hughes, John | Peters, David | Panagioti, Maria | Simon, Chantal | Lewith, George | Cho, Hyun J. | Han, Dongwoon | Choi, Soo J. | Jung, Young S. | Im, Hyea B | Cooley, Kieran | Tummon-Simmons, Laura | Cotton, Sian | Luberto, Christina M. | Wasson, Rachel | Kraemer, Kristen | Sears, Richard | Hueber, Carly | Derk, Gwendolyn | Lill, JR | An, Ruopeng | Steinberg, Lois | Rodriguez, Lourdes Diaz | la Fuente, Francisca García-de | De la Vega, Miguel | Vargas-Román, Keyla | Fernández-Ruiz, Jonatan | Cantarero-Villanueva, Irene | Rodriguez, Lourdes Diaz | García-De la Fuente, Francisca | Jiménez-Guerrero, Fanny | Vargas-Román, Keyla | Fernández-Ruiz, Jonatan | Galiano-Castillo, Noelia | Diaz-Saez, Gualberto | Torres-Jimenez, José I. | Garcia-Gomez, Olga | Hortal-Muñoz, Luis | Diaz-Diez, Camino | Dicen, Demijon | Diezel, Helene | Adams, Jon | Steel, Amie | Wardle, Jon | Diezel, Helene | Steel, Amie | Frawley, Jane | Wardle, Jon | Broom, Alex | Adams, Jon | Dong, Fei | Yu, He | Liu, Tiegang | Ma, Xueyan | Yan, Liyi | Wan, Yuxiang | Zheng, Zian | Gu, Xiaohong | Dong, Fei | Yu, He | Wu, Liqun | Liu, Tiegang | Ma, Xueyan | Ma, Jiaju | Yan, Liyi | Wan, Yuxiang | Zheng, Zian | Zhen, Jianhua | Gu, Xiaohong | Dubois, Julie | Rodondi, Pierre-Yves | Edelhäuser, Friedrich | Schwartze, Sophia | Trapp, Barbara | Cysarz, Dirk
doi:10.1186/s12906-017-1782-4
PMCID: PMC5498855
8.  No clinical utility of KRAS variant rs61764370 for ovarian or breast cancer 
Hollestelle, Antoinette | van der Baan, Frederieke H. | Berchuck, Andrew | Johnatty, Sharon E. | Aben, Katja K. | Agnarsson, Bjarni A. | Aittomäki, Kristiina | Alducci, Elisa | Andrulis, Irene L. | Anton-Culver, Hoda | Antonenkova, Natalia N. | Antoniou, Antonis C. | Apicella, Carmel | Arndt, Volker | Arnold, Norbert | Arun, Banu K. | Arver, Brita | Ashworth, Alan | Baglietto, Laura | Balleine, Rosemary | Bandera, Elisa V. | Barrowdale, Daniel | Bean, Yukie T. | Beckmann, Lars | Beckmann, Matthias W. | Benitez, Javier | Berger, Andreas | Berger, Raanan | Beuselinck, Benoit | Bisogna, Maria | Bjorge, Line | Blomqvist, Carl | Bogdanova, Natalia V. | Bojesen, Anders | Bojesen, Stig E. | Bolla, Manjeet K. | Bonanni, Bernardo | Brand, Judith S. | Brauch, Hiltrud | Brenner, Hermann | Brinton, Louise | Brooks-Wilson, Angela | Bruinsma, Fiona | Brunet, Joan | Brüning, Thomas | Budzilowska, Agnieszka | Bunker, Clareann H. | Burwinkel, Barbara | Butzow, Ralf | Buys, Saundra S. | Caligo, Maria A. | Campbell, Ian | Carter, Jonathan | Chang-Claude, Jenny | Chanock, Stephen J. | Claes, Kathleen B.M. | Collée, J. Margriet | Cook, Linda S. | Couch, Fergus J. | Cox, Angela | Cramer, Daniel | Cross, Simon S. | Cunningham, Julie M. | Cybulski, Cezary | Czene, Kamila | Damiola, Francesca | Dansonka-Mieszkowska, Agnieszka | Darabi, Hatef | de la Hoya, Miguel | deFazio, Anna | Dennis, Joseph | Devilee, Peter | Dicks, Ed M. | Diez, Orland | Doherty, Jennifer A. | Domchek, Susan M. | Dorfling, Cecilia M. | Dörk, Thilo | Santos Silva, Isabel Dos | du Bois, Andreas | Dumont, Martine | Dunning, Alison M. | Duran, Mercedes | Easton, Douglas F. | Eccles, Diana | Edwards, Robert P. | Ehrencrona, Hans | Ejlertsen, Bent | Ekici, Arif B. | Ellis, Steve D. | Engel, Christoph | Eriksson, Mikael | Fasching, Peter A. | Feliubadalo, Lidia | Figueroa, Jonine | Flesch-Janys, Dieter | Fletcher, Olivia | Fontaine, Annette | Fortuzzi, Stefano | Fostira, Florentia | Fridley, Brooke L. | Friebel, Tara | Friedman, Eitan | Friel, Grace | Frost, Debra | Garber, Judy | García-Closas, Montserrat | Gayther, Simon A. | Gentry-Maharaj, Aleksandra | Gerdes, Anne-Marie | Giles, Graham G. | Glasspool, Rosalind | Glendon, Gord | Godwin, Andrew K. | Goodman, Marc T. | Gore, Martin | Greene, Mark H. | Grip, Mervi | Gronwald, Jacek | Kaulich, Daphne Gschwantler | Guénel, Pascal | Guzman, Starr R. | Haeberle, Lothar | Haiman, Christopher A. | Hall, Per | Halverson, Sandra L. | Hamann, Ute | Hansen, Thomas V.O. | Harter, Philipp | Hartikainen, Jaana M. | Healey, Sue | Hein, Alexander | Heitz, Florian | Henderson, Brian E. | Herzog, Josef | Hildebrandt, Michelle A. T. | Høgdall, Claus K. | Høgdall, Estrid | Hogervorst, Frans B.L. | Hopper, John L. | Humphreys, Keith | Huzarski, Tomasz | Imyanitov, Evgeny N. | Isaacs, Claudine | Jakubowska, Anna | Janavicius, Ramunas | Jaworska, Katarzyna | Jensen, Allan | Jensen, Uffe Birk | Johnson, Nichola | Jukkola-Vuorinen, Arja | Kabisch, Maria | Karlan, Beth Y. | Kataja, Vesa | Kauff, Noah | Kelemen, Linda E. | Kerin, Michael J. | Kiemeney, Lambertus A. | Kjaer, Susanne K. | Knight, Julia A. | Knol-Bout, Jacoba P. | Konstantopoulou, Irene | Kosma, Veli-Matti | Krakstad, Camilla | Kristensen, Vessela | Kuchenbaecker, Karoline B. | Kupryjanczyk, Jolanta | Laitman, Yael | Lambrechts, Diether | Lambrechts, Sandrina | Larson, Melissa C. | Lasa, Aadriana | Laurent-Puig, Pierre | Lazaro, Conxi | Le, Nhu D. | Le Marchand, Loic | Leminen, Arto | Lester, Jenny | Levine, Douglas A. | Li, Jingmei | Liang, Dong | Lindblom, Annika | Lindor, Noralane | Lissowska, Jolanta | Long, Jirong | Lu, Karen H. | Lubinski, Jan | Lundvall, Lene | Lurie, Galina | Mai, Phuong L. | Mannermaa, Arto | Margolin, Sara | Mariette, Frederique | Marme, Frederik | Martens, John W.M. | Massuger, Leon F.A.G. | Maugard, Christine | Mazoyer, Sylvie | McGuffog, Lesley | McGuire, Valerie | McLean, Catriona | McNeish, Iain | Meindl, Alfons | Menegaux, Florence | Menéndez, Primitiva | Menkiszak, Janusz | Menon, Usha | Mensenkamp, Arjen R. | Miller, Nicola | Milne, Roger L. | Modugno, Francesmary | Montagna, Marco | Moysich, Kirsten B. | Müller, Heiko | Mulligan, Anna Marie | Muranen, Taru A. | Narod, Steven A. | Nathanson, Katherine L. | Ness, Roberta B. | Neuhausen, Susan L. | Nevanlinna, Heli | Neven, Patrick | Nielsen, Finn C. | Nielsen, Sune F. | Nordestgaard, Børge G. | Nussbaum, Robert L. | Odunsi, Kunle | Offit, Kenneth | Olah, Edith | Olopade, Olufunmilayo I. | Olson, Janet E. | Olson, Sara H. | Oosterwijk, Jan C. | Orlow, Irene | Orr, Nick | Orsulic, Sandra | Osorio, Ana | Ottini, Laura | Paul, James | Pearce, Celeste L. | Pedersen, Inge Sokilde | Peissel, Bernard | Pejovic, Tanja | Pelttari, Liisa M. | Perkins, Jo | Permuth-Wey, Jenny | Peterlongo, Paolo | Peto, Julian | Phelan, Catherine M. | Phillips, Kelly-Anne | Piedmonte, Marion | Pike, Malcolm C. | Platte, Radka | Plisiecka-Halasa, Joanna | Poole, Elizabeth M. | Poppe, Bruce | Pylkäs, Katri | Radice, Paolo | Ramus, Susan J. | Rebbeck, Timothy R. | Reed, Malcolm W.R. | Rennert, Gad | Risch, Harvey A. | Robson, Mark | Rodriguez, Gustavo C. | Romero, Atocha | Rossing, Mary Anne | Rothstein, Joseph H. | Rudolph, Anja | Runnebaum, Ingo | Salani, Ritu | Salvesen, Helga B. | Sawyer, Elinor J. | Schildkraut, Joellen M. | Schmidt, Marjanka K. | Schmutzler, Rita K. | Schneeweiss, Andreas | Schoemaker, Minouk J. | Schrauder, Michael G. | Schumacher, Fredrick | Schwaab, Ira | Scuvera, Giulietta | Sellers, Thomas A. | Severi, Gianluca | Seynaeve, Caroline M. | Shah, Mitul | Shrubsole, Martha | Siddiqui, Nadeem | Sieh, Weiva | Simard, Jacques | Singer, Christian F. | Sinilnikova, Olga M. | Smeets, Dominiek | Sohn, Christof | Soller, Maria | Song, Honglin | Soucy, Penny | Southey, Melissa C. | Stegmaier, Christa | Stoppa-Lyonnet, Dominique | Sucheston, Lara | Swerdlow, Anthony | Tangen, Ingvild L. | Tea, Muy-Kheng | Teixeira, Manuel R. | Terry, Kathryn L. | Terry, Mary Beth | Thomassen, Madas | Thompson, Pamela J. | Tihomirova, Laima | Tischkowitz, Marc | Toland, Amanda Ewart | Tollenaar, Rob A.E.M. | Tomlinson, Ian | Torres, Diana | Truong, Thérèse | Tsimiklis, Helen | Tung, Nadine | Tworoger, Shelley S. | Tyrer, Jonathan P. | Vachon, Celine M. | Van 't Veer, Laura J. | van Altena, Anne M. | Van Asperen, C.J. | van den Berg, David | van den Ouweland, Ans M.W. | van Doorn, Helena C. | Van Nieuwenhuysen, Els | van Rensburg, Elizabeth J. | Vergote, Ignace | Verhoef, Senno | Vierkant, Robert A. | Vijai, Joseph | Vitonis, Allison F. | von Wachenfeldt, Anna | Walsh, Christine | Wang, Qin | Wang-Gohrke, Shan | Wappenschmidt, Barbara | Weischer, Maren | Weitzel, Jeffrey N. | Weltens, Caroline | Wentzensen, Nicolas | Whittemore, Alice S. | Wilkens, Lynne R. | Winqvist, Robert | Wu, Anna H. | Wu, Xifeng | Yang, Hannah P. | Zaffaroni, Daniela | Zamora, M. Pilar | Zheng, Wei | Ziogas, Argyrios | Chenevix-Trench, Georgia | Pharoah, Paul D.P. | Rookus, Matti A. | Hooning, Maartje J. | Goode, Ellen L.
Gynecologic oncology  2015;141(2):386-401.
Objective
Clinical genetic testing is commercially available for rs61764370, an inherited variant residing in a KRAS 3′ UTR microRNA binding site, based on suggested associations with increased ovarian and breast cancer risk as well as with survival time. However, prior studies, emphasizing particular subgroups, were relatively small. Therefore, we comprehensively evaluated ovarian and breast cancer risks as well as clinical outcome associated with rs61764370.
