Clinical Challenges and Consequences of Measurable Residual Disease in Non-APL Acute Myeloid Leukemia
Abstract
:1. Introduction
2. Methods for MRD Detection
2.1. Multiparameter Flow Cytometry
2.2. Quantitative PCR
2.2.1. Core-Binding Factor Leukemia
2.2.2. NPM1 Mutations
2.3. Next Generation Sequencing
3. Practical Challenges
3.1. Peripheral Blood or Bone Marrow?
3.2. Choosing the Optimal Target for MRD Detection
3.3. Timepoint of MRD Evaluation
3.4. MRD Thresholds
4. Clinical Implications of MRD Detection
4.1. Decision towards Allogeneic Transplant
4.2. Pre-Emptive Treatment of Impeding Relapse
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
- Arber, D.A.; Orazi, A.; Hasserjian, R.; Thiele, J.; Borowitz, M.J.; Le Beau, M.M.; Bloomfield, C.D.; Cazzola, M.; Vardiman, J.W. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016, 127, 2391–2405. [Google Scholar] [CrossRef] [PubMed]
- Döhner, H.; Estey, E.; Grimwade, D.; Amadori, S.; Appelbaum, F.R.; Büchner, T.; Dombret, H.; Ebert, B.L.; Fenaux, P.; Larson, R.A.; et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood 2017, 129, 424–447. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Döhner, H.; Weisdorf, D.J.; Bloomfield, C.D. Acute Myeloid Leukemia. N. Engl. J. Med. 2015, 373, 1136–1152. [Google Scholar] [CrossRef] [PubMed]
- Marcucci, G.; Mrózek, K.; Ruppert, A.S.; Archer, K.J.; Pettenati, M.J.; Heerema, N.A.; Carroll, A.J.; Koduru, P.R.; Kolitz, J.E.; Sterling, L.J.; et al. Abnormal Cytogenetics at Date of Morphologic Complete Remission Predicts Short Overall and Disease-Free Survival, and Higher Relapse Rate in Adult Acute Myeloid Leukemia: Results From Cancer and Leukemia Group B Study 8461. J. Clin. Oncol. 2004, 22, 2410–2418. [Google Scholar] [CrossRef] [PubMed]
- Cuneo, A.; Bigoni, R.; Roberti, M.G.; Bardi, A.; Rigolin, G.M.; Piva, N.; Mancini, M.; Nanni, M.; Alimena, G.; Mecucci, C.; et al. Detection and monitoring of trisomy 8 by fluorescence in situ hybridization in acute myeloid leukemia: A multicentric study. Haematologica 1998, 83, 21–26. [Google Scholar]
- Schmidt, H.H.; Pirc-Danoewinata, H.; Linkesch, W.; Strunk, D.; Wieser, R. inv(3)(q21q26) in AML/MDS: Monitoring of the malignant clone with interphase FISH. Haematologica 2003, 88, ECR38. [Google Scholar]
- Beillard, E.; Pallisgaard, N.; van der Velden, V.H.; Bi, W.; Dee, R.; van der Schoot, E.; Delabesse, E.; Macintyre, E.; Gottardi, E.; Saglio, G.; et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR)—A Europe Against Cancer program. Leukemia 2003, 17, 2474–2486. [Google Scholar] [CrossRef]
- Gabert, J.; Beillard, E.; van der Velden, V.H.; Bi, W.; Grimwade, D.; Pallisgaard, N.; Barbany, G.; Cazzaniga, G.; Cayuela, J.M.; Cavé, H.; et al. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia e a Europe Against Cancer program. Leukemia 2003, 17, 2318–2357. [Google Scholar] [CrossRef]
- Grimwade, D.; Jovanovic, J.V.; Hills, R.; Nugent, E.A.; Patel, Y.; Flora, R.; Diverio, D.; Jones, K.; Aslett, H.; Batson, E.; et al. Prospective Minimal Residual Disease Monitoring to Predict Relapse of Acute Promyelocytic Leukemia and to Direct Pre-Emptive Arsenic Trioxide Therapy. J. Clin. Oncol. 2009, 27, 3650–3658. [Google Scholar] [CrossRef]
- Schuurhuis, G.J.; Heuser, M.; Freeman, S.; Bene, M.-C.; Buccisano, F.; Cloos, J.; Grimwade, D.; Haferlach, T.; Hills, R.K.; Hourigan, C.S.; et al. Minimal/measurable residual disease in AML: A consensus document from the European LeukemiaNet MRD Working Party. Blood 2018, 131, 1275–1291. [Google Scholar] [CrossRef]
- Agrawal, M.; Corbacioglu, A.; Paschka, P.; Weber, D.; Gaidzik, V.I.; Jahn, N.; Kündgen, A.; Kindler, T.; Wattad, M.A.; Lübbert, M.; et al. Minimal residual disease monitoring in acute myeloid leukemia (AML) with translocation t(8;21)(q22;q22): Results of the AML Study Group (AMLSG). Blood 2016, 128, 1207. [Google Scholar]
- Weisser, M.; Haferlach, C.; Hiddemann, W.; Schnittger, S. The quality of molecular response to chemotherapy is predictive for the outcome of AML1-ETO-positive AML and is independent of pretreatment risk factors. Leukemia 2007, 21, 1177–1182. [Google Scholar] [CrossRef] [PubMed]
- Ivey, A.; Hills, R.K.; Simpson, M.A.; Jovanovic, J.V.; Gilkes, A.; Grech, A.; Patel, Y.; Bhudia, N.; Farah, H.; Mason, J.; et al. Assessment of Minimal Residual Disease in Standard-Risk AML. N. Engl. J. Med. 2016, 374, 422–433. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Balsat, M.; Renneville, A.; Thomas, X.; de Botton, S.; Caillot, D.; Marceau, A.; Lemasle, E.; Marolleau, J.P.; Nibourel, O.; Berthon, C.; et al. Postinduction minimal residual disease predicts outcome and benefit from allogeneic stem cell transplantation in acute myeloid leukemia with NPM1 mutation: A study be the Acute Leukemia French Association Group. J. Clin. Oncol. 2017, 35, 185–193. [Google Scholar] [CrossRef] [PubMed]
