PURPOSE: Approximately 1-2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients. METHODS: BCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR). RESULTS: In total, 330 blood samples (2-34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios. CONCLUSIONS: Characterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.

3 Medizinische Klinik Medizinische Fakultät Mannheim der Universität Heidelberg Mannheim Germany

Abteilung Hämatologie Onkologie Klinik für Innere Medizin 2 Universitätsklinikum Jena Am Klinikum 1 07747 Jena Germany

Bergonie Institute Cancer Center Bordeaux INSERM U1218 University of Bordeaux Bordeaux France

Department of Experimental Diagnostic and Specialty Medicine Institute of Hematology Lorenzo e Ariosto Seràgnoli University of Bologna Bologna Italy

Department of Hematology and Oncology University of Leipzig Leipzig Germany

Department of Molecular Genetics Institute of Hematology and Blood Transfusion Prague Czech Republic

Faculty of Medicine Imperial College London London UK

Hematology Department Fundeni Clinical Institute University of Medicine and Pharmacy 'Carol Davila' Bucharest Romania

Hematopathology Unit Department of Pathology University of Barcelona Barcelona Spain

School of Medicine University of Southampton Southampton UK

Wessex Regional Genetics Laboratory Salisbury NHS Foundation Trust Salisbury UK

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Arun AK, Senthamizhselvi A, Mani S, Vinodhini K, Janet NB, Lakshmi KM, Abraham A, George B, Srivastava A, Srivastava VM, Mathews V, Balasubramanian P (2017) Frequency of rare PubMed

Awad SA, Hohtari H, Javarappa KK, Brandstoetter T, Kim D, Potdar S, Heckman CA, Kytölä S, Porkka K, Doma E, Sexl V, Kankainen M, Mustjoki S (2019)

Baccarani M, Castagnetti F, Gugliotta G, Rosti G, Soverini S, Albeer A, Pfirrmann M (2019) The proportion of different PubMed

Branford S, Rudzki Z, Hughes TP (2000) A novel PubMed

Cayuela JM, Rousselot P, Nicolini F, Espinouse D, Ollagnier C, Bui-Thi MH, Chabane K, Raffoux E, Callet-Bauchu E, Tigaud I, Magaud JP, Hayette S (2005) Identification of a rare e8a2 PubMed

Colla S, Sammarelli G, Voltolini S, Crugnola M, Sebastio P, Giuliani N (2004) e6a2 PubMed

Cross NC, Melo JV, Feng L, Goldman JM (1994) An optimized multiplex polymerase chain reaction (PCR) for detection of PubMed

Cross NC, White HE, Colomer D, Ehrencrona H, Foroni L, Gottardi E, Lange T, Lion T, Polakova KM, Dulucq S, Martinelli G, Leibundgut EO, Pallisgaard N, Barbany G, Sacha T, Talmaci R, Izzo B, Saglio G, Pane F, Müller MC, Hochhaus A (2015) Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia. Leukemia 29(5):999–1003 PubMed PMC

Duan MH, Li H, Cai H (2017) A rare e13a3 (b2a3) PubMed

Hehlmann R, Hochhaus A, Baccarani M (2007) Chronic myeloid leukaemia. Lancet 370(9584):342–350 PubMed

Hochhaus A, Reiter A, Skladny H, Melo JV, Sick C, Berger U, Guo JQ, Arlinghaus RB, Hehlmann R, Goldman JM, Cross NC (1996) A novel PubMed

Hochhaus A, Baccarani M, Silver RT, Schiffer C, Apperley JF, Cervantes F, Clark RE, Cortes JE, Deininger MW, Guilhot F, Hjorth-Hansen H, Hughes TP, Janssen J, Kantarjian HM, Kim DW, Larson RA, Lipton JH, Mahon FX, Mayer J, Nicolini F, Niederwieser D, Pane F, Radich JP, Rea D, Richter J, Rosti G, Rousselot P, Saglio G, Saußele S, Soverini S, Steegmann JL, Turkina A, Zaritskey A, Hehlmann R (2020) European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia 34(4):966–984 PubMed PMC

Melo JV (1996) The diversity of PubMed

Melo JV (1997) PubMed

Müller MC, Erben P, Saglio G, Gottardi E, Nyvold CG, Schenk T, Ernst T, Lauber S, Kruth J, Hehlmann R, Hochhaus A, European LeukemiaNet (2008) Harmonization of PubMed

Quintás-Cardama A, Cortes J (2009) Molecular biology of PubMed PMC

Rinke J, Schäfer V, Schmidt M, Ziermann J, Kohlmann A, Hochhaus A, Ernst T (2013) Genotyping of 25 leukemia-associated genes in a single work flow by next-generation sequencing technology with low amounts of input template DNA. Clin Chem 59(8):1238–1250 PubMed

Ross DM, O’Hely M, Bartley PA, Dang P, Score J, Goyne JM, Sobrinho-Simoes M, Cross NC, Melo JV, Speed TP, Hughes TP, Morley AA (2013) Distribution of genomic breakpoints in chronic myeloid leukemia: analysis of 308 patients. Leukemia 27(10):2105–2107 PubMed

Sharplin K, Altamura H, Taylor K, Wellwood J, Taylor D, Branford S (2019) Chronic myeloid leukaemia: the dangers of not knowing your PubMed

Snyder DS, McMahon R, Cohen SR, Slovak ML (2004) Chronic myeloid leukemia with an e13a3 PubMed

Verma D, Kantarjian HM, Jones D, Luthra R, Borthakur G, Verstovsek S, Rios MB, Cortes J (2009) Chronic myeloid leukemia (CML) with P190 PubMed PMC

Yu S, Cui M, He X, Jing R, Wang H (2017) A review of the challenge in measuring and standardizing PubMed

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