Methods
Centralized genotyping and analysis were performed for 140,012 women enrolled in the Ovarian Cancer Association Consortium (15,357 ovarian cancer patients; 30,816 controls), the Breast Cancer Association Consortium (33,530 breast cancer patients; 37,640 controls), and the Consortium of Modifiers of BRCA1 and BRCA2 (14,765 BRCA1 and 7904 BRCA2 mutation carriers).
Results
We found no association with risk of ovarian cancer (OR= 0.99, 95% CI 0.94–1.04,p = 0.74) or breast cancer (OR = 0.98, 95% CI 0.94–1.01, p = 0.19) and results were consistent among mutation carriers (BRCA1, ovarian cancer HR = 1.09, 95% CI 0.97–1.23, p = 0.14, breast cancer HR = 1.04, 95% CI 0.97–1.12, p = 0.27; BRCA2, ovarian cancer HR = 0.89, 95% CI 0.71–1.13, p = 0.34, breast cancer HR = 1.06, 95% CI 0.94–1.19, p = 0.35). Null results were also obtained for associations with overall survival following ovarian cancer (HR = 0.94, 95% CI 0.83–1.07, p = 0.38), breast cancer (HR = 0.96, 95% CI 0.87–1.06, p = 0.38), and all other previously-reported associations.
Conclusions
rs61764370 is not associated with risk of ovarian or breast cancer nor with clinical outcome for patients with these cancers. Therefore, genotyping this variant has no clinical utility related to the prediction or management of these cancers.
doi:10.1016/j.ygyno.2015.04.034
PMCID: PMC4630206  PMID: 25940428
KRAS variant; Breast cancer; Ovarian cancer; Genetic association; Clinical outcome
9.  Cellular Immune Responses and Immune Escape Mechanisms in Breast Cancer: Determinants of Immunotherapy 
Breast Care  2016;11(2):102-107.
Summary
More recently, immunotherapy has emerged as a novel potentially effective therapeutic option also for solid malignancies such as breast cancer (BC). Relevant approaches, however, are determined by the 2 main elements of cancer immunoediting - the elimination of nascent transformed cells by immunosurveillance on the one hand and tumor immune escape on the other hand. Correspondingly, we here review the role of the various cellular immune players within the host-protective system and dissect the mechanisms of immune evasion leading to tumor progression. If the immune balance of disseminated BC cell dormancy (equilibrium phase) is lost, distant metastatic relapse may occur. The relevant cellular antitumor responses and translational immunotherapeutic options will also be discussed in terms of clinical benefit and future directions in BC management.
doi:10.1159/000446061
PMCID: PMC4881254  PMID: 27239171
Breast cancer; Cellular immunity; Immune escape; Immunoediting; Immunosurveillance; Immunotherapy
10.  Muscle strength in breast cancer patients receiving different treatment regimes 
Abstract
Background
Muscle dysfunction and sarcopenia have been associated with poor performance status, an increased mortality risk, and greater side effects in oncologic patients. However, little is known about how performance is affected by cancer therapy. We investigated muscle strength in breast cancer patients in different adjuvant treatment settings and also compared it with data from healthy individuals.
Methods
Breast cancer patients (N = 255) from two randomized controlled exercise trials, staged 0–III and aged 54.4 ± 9.4 years, were categorized into four groups according to their treatment status. In a cross‐sectional design, muscle function was assessed bilaterally by isokinetic dynamometry (0°, 60°, 180°/s) as maximal voluntary isometric contraction (MVIC) and maximal isokinetic peak torque (MIPT) in shoulder rotators and knee flexors and extensors. Additionally, muscular fatigue index (FI%) and shoulder flexibility were evaluated. Healthy women (N = 26), aged 53.3 ± 9.8 years, were tested using the same method. Analysis of covariance was used to estimate the impact of different cancer treatments on skeletal muscle function with adjustment for various clinical and socio‐demographic factors.
Results
Consistently, lower muscle strength was measured in shoulder and knee strength in patients after chemotherapy. On average, patients had up to 25% lower strength in lower extremities and 12–16% in upper extremities in MVIC and MIPT during cancer treatment compared with healthy women. No substantial difference between patient groups in shoulder strength, but significantly lower shoulder flexibility in patients with radical mastectomy was measured. Chemotherapy‐treated patients had consistently higher FI%. No serious adverse events were reported.
Conclusions
Breast cancer patients showed markedly impaired muscle strength and joint dysfunctions before and after anticancer treatment. The significant differences between patients and healthy individuals underline the need of exercise therapy as early as possible in order to prevent or counteract the loss of muscle function after curative surgery as well as the consequences of neo‐/adjuvant chemotherapy.
doi:10.1002/jcsm.12165
PMCID: PMC5377413  PMID: 27896952
Isokinetic; Isometric; Multi‐joint; Muscle function; Chemotherapy
11.  Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer 
BMC Cancer  2017;17:124.
Background
Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy.
Methods
Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial. Expression of ESR1, PGR and MKI67 by RT-qPCR was performed on RNA extracted from the same formalin-fixed paraffin-embedded tissue. Concordance between the three methods (vIHC, qIHC and RT-qPCR) was assessed for all 3 markers. The potential of Ki-67 IHC and RT-qPCR to predict pathological complete response (pCR) was evaluated using ROC analysis and non-parametric Mann-Whitney Test.
Results
Correlation between methods (qIHC versus RT-qPCR) was high for ER and PgR (spearman´s r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively) resulting in high levels of concordance using predefined cut-offs. When comparing qIHC of ER and PgR with RT-qPCR of ESR1 and PGR the overall agreement was 96.6 and 91.4%, respectively, while overall agreement of visual IHC with RT-qPCR was slightly lower for ER/ESR1 and PR/PGR (91.2 and 92.9%, respectively). In contrast, only a moderate correlation was observed between qIHC and RT-qPCR continuous data for Ki-67/MKI67 (Spearman’s r = 0.50, p = 0.0001). Up to now no predictive cut-off for Ki-67 assessment by IHC has been established to predict response to neoadjuvant chemotherapy. Setting the desired sensitivity at 100%, specificity for the prediction of pCR (ypT0ypN0) was significantly higher for mRNA than for protein (68.9% vs. 22.2%). Moreover, the proliferation levels in patients achieving a pCR versus not differed significantly using MKI67 RNA expression (Mann-Whitney p = 0.002), but not with qIHC of Ki-67 (Mann-Whitney p = 0.097) or vIHC of Ki-67 (p = 0.131).