- Krönke, J.; Schlenk, R.F.; Jensen, K.-O.; Tschürtz, F.; Corbacioglu, A.; Gaidzik, V.I.; Paschka, P.; Onken, S.; Eiwen, K.; Habdank, M.; et al. Monitoring of Minimal Residual Disease in NPM1-Mutated Acute Myeloid Leukemia: A Study From the German-Austrian Acute Myeloid Leukemia Study Group. J. Clin. Oncol. 2011, 29, 2709–2716. [Google Scholar] [CrossRef]
- Yin, J.A.L.; O’Brien, M.A.; Hills, R.K.; Daly, S.B.; Wheatley, K.; Burnett, A.K.; Yin, J.A.L. Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: Results of the United Kingdom MRC AML-15 trial. Blood 2012, 120, 2826–2835. [Google Scholar] [CrossRef]
- Kern, W.; Schoch, C.; Haferlach, T.; Schnittger, S. Monitoring of minimal residual disease in acute myeloid leukemia. Crit. Rev. Oncol. 2005, 56, 283–309. [Google Scholar] [CrossRef] [Green Version]
- Voskova, D.; Schoch, C.; Schnittger, S.; Hiddemann, W.; Haferlach, T.; Kern, W. Stability of leukemia- associated aberrant immunophenotypes in patients with acute myeloid leukemia between diagnosis and relapse: Comparison with cytomorphologic; cytogenetic; and molecular genetic findings. Cytom. B Clin. Cytom. 2004, 62, 25–38. [Google Scholar] [CrossRef]
- Freeman, S.D.; Hills, R.K.; Virgo, P.; Khan, N.; Couzens, S.; Dillon, R.; Gilkes, A.; Upton, L.; Nielsen, O.J.; Cavenagh, J.D.; et al. Measurable Residual Disease at Induction Redefines Partial Response in Acute Myeloid Leukemia and Stratifies Outcomes in Patients at Standard Risk Without NPM1 Mutations. J. Clin. Oncol. 2018, 36, 1486–1497. [Google Scholar] [CrossRef]
- Terwijn, M.; Van Putten, W.L.J.; Kelder, A.; Van Der Velden, V.H.; Brooimans, R.A.; Pabst, T.; Maertens, J.; Boeckx, N.; De Greef, G.E.; Valk, P.J.; et al. High Prognostic Impact of Flow Cytometric Minimal Residual Disease Detection in Acute Myeloid Leukemia: Data From the HOVON/SAKK AML 42A Study. J. Clin. Oncol. 2013, 31, 3889–3897. [Google Scholar] [CrossRef]
- Ravandi, F.; Jorgensen, J.; Borthakur, G.; Jabbour, E.; Kadia, T.; Pierce, S.; Brandt, M.; Wang, S.; Konoplev, S.; Wang, X.; et al. Persistence of minimal residual disease assessed by multiparameter flow cytometry is highly prognostic in younger patients with acute myeloid leukemia. Cancer 2017, 123, 426–435. [Google Scholar] [CrossRef] [PubMed]
- Walter, R.B.; Gyurkocza, B.; Storer, B.E.; Godwin, C.D.; Pagel, J.M.; Buckley, S.A.; Sorror, M.L.; Wood, B.L.; Storb, R.; Appelbaum, F.R.; et al. Comparison of minimal residual disease as outcome predictor for AML patients in first complete remission undergoing myeloablative or nonmyeloablative allogeneic hematopoietic cell transplantation. Leukemia 2015, 29, 137–144. [Google Scholar] [CrossRef] [PubMed]
- Miguel, J.S.; Martínez, A.; Macedo, A.; Vidriales, M.; López-Berges, C.; González, M.; Caballero, D.; García-Marcos, M.; Ramos, F.; Fernández-Calvo, J.; et al. Immunophenotyping Investigation of Minimal Residual Disease Is a Useful Approach for Predicting Relapse in Acute Myeloid Leukemia Patients. Blood 1997, 90, 2465–2470. [Google Scholar] [CrossRef]
- Freeman, S.D.; Virgo, P.; Couzens, S.; Grimwade, D.; Russell, N.; Hills, R.K.; Burnett, A.K. Prognostic Relevance of Treatment Response Measured by Flow Cytometric Residual Disease Detection in Older Patients with Acute Myeloid Leukemia. J. Clin. Oncol. 2013, 31, 4123–4131. [Google Scholar] [CrossRef]
- Chen, X.; Xie, H.; Wood, B.L.; Walter, R.; Pagel, J.M.; Becker, P.S.; Sandhu, V.K.; Abkowitz, J.L.; Appelbaum, F.R.; Estey, E.H. Relation of Clinical Response and Minimal Residual Disease and Their Prognostic Impact on Outcome in Acute Myeloid Leukemia. J. Clin. Oncol. 2015, 33, 1258–1264. [Google Scholar] [CrossRef]
- Maurillo, L.; Buccisano, F.; Del Principe, M.I.; Del Poeta, G.; Spagnoli, A.; Panetta, P.; Ammatuna, E.; Neri, B.; Ottaviani, L.; Sarlo, C.; et al. Toward Optimization of Postremission Therapy for Residual Disease–Positive Patients with Acute Myeloid Leukemia. J. Clin. Oncol. 2008, 26, 4944–4951. [Google Scholar] [CrossRef]
- Araki, D.; Wood, B.L.; Othus, M.; Radich, J.P.; Halpern, A.B.; Zhou, Y.; Mielcarek, M.; Estey, E.H.; Appelbaum, F.R.; Walter, R.B. Allogeneic Hematopoietic Cell Transplantation for Acute Myeloid Leukemia: Time to Move Toward a Minimal Residual Disease–Based Definition of Complete Remission? J. Clin. Oncol. 2016, 34, 329–336. [Google Scholar] [CrossRef]
