title: High applicability of ASO-RQPCR for detection of minimal residual disease in multiple myeloma by entirely patient-specific primers/probes.
abstract: Allele-specific oligonucleotide real-time quantitative PCR (ASO-RQPCR) is a standardized technique for detection and monitoring of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) but not multiple myeloma (MM) due to a low applicability inherent with presence of somatic hypermutation. Herein, by a staged PCR approach and sequencing, clonality and tumor-specific complementarity-determining region 3 (CDR3) sequence were identified in 13/13 MM by sequential PCR of IgH VDJ (n = 10), IgH DJ (n = 2), or IgK VJ (n = 1). Using consensus primers/probes conventionally employed in ALL, ASO-RQPCR worked in three (23.1 %) cases only. Conversely, using entirely patient-specific primers/probes, ASO-RQPCR was applicable in eight (61.5 %) cases with a sensitivity of 5 × 10-4-10-5. Moreover, using standard curves constructed by serial dilution of plasmids cloned with patient-specific CDR3, ASO-RQPCR was successful in 12 (92.3 %) cases with a sensitivity of 10-4-10-5, but not in a case lacking an N region, in which design of a tumor-specific ASO primer was precluded. Finally, in a patient in complete response (CR), further reduction of MRD after autologous stem cell transplantation (ASCT) was demonstrated. In summary, using entirely patient-specific primers/probes, ASO-RQPCR was applicable in >90 % MM patients and enabled detection of dynamic changes of MRD before and after ASCT despite conventional CR.
authors.affiliation: Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Yinlei
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Kwan Yeung
authors.initials: KY
authors.lastname: Wong
authors.affiliation: Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Tsz Kin
authors.initials: TK
authors.lastname: Fung
authors.affiliation: Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong. jcschim@hku.hk.
authors.forename: Chor Sang
authors.initials: CS
authors.lastname: Chim
mesh_terms: D000483:Alleles; D022801:Complementarity Determining Regions / Q000235:genetics; D017931:DNA Primers; D015342:DNA Probes; D006801:Humans; D009101:Multiple Myeloma / Q000175:diagnosis*; D018365:Neoplasm, Residual / Q000175:diagnosis*; D057285:Precision Medicine / Q000379:methods* / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D022801:Complementarity Determining Regions; D017931:DNA Primers; D015342:DNA Probes
keywords: Minimal residual disease; Multiple myeloma; Patient-specific ASO-RQPCR