Conclusion
Digital image analysis can successfully be implemented for assessing ER, PR and Ki-67. IHC for ER and PR reveals high concordance with RT-qPCR. However, RT-qPCR displays a broader dynamic range and higher sensitivity than IHC. Moreover, correlation between Ki-67 qIHC and RT-qPCR is only moderate and RT-qPCR with MammaTyper® outperforms qIHC in predicting pCR. Both methods yield improvements to error-prone manual scoring of Ki-67. However, RT-qPCR was significantly more specific.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-017-3111-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-017-3111-1
PMCID: PMC5307758  PMID: 28193205
Image analysis; Breast cancer; Ki67; mRNA; RT-qPCR; Prediction; Pathologic complete response; neoadjuvant; Immunohistochemistry (IHC); MammaTyper®
12.  In silico SNP analysis of the breast cancer antigen NY-BR-1 
BMC Cancer  2016;16:901.
Background
Breast cancer is one of the most common malignancies with increasing incidences every year and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic disease. The novel breast cancer associated antigen NY-BR-1 was identified by SEREX analysis and is expressed in the majority (>70%) of breast tumors as well as metastases, in normal breast tissue, in testis and occasionally in prostate tissue. The biological function and regulation of NY-BR-1 is up to date unknown.
Methods
We performed an in silico analysis on the genetic variations of the NY-BR-1 gene using data available in public SNP databases and the tools SIFT, Polyphen and Provean to find possible functional SNPs. Additionally, we considered the allele frequency of the found damaging SNPs and also analyzed data from an in-house sequencing project of 55 breast cancer samples for recurring SNPs, recorded in dbSNP.
Results
Over 2800 SNPs are recorded in the dbSNP and NHLBI ESP databases for the NY-BR-1 gene. Of these, 65 (2.07%) are synonymous SNPs, 191 (6.09%) are non-synoymous SNPs, and 2430 (77.48%) are noncoding intronic SNPs. As a result, 69 non-synoymous SNPs were predicted to be damaging by at least two, and 16 SNPs were predicted as damaging by all three of the used tools. The SNPs rs200639888, rs367841401 and rs377750885 were categorized as highly damaging by all three tools. Eight damaging SNPs are located in the ankyrin repeat domain (ANK), a domain known for its frequent involvement in protein-protein interactions. No distinctive features could be observed in the allele frequency of the analyzed SNPs.
Conclusion
Considering these results we expect to gain more insights into the variations of the NY-BR-1 gene and their possible impact on giving rise to splice variants and therefore influence the function of NY-BR-1 in healthy tissue as well as in breast cancer.
doi:10.1186/s12885-016-2924-7
PMCID: PMC5116164  PMID: 27863482
NY-BR-1; Breast cancer; Antigen; SNPs; In silico
13.  rs2735383, located at a microRNA binding site in the 3’UTR of NBS1, is not associated with breast cancer risk 
Liu, Jingjing | Lončar, Ivona | Collée, J. Margriet | Bolla, Manjeet K. | Dennis, Joe | Michailidou, Kyriaki | Wang, Qin | Andrulis, Irene L. | Barile, Monica | Beckmann, Matthias W. | Behrens, Sabine | Benitez, Javier | Blomqvist, Carl | Boeckx, Bram | Bogdanova, Natalia V. | Bojesen, Stig E. | Brauch, Hiltrud | Brennan, Paul | Brenner, Hermann | Broeks, Annegien | Burwinkel, Barbara | Chang-Claude, Jenny | Chen, Shou-Tung | Chenevix-Trench, Georgia | Cheng, Ching Y. | Choi, Ji-Yeob | Couch, Fergus J. | Cox, Angela | Cross, Simon S. | Cuk, Katarina | Czene, Kamila | Dörk, Thilo | dos-Santos-Silva, Isabel | Fasching, Peter A. | Figueroa, Jonine | Flyger, Henrik | García-Closas, Montserrat | Giles, Graham G. | Glendon, Gord | Goldberg, Mark S. | González-Neira, Anna | Guénel, Pascal | Haiman, Christopher A. | Hamann, Ute | Hart, Steven N. | Hartman, Mikael | Hatse, Sigrid | Hopper, John L. | Ito, Hidemi | Jakubowska, Anna | Kabisch, Maria | Kang, Daehee | Kosma, Veli-Matti | Kristensen, Vessela N. | Le Marchand, Loic | Lee, Eunjung | Li, Jingmei | Lophatananon, Artitaya | Jan Lubinski,  | Mannermaa, Arto | Matsuo, Keitaro | Milne, Roger L. | Neuhausen, Susan L. | Nevanlinna, Heli | Orr, Nick | Perez, Jose I. A. | Peto, Julian | Putti, Thomas C. | Pylkäs, Katri | Radice, Paolo | Sangrajrang, Suleeporn | Sawyer, Elinor J. | Schmidt, Marjanka K. | Schneeweiss, Andreas | Shen, Chen-Yang | Shrubsole, Martha J. | Shu, Xiao-Ou | Simard, Jacques | Southey, Melissa C. | Swerdlow, Anthony | Teo, Soo H. | Tessier, Daniel C. | Thanasitthichai, Somchai | Tomlinson, Ian | Torres, Diana | Truong, Thérèse | Tseng, Chiu-Chen | Vachon, Celine | Winqvist, Robert | Wu, Anna H. | Yannoukakos, Drakoulis | Zheng, Wei | Hall, Per | Dunning, Alison M. | Easton, Douglas F. | Hooning, Maartje J. | van den Ouweland, Ans M. W. | Martens, John W. M. | Hollestelle, Antoinette
Scientific Reports  2016;6:36874.