- Zhou, Y.; Othus, M.; Araki, D.; Wood, B.L.; Radich, J.P.; Halpern, A.B.; Mielcarek, M.; Estey, E.H.; Appelbaum, F.R.; Walter, R.B. Pre- and post-transplant quantification of measurable (’minimal’) residual disease via multiparameter flow cytometry in adult acute myeloid leukemia. Leukemia 2016, 30, 1456–1464. [Google Scholar] [CrossRef]
- Norkin, M.; Katragadda, L.; Zou, F.; Xiong, S.; Chang, M.; Dai, Y.; Hsu, J.W.; Moreb, J.S.; Leather, H.; Murthy, H.S.; et al. Minimal residual disease by either flow cytometry or cytogenetics prior to an allogeneic hematopoietic stem cell transplant is associated with poor outcome in acute myeloid leukemia. Blood Cancer J. 2017, 7, 634. [Google Scholar] [CrossRef]
- Getta, B.M.; Devlin, S.M.; Levine, R.L.; Arcila, M.E.; Mohanty, A.S.; Zehir, A.; Tallman, M.S.; Giralt, S.A.; Roshal, M. Multicolor Flow Cytometry and Multigene Next-Generation Sequencing Are Complementary and Highly Predictive for Relapse in Acute Myeloid Leukemia after Allogeneic Transplantation. Boil. Blood Marrow Transplant. 2017, 23, 1064–1071. [Google Scholar] [CrossRef] [Green Version]
- Cilloni, D.; Renneville, A.; Hermitte, F.; Hills, R.K.; Daly, S.; Jovanovic, J.V.; Gottardi, E.; Fava, M.; Schnittger, S.; Weiss, T.; et al. Real-Time Quantitative Polymerase Chain Reaction Detection of Minimal Residual Disease by Standardized WT1 Assay to Enhance Risk Stratification in Acute Myeloid Leukemia: A European LeukemiaNet Study. J. Clin. Oncol. 2009, 27, 5195–5201. [Google Scholar] [CrossRef] [PubMed]
- Grimwade, D.; Mrózek, K. Diagnostic and Prognostic Value of Cytogenetics in Acute Myeloid Leukemia. Hematol. Clin. N. Am. 2011, 25, 1135–1161. [Google Scholar] [CrossRef]
- Grimwade, D.; Freeman, S.D. Defining minimal residual disease in acute myeloid leukemia: Which platforms are ready for “prime time”? Blood 2014, 124, 3345–3355. [Google Scholar] [CrossRef] [PubMed]
- Takatsuki, H.; Yufu, Y.; Tachikawa, Y.; Uike, N. Monitoring minimal residual disease in patients with MLL-AF6 fusion transcript-positive acute myeloid leukemia following allogeneic bone marrow transplantation. Int. J. Hematol. 2002, 75, 298–301. [Google Scholar] [CrossRef] [PubMed]
- Scholl, C.; Schlenk, R.F.; Eiwen, K.; Döhner, H.; Fröhling, S.; Döhner, K. The prognostic value of MLL-AF9 detection in patients with t(9;11)(p22;q23)-positive acute myeloid leukemia. Haematologica 2005, 90, 1626–1634. [Google Scholar] [PubMed]
- Østergaard, M.; Olesen, L.H.; Hasle, H.; Kjeldsen, E.; Hokland, P. WT1 gene expression: An excellent tool for monitoring minimal residual disease in 70% of acute myeloid leukaemia patients—Results from a single-centre study. Br. J. Haematol. 2004, 125, 590–600. [Google Scholar] [CrossRef] [PubMed]
- Lange, T.; Hubmann, M.; Burkhardt, R.; Franke, G.N.; Cross, M.; Scholz, M.; Leiblein, S.; Al-Ali, H.K.; Edelmann, J.; Thiery, J.; et al. Monitoring of WT1 expression in PB and CD34(+) donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning. Leukemia 2011, 25, 498–505. [Google Scholar] [CrossRef] [PubMed]
- Duléry, R.; Nibourel, O.; Gauthier, J.; Elsermans, V.; Behal, H.; Coiteux, V.; Magro, L.; Renneville, A.; Marceau, A.; Boyer, T.; et al. Impact of Wilms’ tumor 1 expression on outcome of patients undergoing allogeneic stem cell transplantation for AML. Bone Marrow Transplant. 2017, 52, 539–543. [Google Scholar] [CrossRef]
- Weber, S.; Alpermann, T.; Dicker, F.; Jeromin, S.; Nadarajah, N.; Eder, C.; Fasan, A.; Kohlmann, A.; Meggendorfer, M.; Haferlach, C.; et al. BAALC expression: A suitable marker for prognostic risk stratification and detection of residual disease in cytogenetically normal acute myeloid leukemia. Blood Cancer J. 2014, 4, e173. [Google Scholar] [CrossRef]
- Jentzsch, M.; Bill, M.; Grimm, J.; Schulz, J.; Goldmann, K.; Beinicke, S.; Häntschel, J.; Pönisch, W.; Franke, G.N.; Vucinic, V.; et al. High Blood BAALC Copy Numbers at Allogeneic Transplantation Predict Early Relapse in Patients with Acute Myeloid Leukemia. Oncotarget 2017, 8, 87944–87954. [Google Scholar] [CrossRef]
- Carturan, S.; Petiti, J.; Rosso, V.; Calabrese, C.; Signorino, E.; Bot-Sartor, G.; Nicoli, P.; Gallo, D.; Bracco, E.; Morotti, A.; et al. Variable but consistent pattern of Meningioma 1 gene (MN1) expression in different genetic subsets of acute myelogenous leukaemia and its potential use as a marker for minimal residual disease detection. Oncotarget 2016, 7, 74082–74096. [Google Scholar] [CrossRef] [PubMed]
- Jentzsch, M.; Bill, M.; Grimm, J.; Schulz, J.; Beinicke, S.; Häntschel, J.; Goldmann, K.; Pönisch, W.; Franke, G.-N.; Vucinic, V.; et al. Prognostic Impact of Blood MN1 Copy Numbers Before Allogeneic Stem Cell Transplantation in Patients With Acute Myeloid Leukemia. HemaSphere 2019, 3, e167. [Google Scholar] [CrossRef]
