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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0336-4) contains supplementary material, which is available to authorized users.

Molecular detection of minimal residual disease in multiple myeloma

“…In this regard, consensus primers developed by the BIOMED‐2 group in the form of several multiplex primer sets are the most widely used (van Dongen et al , ; Sarasquete et al , ; Martinez‐Lopez et al , ; Puig et al , ; Silvennoinen et al , ). Multiple immunoglobulin gene rearrangements are used as MRD targets in MM, including IGH complete VDJ and IGH incomplete DJ rearrangements, IGK deletion (Kde), IGK VJ and IGL VJ rearrangements (Gonzalez et al , ; Puig et al , ; Silvennoinen et al , ; Bai et al , ). Clonal PCR bands are sequenced by Sanger sequencing, and sequencing data is analysed with the ImMunoGeneTics (IMGT) V‐QUEST system (http://www.imgt.org) or the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST; https://blast.ncbi.nlm.nih.gov/) to identify the patient‐specific sequence of the corresponding tumour PC CDR3 region.…”

“…One is the fluorescent techniques used in Ig‐based RQ‐PCR MRD. Taqman probes are widely employed in MM and other haematological malignancies due to their high specificity (Gerard et al , ; Ladetto et al , ; Rasmussen et al , ; Verhagen et al , ; Sarasquete et al , ; Puig et al , ; Bai et al , ), although SYBR Green has been explored in some studies in ALL and a very few reports in MM (Nakao et al , ; Li et al , ; Pine et al , ; Pekova et al , ; Putkonen et al , ). Another important point is the location/mapping of the primers and probes in the RQ‐PCR step.…”

“…Indeed, several studies have demonstrated the influence of SHM on the performance of RQ‐PCR. For instance, in a recent study, we showed that ASO RQ‐PCR using consensus primers and probes was only applicable to 23% ( n = 3) of MM patients (Bai et al , ). In the 13 cases analysed, between one and eight mismatches against consensus primers/probes were found in eight (80%) patients with IGH VDJ rearrangements and one of two patients with IGH DJ rearrangements (Bai et al , ).…”

“…For instance, in a recent study, we showed that ASO RQ‐PCR using consensus primers and probes was only applicable to 23% ( n = 3) of MM patients (Bai et al , ). In the 13 cases analysed, between one and eight mismatches against consensus primers/probes were found in eight (80%) patients with IGH VDJ rearrangements and one of two patients with IGH DJ rearrangements (Bai et al , ). Similarly, Gonzalez et al () showed that 75% of IGH VDJ rearrangements in MM had mismatches against consensus primers/probes designed for ALL, proving that a total mismatch of ≥3 bases would preclude successful RQ‐PCR.…”

Standardisation of minimal residual disease in multiple myeloma

“…Chinese group published data about a possible increase in ASO-qPCR applicability, from 61.5 to 92.3%, using stepwise PCR approach, mismatch analysis, and adding CDR3-cloned plasmid DNA to build the standard curve by entirely patient-specific primers/probes (Bai, Wong, Fung, & Chim, 2016).…”