NBS1, also known as NBN, plays an important role in maintaining genomic stability. Interestingly, rs2735383 G > C, located in a microRNA binding site in the 3′-untranslated region (UTR) of NBS1, was shown to be associated with increased susceptibility to lung and colorectal cancer. However, the relation between rs2735383 and susceptibility to breast cancer is not yet clear. Therefore, we genotyped rs2735383 in 1,170 familial non-BRCA1/2 breast cancer cases and 1,077 controls using PCR-based restriction fragment length polymorphism (RFLP-PCR) analysis, but found no association between rs2735383CC and breast cancer risk (OR = 1.214, 95% CI = 0.936–1.574, P = 0.144). Because we could not exclude a small effect size due to a limited sample size, we further analyzed imputed rs2735383 genotypes (r2 > 0.999) of 47,640 breast cancer cases and 46,656 controls from the Breast Cancer Association Consortium (BCAC). However, rs2735383CC was not associated with overall breast cancer risk in European (OR = 1.014, 95% CI = 0.969–1.060, P = 0.556) nor in Asian women (OR = 0.998, 95% CI = 0.905–1.100, P = 0.961). Subgroup analyses by age, age at menarche, age at menopause, menopausal status, number of pregnancies, breast feeding, family history and receptor status also did not reveal a significant association. This study therefore does not support the involvement of the genotype at NBS1 rs2735383 in breast cancer susceptibility.
doi:10.1038/srep36874
PMCID: PMC5109293  PMID: 27845421
14.  DNA methylation array analysis identifies breast cancer associated RPTOR, MGRN1 and RAPSN hypomethylation in peripheral blood DNA 
Oncotarget  2016;7(39):64191-64202.
DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 × 10−6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.
doi:10.18632/oncotarget.11640
PMCID: PMC5325435  PMID: 27577081
breast cancer; DNA methylation; MGRN1; RAPSN; RPTOR
15.  Prognosis of breast cancer molecular subtypes in routine clinical care: A large prospective cohort study 
BMC Cancer  2016;16:734.
Background
In Germany, most breast cancer patients are treated in specialized breast cancer units (BCU), which are certified, and routinely monitored. Herein, we evaluate up-to-date oncological outcome of breast cancer (BC) molecular subtypes in routine clinical care of a specialized BCU.
Methods
The study was a prospectively single-center cohort study of 4102 female cases with primary, unilateral, non-metastatic breast cancer treated between 01 January 2003 and 31 December 2012. The five routinely used molecular subtypes (Luminal A-like, Luminal B/HER2 negative-like, Luminal B/HER2 positive-like, HER2-type, Triple negative) were analyzed. The median follow-up time of the whole cohort was 55 months. We calculated estimates for local control rate (LCR), disease-free survival (DFS), distant disease-free survival (DDFS), overall survival (OS), and relative overall survival (ROS).
Results
Luminal A-like tumors were the most frequent (44.7 %) and showed the best outcome with LCR of 99.1 % (95 % CI 98.5; 99.7), OS of 95.1 % (95 % CI 93.7; 96.5), and ROS of 100.0 % (95 % CI 98.5; 101.5). Triple negative tumors (12.3 %) presented the poorest outcome with LCR of 89.6 % (95 % CI 85.8; 93.4), OS of 78.5 % (95 % CI 73.8; 83.3), and ROS of 80.1 % (95 % CI 73.8; 83.2).
Conclusions
Patients with a favorable subtype can expect an OS above 95 % and an LCR of almost 100 % over 5 years. On the other hand the outcome of patients with HER2 and Triple negative subtypes remains poor, thus necessitating more intensified research and care.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-016-2766-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-016-2766-3
PMCID: PMC5024419  PMID: 27634735
Breast cancer; Molecular subtypes; Outcome; Breast care unit
16.  Intravenous pamidronate versus oral and intravenous clodronate in bone metastatic breast cancer: a randomized, open-label, non-inferiority Phase III trial 
OncoTargets and therapy  2016;9:4173-4180.
Purpose
Patients with metastasized breast cancer often suffer from discomfort caused by metastatic bone disease. Thus, osteoprotection is an important part of therapy in breast cancer metastasized to bone, and bisphosphonates (BPs) are a major therapeutic option. In this study, our objectives were to compare the side effects of oral versus intravenous BP treatment and to assess their clinical effectiveness.
Patients and methods
In this prospective randomized, open-label, non-inferiority trial, we enrolled breast cancer patients with at least one bone metastasis and an Eastern Cooperative Oncology Group performance status of 0–2. Patients were randomly assigned to one of the three treatment groups: A, 60 mg pamidronate intravenously q3w; B-iv, 900 mg clodronate intravenously q3w; and B-o, 2,400 mg oral clodronate daily. Assessments were performed at baseline and every 3 months thereafter.
Results
Between 1995 and 1999, 321 patients with confirmed bone metastases from breast cancer were included in the study. At first follow-up, gastrointestinal (GI) tract side effects were most common, and adverse effects on the GI tract were more frequent in the oral treatment group (P=0.002 and P<0.001, respectively). There were no statistically significant differences among the treatment cohorts for other documented side effects (skin, serum electrolytes, urinary tract, immune system, and others). No significant differences in clinical effectiveness of BP treatment, as assessed by pain score, were detected among the groups; however, pathologic fractures were more effectively prevented by intravenous than oral BP administration (P=0.03). Noncompliance rates were similar among the study cohorts.
Conclusion
We conclude that oral BP treatment is significantly associated with higher rates of adverse GI side effects. Additionally, our data indicate that intravenous BP administration is more effective than oral treatment in prevention of pathologic fractures; hence, oral administration should be considered with caution.
doi:10.2147/OTT.S103130
PMCID: PMC4944913  PMID: 27468239
metastatic bone disease; bisphosphonates; adverse effects; clinical effectiveness
17.  Detection of circulating tumor cells using manually performed immunocytochemistry (MICC) does not correlate with outcome in patients with early breast cancer – Results of the German SUCCESS-A- trial 
BMC Cancer  2016;16:401.
Background
Recently, the prognostic significance of circulating tumor cells (CTCs) in primary breast cancer as assessed using the Food-and-Drug-Administration-approved CellSearch® system has been demonstrated. Here, we evaluated the prognostic relevance of CTCs, as determined using manually performed immunocytochemistry (MICC) in peripheral blood at primary diagnosis, in patients from the prospectively randomized multicenter SUCCESS-A trial (EudraCT2005000490-21).
Methods
We analyzed 23 ml of blood from 1221 patients with node-positive or high risk node-negative breast cancer before adjuvant taxane-based chemotherapy. Cells were separated using a density gradient followed by epithelial cell labeling with the anti-cytokeratin-antibody A45-B/B3, immunohistochemical staining with new fuchsin, and cytospin preparation. All cytospins were screened for CTCs, and the cutoff for positivity was at least one CTC. The prognostic value of CTCs with regard to disease-free survival (DFS), distant disease-free survival (DDFS), breast-cancer-specific survival (BCSS), and overall survival (OS) was assessed using both univariate analyses applying the Kaplan–Meier method and log-rank tests, and using multivariate Cox regressions adjusted for other predictive factors.