- Mencia-Trinchant, N.; Hu, Y.; Alas, M.A.; Ali, F.; Wouters, B.J.; Lee, S.; Ritchie, E.K.; Desai, P.; Guzman, M.L.; Roboz, G.J.; et al. Minimal Residual Disease Monitoring of Acute Myeloid Leukemia by Massively Multiplex Digital PCR in Patients with NPM1 Mutations. J. Mol. Diagn. 2017, 19, 537–548. [Google Scholar] [CrossRef] [PubMed]
- Bill, M.; Grimm, J.; Jentzsch, M.; Kloss, L.; Goldmann, K.; Schulz, J.; Beinicke, S.; Häntschel, J.; Cross, M.; Vucinic, V.; et al. Digital droplet PCR-based absolute quantification of pre-transplant NPM1 mutation burden predicts relapse in acute myeloid leukemia patients. Ann. Hematol. 2018, 97, 1757–1765. [Google Scholar] [CrossRef] [PubMed]
- Hindson, C.M.; Chevillet, J.R.; A Briggs, H.; Gallichotte, E.N.; Ruf, I.K.; Hindson, B.J.; Vessella, R.L.; Tewari, M. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat. Methods 2013, 10, 1003–1005. [Google Scholar] [CrossRef] [PubMed]
- Elmaagacli, A.H.; Beelen, D.W.; Kroll, M.; Trzensky, S.; Stein, C.; Schaefer, U.W. Detection of CBFbeta/MYH11 fusion transcripts in patients with inv(16) acute myeloid leukemia after allogeneic bone marrow or peripheral blood progenitor cell transplantation. Bone Marrow Transplant. 1998, 21, 159–166. [Google Scholar] [CrossRef] [PubMed]
- Morschhauser, F.; Cayuela, J.M.; Martini, S.; Baruchel, A.; Rousselot, P.; Socié, G.; Berthou, P.; Jouet, J.P.; Straetmans, N.; Sigaux, F.; et al. Evaluation of minimal residual disease using reverse-transcriptase polymerase chain reaction in t(8;21) acute myeloid leukemia; a multicentre study of 51 patients. J. Clin. Oncol. 2000, 18, 788–794. [Google Scholar] [CrossRef]
- Rücker, F.G.; Agrawal, M.; Corbacioglu, A.; Weber, D.; Kapp-Schwoerer, S.; Gaidzik, V.I.; Jahn, N.; Schroeder, T.; Wattad, M.; Lübbert, M.; et al. Measurable Residual Disease Monitoring in Acute Myeloid Leukemia with t(8;21)(q22;q22.1): Results of the AML Study Group. Blood 2019, 2019, 1425. [Google Scholar] [CrossRef]
- Zhu, H.-H.; Zhang, X.-H.; Qin, Y.-Z.; Liu, D.-H.; Jiang, H.; Chen, H.; Jiang, Q.; Xu, L.-P.; Lu, J.; Han, W.; et al. MRD-directed risk stratification treatment may improve outcomes of t(8;21) AML in the first complete remission: Results from the AML05 multicenter trial. Blood 2013, 121, 4056–4062. [Google Scholar] [CrossRef]
- Schnittger, S.; Weisser, M.; Schoch, C.; Hiddemann, W.; Haferlach, T.; Kern, W. New score predicting for prognosis in PML-RARA1; AML1-ETO1; or CBFB-MYH111 acute myeloid leukemia based on quantification of fusion transcripts. Blood 2003, 102, 2746–2755. [Google Scholar] [CrossRef]
- Jourdan, E.; Boissel, N.; Chevret, S.; Delabesse, E.; Renneville, A.; Cornillet, P.; Blanchet, O.; Cayuela, J.M.; Recher, C.; Raffoux, E.; et al. French AML Intergroup. Prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia. Blood 2013, 121, 2213–2223. [Google Scholar] [CrossRef] [PubMed]
- Willeckens, C.; Blanchet, O.; Renneville, A.; Cornillet-Lefebvre, P.; Pautas, C.; Guieze, R.; Ifrah, N.; Dombret, H.; Jourdan, E.; Preudhomme, C.; et al. Prospective long-term minimal residual disease monitoring using RQ-PCR in RUNX1-RUNX1T1-positive acute myeloid leukemia: Results of the French CBF-2006 trial. Haematologica 2016, 101, 328–335. [Google Scholar] [CrossRef] [PubMed]
- Yin, J.A.L.; Frost, L. Monitoring AML1-ETO and CBFbeta-MYH11 transcripts in acute myeloid leukemia. Curr. Oncol. Rep. 2003, 5, 399–404. [Google Scholar] [CrossRef] [PubMed]
- Falini, B.; Mecucci, C.; Tiacci, E.; Alcalay, M.; Rosati, R.; Pasqualucci, L.; La Starza, R.; Diverio, D.; Colombo, E.; Santucci, A.; et al. Cytoplasmic Nucleophosmin in Acute Myelogenous Leukemia with a Normal Karyotype. N. Engl. J. Med. 2005, 352, 254–266. [Google Scholar] [CrossRef] [PubMed]
- Hubmann, M.; Köhnke, T.; Hoster, E.; Schneider, S.; Dufour, A.; Zellmeier, E.; Fiegl, M.; Braess, J.; Bohlander, S.K.; Subklewe, M.; et al. Molecular response assessment by quantitative real-time polymerase chain reaction after induction therapy in NPM1-mutated patients identifies those at high risk of relapse. Haematologica 2014, 99, 1317–1325. [Google Scholar] [CrossRef]
- Schnittger, S.; Kern, W.; Tschulik, C.; Weiss, T.; Dicker, F.; Falini, B.; Haferlach, C.; Haferlach, T. Minimal residual disease levels assessed by NPM1 mutation–specific RQ-PCR provide important prognostic information in AML. Blood 2009, 114, 2220–2231. [Google Scholar] [CrossRef]
- Chou, W.-C.; Tang, J.-L.; Wu, S.-J.; Tsay, W.; Yao, M.; Huang, S.-Y.; Huang, K.-C.; Chen, C.-Y.; Huang, C.-F.; Tien, H.-F. Clinical implications of minimal residual disease monitoring by quantitative polymerase chain reaction in acute myeloid leukemia patients bearing nucleophosmin (NPM1) mutations. Leukemia 2007, 21, 998–1004. [Google Scholar] [CrossRef]