Results
In 20.6 % of all patients (n = 251) a median of 1 (range, 1–256) CTC was detected, while 79.4 % of the patients (n = 970) were negative for CTCs before adjuvant chemotherapy. A pT1 tumor was present in 40.0 % of patients, 4.8 % had G1 grading and 34.6 % were node-negative. There was no association between CTC positivity and tumor stage, nodal status, grading, histological type, hormone receptor status, Her2 status, menopausal status or treatment. Univariate survival analyses based on a median follow-up of 64 months revealed no significant differences between CTC-positive and CTC-negative patients with regard to DFS, DDFS, BCSS, or OS. This was confirmed by fully adjusted multivariate Cox regressions, showing that the presence of CTCs (yes/no) as assessed by MICC did not predict DFS, DDFS, BCSS or OS.
Conclusions
We could not demonstrate prognostic relevance regarding CTCs that were quantified using the MICC method at the time of primary diagnosis in our cohort of early breast cancer patients. Further studies are necessary to evaluate if the presence of CTCs assessed using MICC has prognostic relevance, or can be used for risk stratification and treatment monitoring in adjuvant breast cancer.
Trial registration
The ClinicalTrial.gov registration ID of this prospectively randomized trial is NCT02181101; the (retrospective) registration date was June 2014 (study start date September 2005).
doi:10.1186/s12885-016-2454-3
PMCID: PMC4936301  PMID: 27387743
Breast cancer; Circulating tumor cells; Manual immunocytochemistry; Disease-free survival; Overall survival; Neoplasm; Neoplasm recurrence; Translational research; Detection method
18.  Targeted Therapies in Triple-Negative Breast Cancer 
Breast Care  2015;10(3):159-166.
Summary
Triple-negative breast cancer (TNBC) is a heterogeneous disease comprised of several biologically distinct subtypes. However, treatment is currently mainly relying on chemotherapy as there are no targeted therapies specifically approved for TNBC. Despite initial responses to chemotherapy, resistance frequently and rapidly develops and metastatic TNBC has a poor prognosis. New targeted approaches are, therefore, urgently needed. Currently, bevacizumab, a monoclonal anti-vascular endothelial growth factor (VEGF)-A antibody, is the only targeted agent with an approval for the therapy of metastatic breast cancer, but does not provide a specific benefit in the TNBC subtype. This review discusses the current clinical developments in targeted approaches for TNBC, including anti-angiogenic therapies, epidermal growth factor receptor (EGFR)-targeted therapies, poly(ADP-ribose) polymerase (PARP) inhibitors and platinum salts, as well as novel strategies using immune-checkpoint inhibitors, which have recently demonstrated first promising results. Strategies focusing on specific subtypes of TNBC like anti-androgenic therapies for the luminal androgen receptor subtype (LAR) and others are also discussed.
doi:10.1159/000433622
PMCID: PMC4569194  PMID: 26557820
Triple-negative breast cancer; Subtypes; PARP inhibitors; Immune-checkpoint inhibitors; Targeted therapy; Bevacizumab
19.  14th St. Gallen International Breast Cancer Conference 2015: Evidence, Controversies, Consensus – Primary Therapy of Early Breast Cancer: Opinions Expressed by German Experts 
Breast Care  2015;10(3):211-219.
Summary
The key topics of this year's 14th St. Gallen Consensus Conference on the diagnosis and therapy of primary breast cancer were again questions about breast surgery and axillary surgery, radio-oncology and systemic therapy options in consideration of tumor biology, and the clinical application of multigene assays. This year, the consensus conference took place in Vienna. From a German perspective, it makes sense to substantiate the results of the vote of the international panel representing 19 countries in light of the updated national therapy recommendations of the AGO (Arbeitsgemeinschaft Gynäkologische Onkologie). Therefore, 14 German breast cancer experts, 3 of whom are members of the International St. Gallen Panel, have commented on the voting results of the St. Gallen Consensus Conference 2015 in relation to clinical routine in Germany.
doi:10.1159/000433590
PMCID: PMC4569213  PMID: 26557827
St. Gallen Consensus 2015; Systemic therapy; Local therapy; Sentinel node biopsy; Endocrine therapy; Targeted substances; Multigene assay
20.  Non-pegylated liposomal doxorubicin for patients with recurrent ovarian cancer: A multicentric phase II trial 
Oncology Letters  2016;12(2):1211-1215.
Patients with non-platinum-sensitive recurrent ovarian cancer have a poor prognosis. Non-pegylated liposomal doxorubicin (Myocet®) is a promising drug that may be able to improve treatment for such patients. In the current study, patients with recurrent ovarian cancer relapsing within 12 months after primary treatment received non-pegylated liposomal doxorubicin at 75 mg/m2 d1q22 and 60 mg/m2 d1q22 after study dose modification, respectively. There were 29 patients enrolled in the trial, and 124 cycles of non-pegylated liposomal doxorubicin were administered in total. All 29 patients were evaluable for toxicity. The clinical benefit rate (defined as the proportion of patients with either complete remission or partial remission, or with stable disease for >6 months) was 50%. The predominant non-hematological toxicity was nausea and vomiting (18 patients, grade I/II), whilst no palmar plantar erythrodysesthesia was observed. In 3 patients, a grade III hematological toxicity occurred, and the treatment schedule was consequently modified to 60 mg/m2 d1q22. The findings suggest that non-pegylated liposomal doxorubicin administered in a schedule of 60 mg/m2 d1q22 is well-manageable and is associated with tolerable non-hematological toxicities (predominantly nausea).
doi:10.3892/ol.2016.4740
PMCID: PMC4950884  PMID: 27446420
epithelial ovarian cancer; platinum-resistant recurrence; non-pegylated liposomal doxorubicin
21.  Mucin 1-specific B cell immune responses and their impact on overall survival in breast cancer patients 
Oncoimmunology  2015;5(1):e1057387.
Considering the diverse functions of B cells, responses to tumor-associated antigens (TAA) have been thought to be the main source of B cell-mediated antitumor immunity. Polymorphic epithelial mucin (MUC1) is considered one of the most specific TAA in patients with breast cancer. The present study aims to dissect the level and subclasses of naturally occurring anti-MUC1 antibodies in regard to tumor biologic parameters, clinical characteristics and overall survival. In 288 primary, non-metastatic breast cancer patients, pretreatment serum levels of anti-MUC1 immunoglobulin G (IgG) and its subclasses G1–4 as well as immunoglobulin M (IgM) were analyzed via ELISA. With respect to overall survival (Kaplan–Meier analysis), tumor biologic parameters as hormone receptor status, human epidermal growth factor receptor 2 (Her2), Ki-67 expression and tumor grading have been correlated as well as clinical characteristics as nodal involvement, tumor stage and patients' age at the time of diagnosis. Median follow-up time was 148 mo (IQR: 73.1–158.5 mo). A significant increase in IgG antibody titers was correlated highly significantly with an improved overall survival of patients. In multivariate analysis, total IgG proved to be an independent prognostic marker for overall survival (p = 0.002). IgG subclass analysis did not reveal any correlation of IgG1, IgG3 and IgG4 levels with overall survival, while increased immunoglobulin G2 (IgG2) values, although statistically not significant, tended to correlate with prolonged patient survival. MUC1-specific IgM antibodies were shown not to be predictive of overall survival. Altogether, humoral immune responses appear to play a crucial part in the tumor immunity of breast cancer patients. The present data confirms the positive impact of tumor-specific IgG on prolonged overall survival in breast cancer patients. MUC1-antibody testing might be a useful tool to identify high-risk patients who may need adjuvant therapy and potentially might benefit from MUC1-directed immunotherapy.
doi:10.1080/2162402X.2015.1057387
PMCID: PMC4760286  PMID: 26942066
ADCC; B cells; breast cancer; immunoglobulin G; MUC1-specific antibodies; prognosis
22.  Pre-Osteoblasts Stimulate Migration of Breast Cancer Cells via the HGF/MET Pathway 
PLoS ONE  2016;11(3):e0150507.
Introduction
The occurrence of skeletal metastases in cancer, e.g. breast cancer (BC), deteriorates patient life expectancy and quality-of-life. Current treatment options against tumor-associated bone disease are limited to anti-resorptive therapies and aimed towards palliation. There remains a lack of therapeutic approaches, which reverse or even prevent the development of bone metastases. Recent studies demonstrate that not only osteoclasts (OCs), but also osteoblasts (OBs) play a central role in the pathogenesis of skeletal metastases, partly by producing hepatocyte growth factor (HGF), which promotes tumor cell migration and seeding into the bone. OBs consist of a heterogeneous cell pool with respect to their maturation stage and function. Recent studies highlight the critical role of pre-OBs in hematopoiesis. Whether the development of bone metastases can be attributed to a particular OB maturation stage is currently unknown.
Methods and Results
Pre-OBs were generated from healthy donor (HD)-derived bone marrow stromal cells (BMSC) as well as the BMSC line KM105 and defined as ALPlow OPNlow RUNX2high OSX high CD166high. Conditioned media (CM) of pre-OBs, but not of undifferentiated cells or mature OBs, enhanced migration of metastatic BC cells. Importantly, HGF mRNA was significantly up-regulated in pre-OBs versus mature OBs, and CM of pre-OBs activated the MET signaling pathway. Highlighting a key role for HGF, CM from HGF-negative pre-OBs derived from the BMSC line HS27A did not support migration of BC cells. Genetically (siMET) or pharmacologically (INCB28060) targeting MET inhibited both HGF- and pre-OB CM- mediated BC cell migration.
Conclusions
Our data demonstrate for the first time a role for pre-OBs in mediating HGF/MET- dependent migration of BC cells and strongly support the clinical evaluation of INCB28060 and other MET inhibitors to limit and/or prevent BC-associated bone metastases.
doi:10.1371/journal.pone.0150507
PMCID: PMC4774929  PMID: 26934743
23.  Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: therapeutic implications 
Background
Molecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown. The anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) is frequently amplified in BC; and elevated Mcl-1 levels have been correlated with poor prognosis. Here we investigated the pathophysiologic role of Mcl-1 in Her2-positive BC cells under hypoxic conditions.
Methods
RNA interference and a novel small molecule inhibitor, EU-5346, were used to examine the role of Mcl-1 in Her2-positive BC cell lines and primary BC cells (sensitive or intrinsically resistant to Her2 inhibitors) under hypoxic conditions (using a hypoxic incubation chamber). Mechanisms-of-action were investigated by RT-PCR, mitochondrial isolation, as well as immunoprecipitation/blotting analysis, and microscopy. The specificity against Mcl-1 of the novel small molecule inhibitor EU5346 was verified in Mcl-1Δ/nullversus Mcl-1wt/wt Murine Embryonic Fibroblasts (MEFs). Proliferation, survival, and spheroid formation were assessed in response to Mcl-1 and Her2 inhibition.
Results
We demonstrate for a strong correlation between high Mcl-1 protein levels and hypoxia, predominantly in Her2-positive BC cells. Surprisingly, genetic depletion of Mcl-1 decreased Her2 and Hif-1α levels followed by inhibition of BC cell survival. In contrast, Mcl-1 protein levels were not downregulated after genetic depletion of Her2 indicating a regulatory role of Mcl-1 upstream of Her2. Indeed, Mcl-1 and Her2 co-localize within the mitochondrial fraction and form a Mcl-1/Her2- protein complex. Similar to genetically targeting Mcl-1 the novel small molecule Mcl-1 inhibitor EU-5346 induced cell death and decreased spheroid formation in Her2-positive BC cells. Of interest, EU-5346 induced ubiquitination of Mcl-1- bound Her2 demonstrating a previously unknown role for Mcl-1 to stabilize Her2 protein levels. Importantly, targeting Mcl-1 was also active in Her2-positive BC cells resistant to Her2 inhibitors, including a brain-primed Her2-positive cell line.
Conclusion
Our data demonstrate a critical role of Mcl-1 in Her2-positive BC cell survival under hypoxic conditions and provide the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient outcome in BC.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-016-0686-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-016-0686-4
PMCID: PMC4769490  PMID: 26921175
Breast cancer; Myeloid cell leukemia-1; Hypoxia; Apoptosis
24.  The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features 
Introduction
Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells.