- Karas, M.; Steinerova, K.; Lysak, D.; Sramek, J.; Jindra, P.; Hrabetova, M.; Jungova, A.; Polivka, J.; Holubec, L. Pre-transplant Quantitative Determination of NPM1 Mutation Significantly Predicts Outcome of AIlogeneic Hematopoietic Stem Cell Transplantation in Patients with Normal Karyotype AML in Complete Remission. Anticancer Res. 2016, 36, 5487–5498. [Google Scholar] [CrossRef]
- Kayser, S.; Benner, A.; Thiede, C.; Martens, U.; Huber, J.; Stadtherr, P.; Janssen, J.W.G.; Röllig, C.; Uppenkamp, M.J.; Bochtler, T.; et al. Pretransplant NPM1 MRD levels predict outcome after allogeneic hematopoietic stem cell transplantation in patients with acute myeloid leukemia. Blood Cancer J. 2016, 6, e449. [Google Scholar] [CrossRef]
- Shayegi, N.; Kramer, M.; Bornhäuser, M.; Schaich, M.; Schetelig, J.; Platzbecker, U.; Röllig, C.; Heiderich, C.; Landt, O.; Ehninger, G.; et al. The level of residual disease based on mutant NPM1 is an independent prognostic factor for relapse and survival in AML. Blood 2013, 122, 83–92. [Google Scholar] [CrossRef]
- Griffith, M.; Miller, C.A.; Griffith, O.L.; Krysiak, K.; Skidmore, Z.L.; Ramu, A.; Walker, J.R.; Dang, H.X.; Trani, L.; Larson, D.E.; et al. Optimizing cancer genome sequencing and analysis. Cell Syst. 2015, 1, 210–223. [Google Scholar] [CrossRef] [PubMed]
- Thol, F.; Gabdoulline, R.; Liebich, A.; Klement, P.; Schiller, J.; Kandziora, C.; Hambach, L.; Stadler, M.; Koenecke, C.; Flintrop, M.; et al. Measurable residual disease monitoring by NGS before allogeneic hematopoietic cell transplantation in AML. Blood 2018, 132, 1703–1713. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Shumilov, E.; Flach, J.; Joncourt, R.; Porret, N.; Wiedemann, G.; Angelillo-Scherrer, A.; Trümper, L.; Fiedler, M.; Jeker, B.; Amstutz, U.; et al. Critical evaluation of current molecular MRD strategies including NGS for the management of AML patients with multiple mutations. Hematol. Oncol. 2019, 37, 319–322. [Google Scholar] [CrossRef] [PubMed]
- Genovese, G.; Kähler, A.K.; Handsaker, R.E.; Lindberg, J.; Rose, S.A.; Bakhoum, S.F.; Chambert, K.; Mick, E.; Neale, B.M.; Fromer, M.; et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N. Engl. J. Med. 2014, 371, 2477–2487. [Google Scholar] [CrossRef] [PubMed]
- Bhatnagar, B.; Eisfeld, A.K.; Nicolet, D.; Mrózek, K.; Blachly, J.S.; Orwick, S.; Lucas, D.M.; Kohlschmidt, J.; Blum, W.; Kolitz, J.E.; et al. Persistence of DNMT3A R882 mutations during remission does not adversely affect outcomes of patients with acute myeloid leukaemia. Br. J. Haematol. 2016, 175, 226–236. [Google Scholar] [CrossRef] [PubMed]
- Grimm, J.; Bill, M.; Jentzsch, M.; Beinicke, S.; Häntschel, J.; Goldmann, K.; Schulz, J.; Cross, M.; Franke, G.N.; Behre, G.; et al. Clinical Impact of Clonal Hematopoiesis in Acute Myeloid Leukemia Patients Receiving Allogeneic Transplantation. Bone Marrow Transplant. 2019, 54, 1189–1197. [Google Scholar] [CrossRef] [PubMed]
- Jongen-Lavrencic, M.; Grob, T.; Kavelaars, F.G.; Al Hinai, A.; Zeilemaker, A.; Erpelinck-Verschueren, C.A.; Gradowska, P.L.; Meijer, R.; Biemond, B.J.; Kooy, M.V.M.; et al. Molecular Minimal Residual Disease in Acute Myeloid Leukemia. New Engl. J. Med. 2018, 378, 1189–1199. [Google Scholar] [CrossRef]
- Kim, T.; Moon, J.H.; Ahn, J.-S.; Kim, Y.-K.; Lee, S.-S.; Ahn, S.-Y.; Jung, S.-H.; Yang, D.-H.; Lee, J.-J.; Choi, S.H.; et al. Next-generation sequencing–based posttransplant monitoring of acute myeloid leukemia identifies patients at high risk of relapse. Blood 2018, 132, 1604–1613. [Google Scholar] [CrossRef]
- Morita, K.; Kantarjian, H.M.; Wang, F.; Yan, Y.; Bueso-Ramos, C.; Sasaki, K.; Issa, G.C.; Wang, S.; Jorgensen, J.; Song, X.; et al. Clearance of Somatic Mutations at Remission and the Risk of Relapse in Acute Myeloid Leukemia. J. Clin. Oncol. 2018, 36, 1788–1797. [Google Scholar] [CrossRef]
- Klco, J.M.; Miller, C.A.; Griffith, M.; Petti, A.; Spencer, D.H.; Ketkar-Kulkarni, S.; Wartman, L.D.; Christopher, M.; Lamprecht, T.L.; Helton, N.M.; et al. Association Between Mutation Clearance After Induction Therapy and Outcomes in Acute Myeloid Leukemia. JAMA 2015, 314, 811–822. [Google Scholar] [CrossRef]
- Kim, T.; Ahn, J.S.; Jung, S.H.; Ahn, S.Y.; Jung, S.Y.; Yang, D.H.; Lee, J.J.; Choi, S.; Lee, J.Y.; Lee, H.Y.; et al. Allogeneic hematopoietic cell transplantation can abrogate increasing risk of relapse from persistent mutations measured by targeted sequencing at remission in normal karyotype acute myeloid leukemia. HemaSphere 2019, 3, 743–744. [Google Scholar] [CrossRef]