Methods
We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations.
Results
High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial–mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy.
Conclusions
In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-015-0652-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-015-0652-6
PMCID: PMC4660783  PMID: 26607327
25.  Genetic variation in mitotic regulatory pathway genes is associated with breast tumor grade 
Purrington, Kristen S. | Slettedahl, Seth | Bolla, Manjeet K. | Michailidou, Kyriaki | Czene, Kamila | Nevanlinna, Heli | Bojesen, Stig E. | Andrulis, Irene L. | Cox, Angela | Hall, Per | Carpenter, Jane | Yannoukakos, Drakoulis | Haiman, Christopher A. | Fasching, Peter A. | Mannermaa, Arto | Winqvist, Robert | Brenner, Hermann | Lindblom, Annika | Chenevix-Trench, Georgia | Benitez, Javier | Swerdlow, Anthony | Kristensen, Vessela | Guénel, Pascal | Meindl, Alfons | Darabi, Hatef | Eriksson, Mikael | Fagerholm, Rainer | Aittomäki, Kristiina | Blomqvist, Carl | Nordestgaard, Børge G. | Nielsen, Sune F. | Flyger, Henrik | Wang, Xianshu | Olswold, Curtis | Olson, Janet E. | Mulligan, Anna Marie | Knight, Julia A. | Tchatchou, Sandrine | Reed, Malcolm W.R. | Cross, Simon S. | Liu, Jianjun | Li, Jingmei | Humphreys, Keith | Clarke, Christine | Scott, Rodney | Fostira, Florentia | Fountzilas, George | Konstantopoulou, Irene | Henderson, Brian E. | Schumacher, Fredrick | Le Marchand, Loic | Ekici, Arif B. | Hartmann, Arndt | Beckmann, Matthias W. | Hartikainen, Jaana M. | Kosma, Veli-Matti | Kataja, Vesa | Jukkola-Vuorinen, Arja | Pylkäs, Katri | Kauppila, Saila | Dieffenbach, Aida Karina | Stegmaier, Christa | Arndt, Volker | Margolin, Sara | Balleine, Rosemary | Arias Perez, Jose Ignacio | Pilar Zamora, M. | Menéndez, Primitiva | Ashworth, Alan | Jones, Michael | Orr, Nick | Arveux, Patrick | Kerbrat, Pierre | Truong, Thérèse | Bugert, Peter | Toland, Amanda E. | Ambrosone, Christine B. | Labrèche, France | Goldberg, Mark S. | Dumont, Martine | Ziogas, Argyrios | Lee, Eunjung | Dite, Gillian S. | Apicella, Carmel | Southey, Melissa C. | Long, Jirong | Shrubsole, Martha | Deming-Halverson, Sandra | Ficarazzi, Filomena | Barile, Monica | Peterlongo, Paolo | Durda, Katarzyna | Jaworska-Bieniek, Katarzyna | Tollenaar, Robert A.E.M. | Seynaeve, Caroline | Brüning, Thomas | Ko, Yon-Dschun | Van Deurzen, Carolien H.M. | Martens, John W.M. | Kriege, Mieke | Figueroa, Jonine D. | Chanock, Stephen J. | Lissowska, Jolanta | Tomlinson, Ian | Kerin, Michael J. | Miller, Nicola | Schneeweiss, Andreas | Tapper, William J. | Gerty, Susan M. | Durcan, Lorraine | Mclean, Catriona | Milne, Roger L. | Baglietto, Laura | dos Santos Silva, Isabel | Fletcher, Olivia | Johnson, Nichola | Van'T Veer, Laura J. | Cornelissen, Sten | Försti, Asta | Torres, Diana | Rüdiger, Thomas | Rudolph, Anja | Flesch-Janys, Dieter | Nickels, Stefan | Weltens, Caroline | Floris, Giuseppe | Moisse, Matthieu | Dennis, Joe | Wang, Qin | Dunning, Alison M. | Shah, Mitul | Brown, Judith | Simard, Jacques | Anton-Culver, Hoda | Neuhausen, Susan L. | Hopper, John L. | Bogdanova, Natalia | Dörk, Thilo | Zheng, Wei | Radice, Paolo | Jakubowska, Anna | Lubinski, Jan | Devillee, Peter | Brauch, Hiltrud | Hooning, Maartje | García-Closas, Montserrat | Sawyer, Elinor | Burwinkel, Barbara | Marmee, Frederick | Eccles, Diana M. | Giles, Graham G. | Peto, Julian | Schmidt, Marjanka | Broeks, Annegien | Hamann, Ute | Chang-Claude, Jenny | Lambrechts, Diether | Pharoah, Paul D.P. | Easton, Douglas | Pankratz, V. Shane | Slager, Susan | Vachon, Celine M. | Couch, Fergus J.
Human Molecular Genetics  2014;23(22):6034-6046.
Mitotic index is an important component of histologic grade and has an etiologic role in breast tumorigenesis. Several small candidate gene studies have reported associations between variation in mitotic genes and breast cancer risk. We measured associations between 2156 single nucleotide polymorphisms (SNPs) from 194 mitotic genes and breast cancer risk, overall and by histologic grade, in the Breast Cancer Association Consortium (BCAC) iCOGS study (n = 39 067 cases; n = 42 106 controls). SNPs in TACC2 [rs17550038: odds ratio (OR) = 1.24, 95% confidence interval (CI) 1.16–1.33, P = 4.2 × 10−10) and EIF3H (rs799890: OR = 1.07, 95% CI 1.04–1.11, P = 8.7 × 10−6) were significantly associated with risk of low-grade breast cancer. The TACC2 signal was retained (rs17550038: OR = 1.15, 95% CI 1.07–1.23, P = 7.9 × 10−5) after adjustment for breast cancer risk SNPs in the nearby FGFR2 gene, suggesting that TACC2 is a novel, independent genome-wide significant genetic risk locus for low-grade breast cancer. While no SNPs were individually associated with high-grade disease, a pathway-level gene set analysis showed that variation across the 194 mitotic genes was associated with high-grade breast cancer risk (P = 2.1 × 10−3). These observations will provide insight into the contribution of mitotic defects to histological grade and the etiology of breast cancer.
doi:10.1093/hmg/ddu300
PMCID: PMC4204763  PMID: 24927736

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