- Maurillo, L.; Buccisano, F.; Spagnoli, A.; Del Poeta, G.; Panetta, P.; Neri, B.; Del Principe, M.I.; Mazzone, C.; Consalvo, M.I.; Tamburini, A.; et al. Monitoring of minimal residual disease in adult acute myeloid leukemia using peripheral blood as an alternative source to bone marrow. Haematologica 2007, 92, 605–611. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zeijlemaker, W.; Kelder, A.; Oussoren-Brockhoff, Y.J.M.; Scholten, W.J.; Snel, A.N.; Veldhuizen, D.; Cloos, J.; Ossenkoppele, G.J.; Schuurhuis, G.J. Peripheral blood minimal residual disease may replace bone marrow minimal residual disease as an immunophenotypic biomarker for impending relapse in acute myeloid leukemia. Leukemia 2016, 30, 708–715. [Google Scholar] [CrossRef] [PubMed]
- Corbacioglu, A.; Scholl, C.; Schlenk, R.F.; Eiwen, K.; Du, J.; Bullinger, L.; Fröhling, S.; Reimer, P.; Rummel, M.; Derigs, H.-G.; et al. Prognostic Impact of Minimal Residual Disease in CBFB-MYH11-Positive Acute Myeloid Leukemia. J. Clin. Oncol. 2010, 28, 3724–3729. [Google Scholar] [CrossRef] [PubMed]
- Ommen, H.B.; Schnittger, S.; Jovanovic, J.V.; Ommen, I.B.; Hasle, H.; Østergaard, M.; Grimwade, D.; Hokland, P. Strikingly different molecular relapse kinetics in NPM1c, PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 acute myeloid leukemias. Blood 2010, 115, 198–205. [Google Scholar] [CrossRef]
- Boeckx, N.; De Roover, J.; Van Der Velden, V.H.J.; Maertens, J.; Uyttebroeck, A.; Vandenberghe, P.; Van Dongen, J.J.M. Quantification of CBFB-MYH11 fusion gene levels in paired peripheral blood and bone marrow samples by real-time PCR. Leukemia 2005, 19, 1988–1990. [Google Scholar] [CrossRef] [Green Version]
- Papaemmanuil, E.; Gerstung, M.; Bullinger, L.; Gaidzik, V.I.; Paschka, P.; Roberts, N.D.; Potter, N.E.; Heuser, M.; Thol, F.; Bolli, N.; et al. Genomic Classification and Prognosis in Acute Myeloid Leukemia. N. Engl. J. Med. 2016, 374, 2209–2221. [Google Scholar] [CrossRef]
- Jaiswal, S.; Natarajan, P.; Silver, A.J.; Gibson, C.J.; Bick, A.G.; Shvartz, E.; McConkey, M.; Gupta, N.; Gabriel, S.; Ardissino, D.; et al. Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease. N. Engl. J. Med. 2017, 377, 111–121. [Google Scholar] [CrossRef]
- Pløen, G.G.; Nederby, L.; Guldberg, P.; Hansen, M.; Ebbesen, L.H.; Jensen, U.B.; Hokland, P.; Aggerholm, A. Persistence of DNMT3A mutations at long-term remission in adult patients with AML. Br. J. Haematol. 2014, 167, 478–486. [Google Scholar] [CrossRef]
- Rothenberg-Thurley, M.; Amler, S.; Goerlich, D.; Kohnke, T.; Konstandin, N.P.; Schneider, S.; Sauerland, M.C.; Herold, T.; Hubmann, M.; Ksienzyk, B.; et al. Persistence of pre-leukemic clones during first remission and risk of relapse in acute myeloid leukemia. Leukemia 2018, 32, 1598–1608. [Google Scholar] [CrossRef] [Green Version]
- Porter, C.C. Germ line mutations associated with leukemias. ASH Educ. Program Book 2016, 2016, 302–308. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bornhäuser, M.; Oelschlaegel, U.; Platzbecker, U.; Bug, G.; Lutterbeck, K.; Kiehl, M.G.; Schetelig, J.; Kiani, A.; Illmer, T.; Schaich, M.; et al. Monitoring of donor chimerism in sorted CD34+ peripheral blood cells allows the sensitive detection of imminent relapse after allogeneic stem cell transplantation. Haematologica 2009, 94, 1613–1617. [Google Scholar] [CrossRef] [PubMed]
- Jovanovic, J.V.; Ivey, A.; Vannucchi, A.M.; Lippert, E.; Oppliger Leibundgut, E.; Cassinat, B.; Pallisgaard, N.; Maroc, N.; Hermouet, S.; Nickless, G.; et al. Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2- V617F-associated myeloproliferative neoplasms: A joint European LeukemiaNet/MPN & MPNr-EuroNet (COST action BM0902) study. Leukemia 2013, 27, 2032–2039. [Google Scholar] [PubMed]
- Zeijlemaker, W.; Grob, T.; Meijer, R.; Hanekamp, D.; Kelder, A.; Carbaat-Ham, J.C.; Oussoren-Brockhoff, Y.J.M.; Snel, A.N.; Veldhuizen, D.; Scholten, W.J.; et al. CD34+CD38- leukemic stem cell frequency to predict outcome in acute myeloid leukemia. Leukemia 2019, 33, 1102–1112. [Google Scholar] [CrossRef] [PubMed]
- Köhnke, T.; Sauter, D.; Ringel, K.; Hoster, E.; Laubender, R.P.; Hubmann, M.; Bohlander, S.K.; Kakadia, P.M.; Schneider, S.; Dufour, A.; et al. Early assessment of minimal residual disease in AML by flow cytometry during aplasia identifies patients at increased risk of relapse. Leukemia 2015, 29, 377–386. [Google Scholar] [CrossRef]
- Buckley, S.A.; Wood, B.L.; Othus, M.; Hourigan, C.S.; Ustun, C.; Linden, M.A.; DeFor, T.E.; Malagola, M.; Anthias, C.; Valkova, V.; et al. Minimal residual disease prior to allogeneic hematopoietic cell transplantation in acute myeloid leukemia: A meta-analysis. Haematologica 2017, 102, 865–873. [Google Scholar] [CrossRef]
- Hokland, P.; Ommen, H.B. Towards individualized follow-up in adult acute myeloid leukemia in remission. Blood 2011, 117, 2577–2584. [Google Scholar] [CrossRef] [Green Version]
- Ravandi, F.; Walter, R.B.; Freeman, S.D. Evaluating measurable residual disease in acute myeloid leukemia. Blood Adv. 2018, 2, 1356–1366. [Google Scholar] [CrossRef] [Green Version]
- Ossenkoppele, G.; Schuurhuis, G.J. MRD in AML: Does it already guide therapy decision-making? ASH Educ. Program Book 2016, 2016, 356–365. [Google Scholar] [CrossRef]
- Hourigan, C.S.; Gale, R.P.; Gormley, N.J.; Ossenkoppele, G.J.; Walter, R.B. Measurable residual disease testing in acute myeloid leukaemia. Leukemia 2017, 31, 1482–1490. [Google Scholar] [CrossRef]
- Esteve, J.; Escoda, L.; Martin, G.; Rubio, V.; Díaz-Mediavilla, J.; Gonzalez, M.; Rivas, C.; Alvarez, C.; Miguel, J.D.G.S.; Brunet, S.; et al. Outcome of patients with acute promyelocytic leukemia failing to front-line treatment with all-trans retinoic acid and anthracycline-based chemotherapy (PETHEMA protocols LPA96 and LPA99): Benefit of an early intervention. Leukemia 2007, 21, 446–452. [Google Scholar] [CrossRef] [PubMed]
- Coco, F.L.; Diverio, D.; Avvisati, G.; Petti, M.C.; Meloni, G.; Pogliani, E.M.; Biondi, A.; Rossi, G.; Carlo-Stella, C.; Selleri, C.; et al. Therapy of Molecular Relapse in Acute Promyelocytic Leukemia. Blood 1999, 94, 2225–2229. [Google Scholar] [CrossRef] [PubMed]
- Platzbecker, U.; Middeke, J.M.; Sockel, K.; Herbst, R.; Wolf, D.; Baldus, C.D.; Oelschlägel, U.; Mütherig, A.; Fransecky, L.; Noppeney, R.; et al. Measurable residual disease-guided treatment with azacitidine to prevent haematological relapse in patients with myelodysplastic syndrome and acute myeloid leukaemia (RELAZA2): An open-label; multicentre; phase 2 trial. Lancet Oncol. 2018, 19, 1668–1679. [Google Scholar] [CrossRef]
- Burnett, A.K.; Goldstone, A.; Hills, R.K.; Milligan, D.; Yin, J.; Wheatley, K.; Hunter, A.; Russell, N.; Prentice, A. Curability of Patients With Acute Myeloid Leukemia Who Did Not Undergo Transplantation in First Remission. J. Clin. Oncol. 2013, 31, 1293–1301. [Google Scholar] [CrossRef] [PubMed]
- Estey, E.H. Acute myeloid leukemia: 2019 update on risk-stratification and management. Am. J. Hematol. 2018, 93, 1267–1291. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Appelbaum, F.R. Hematopoietic cell transplantation as treatment of patients with acute myeloid leukemia with measurable residual disease after consolidation therapy. Best Pract. Res. Clin. Haematol. 2018, 31, 405–409. [Google Scholar] [CrossRef]
- Nguyen, S.; Leblanc, T.; Fenaux, P.; Witz, F.; Blaise, D.; Pigneux, A.; Thomas, X.; Rigal-Huguet, F.; Lioure, B.; Auvrignon, A.; et al. A white blood cell index as the main prognostic factor in t(8;21) acute myeloid leukemia (AML): A survey of 161 cases from the French AML Intergroup. Blood 2002, 99, 3517–3523. [Google Scholar] [CrossRef]
- Schlenk, R.; Benner, A.; Krauter, J.; Büchner, T.; Sauerland, C.; Ehninger, G.; Schaich, M.; Mohr, B.; Niederwieser, D.; Krahl, R.; et al. Individual Patient Data–Based Meta-Analysis of Patients Aged 16 to 60 Years With Core Binding Factor Acute Myeloid Leukemia: A Survey of the German Acute Myeloid Leukemia Intergroup. J. Clin. Oncol. 2004, 22, 3741–3750. [Google Scholar] [CrossRef]
- Marcucci, G.; Mrózek, K.; Ruppert, A.S.; Maharry, K.; Kolitz, J.E.; Moore, J.O.; Mayer, R.J.; Pettenati, M.J.; Powell, B.L.; Edwards, C.G.; et al. Prognostic Factors and Outcome of Core Binding Factor Acute Myeloid Leukemia Patients With t(8;21) Differ From Those of Patients With inv(16): A Cancer and Leukemia Group B Study. J. Clin. Oncol. 2005, 23, 5705–5717. [Google Scholar] [CrossRef]
- Schlenk, R.F.; Pasquini, M.C.; Perez, W.S.; Zhang, M.J.; Krauter, J.; Antin, J.H.; Bashey, A.; Bolwell, B.J.; Büchner, T.; Cahn, J.Y.; et al. CIBMTR Acute Leukemia Working Committee. HLA-identical sibling allogeneic transplants versus chemotherapy in acute myelogenous leukemia with t(8;21) in first complete remission: Collaborative study between the German AML Intergroup and CIBMTR. Biol. Blood Marrow Transplant. 2008, 14, 187–196. [Google Scholar] [CrossRef]
- Venditti, A.; Piciocchi, A.; Candoni, A.; Melillo, L.; Calafiore, V.; Cairoli, R.; De Fabritiis, P.; Storti, G.; Salutari, P.; Lanza, F.; et al. GIMEMA AML1310 trial of risk-adapted, MRD-directed therapy for young adults with newly diagnosed acute myeloid leukemia. Blood 2019, 134, 935–945. [Google Scholar] [CrossRef] [PubMed]
- Versluis, J.; Kalin, B.; Zeijlemaker, W.; Passweg, J.; Graux, C.; Manz, M.G.; Vekemans, M.-C.; Biemond, B.J.; Legdeur, M.-C.J.; Kooy, M.V.M.; et al. Graft-Versus-Leukemia Effect of Allogeneic Stem-Cell Transplantation and Minimal Residual Disease in Patients with Acute Myeloid Leukemia in First Complete Remission. JCO Precis. Oncol. 2017, 1, 1–13. [Google Scholar] [CrossRef]
- Brunstein, C.G.; Gutman, J.A.; Weisdorf, D.J.; Woolfrey, A.E.; DeFor, T.E.; Gooley, T.A.; Verneris, M.R.; Appelbaum, F.R.; Wagner, J.E.; Delaney, C. Allogeneic hematopoietic cell transplantation for hematologic malignancy: Relative risks and benefits of double umbilical cord blood. Blood 2010, 116, 4693–4699. [Google Scholar] [CrossRef] [PubMed]
- Milano, F.; Gooley, T.; Wood, B.; Woolfrey, A.; Flowers, M.E.; Doney, K.; Witherspoon, R.; Mielcarek, M.; Deeg, J.H.; Sorror, M.; et al. Cord-Blood Transplantation in Patients with Minimal Residual Disease. N. Engl. J. Med. 2016, 375, 944–953. [Google Scholar] [CrossRef]
- Topp, M.S.; Gökbuget, N.; Zugmaier, G.; Degenhard, E.; Goebeler, M.-E.; Klinger, M.; Neumann, S.A.; Horst, H.A.; Raff, T.; Viardot, A.; et al. Long-term follow-up of hematologic relapse-free survival in a phase 2 study of blinatumomab in patients with MRD in B-lineage ALL. Blood 2012, 120, 5185–5187. [Google Scholar] [CrossRef] [Green Version]
- Goodyear, O.C.; Dennis, M.; Jilani, N.Y.; Loke, J.; Siddique, S.; Ryan, G.; Nunnick, J.; Khanum, R.; Raghavan, M.; Cook, M.; et al. Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML). Blood 2012, 119, 3361–3369. [Google Scholar] [CrossRef]
- Dinardo, C.D.; Stein, E.M.; De Botton, S.; Roboz, G.J.; Altman, J.K.; Mims, A.S.; Swords, R.; Collins, R.H.; Mannis, G.N.; Pollyea, D.A.; et al. Durable Remissions with Ivosidenib in IDH1-Mutated Relapsed or Refractory AML. N. Engl. J. Med. 2018, 378, 2386–2398. [Google Scholar] [CrossRef]
- Stein, E.M.; Dinardo, C.D.; Fathi, A.T.; Pollyea, D.A.; Stone, R.M.; Altman, J.K.; Roboz, G.J.; Patel, M.R.; Collins, R.; Flinn, I.W.; et al. Molecular remission and response patterns in patients with mutant-IDH2 acute myeloid leukemia treated with enasidenib. Blood 2019, 133, 676–687. [Google Scholar] [CrossRef]
- Sandmaier, B.M.; Khaled, S.; Oran, B.; Gammon, G.; Trone, D.; Frankfurt, O. Results of a phase 1 study of quizartinib as maintenance therapy in subjects with acute myeloid leukemia in remission following allogeneic hematopoietic stem cell transplant. Am. J. Hematol. 2018, 93, 222–231. [Google Scholar] [CrossRef]
- Perl, A.; Martinelli, G.; Cortes, J.; Neubauer, A.; Berman, E.; Paolini, S.; Montesinos, P.; Baer, M.; Larson, R.; Ustun, C.; et al. Gilteritinib significantly prolongs overall survival in patients with FLT3-mutated (FLT3mut+) relapsed/refractory acute myeloid leukemia (AML): Results from the phase 3 Admital trial. Hemasphere 2019, 3, 392–393. [Google Scholar] [CrossRef]
- Godwin, C.D.; Gale, R.P.; Walter, R.B. Gemtuzumab ozogamicin in acute myeloid leukemia. Leukemia 2017, 31, 1855–1868. [Google Scholar] [CrossRef] [PubMed]
- Van de Loosdrecht, A.A.; van Wetering, S.; Santegoets, S.J.A.M.; Singh, S.K.; Eeltink, C.M.; den Hartog, Y.; Koppes, M.; Kaspers, J.; Ossenkoppele, G.J.; Kruisbeek, A.M.; et al. A novel allogeneic off-the-shelf dendritic cell vaccine for post-remission treatment of elderly patients with acute myeloid leukemia. Cancer Immunol. Immunother. 2018, 67, 1505–1518. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Confounding Factors | False Negative Results | False Positive Results |
---|---|---|
patient or disease related factors |
|
|
sampling related factors |
|
|
assay related factors |
|
|
Method | Sensitivity | Advantage | Disadvantage |
---|---|---|---|
Conventional Morphology: blast count | 1 in 20 cells | - | - |
FISH: numeric and structural cytogenetic aberrations | 1 in 100–500 cells |
|
|
MFC: LAIP, DfN | 1 in 1000–100,000 |
|
|
qRT-PCR: molecular aberrations | 1 in 100,000–1,000,000 cells |
|
|
NGS: molecular aberrations | 1 in 100,000–1,000,000 cells |
|
|
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Jentzsch, M.; Schwind, S.; Bach, E.; Stasik, S.; Thiede, C.; Platzbecker, U. Clinical Challenges and Consequences of Measurable Residual Disease in Non-APL Acute Myeloid Leukemia. Cancers 2019, 11, 1625. https://doi.org/10.3390/cancers11111625
Jentzsch M, Schwind S, Bach E, Stasik S, Thiede C, Platzbecker U. Clinical Challenges and Consequences of Measurable Residual Disease in Non-APL Acute Myeloid Leukemia. Cancers. 2019; 11(11):1625. https://doi.org/10.3390/cancers11111625
Chicago/Turabian StyleJentzsch, Madlen, Sebastian Schwind, Enrica Bach, Sebastian Stasik, Christian Thiede, and Uwe Platzbecker. 2019. "Clinical Challenges and Consequences of Measurable Residual Disease in Non-APL Acute Myeloid Leukemia" Cancers 11, no. 11: 1625. https://doi.org/10.3390/cancers11111625