Comparative analysis between RQ‐PCR and digital‐droplet‐PCR of immunoglobulin/T‐cell receptor gene rearrangements to monitor minimal residual disease in acute lymphoblastic leukaemia

Starza, Irene Della; Nunes, Vittorio; Cavalli, Marzia; Novi, Lucia Anna De; Ilari, Caterina; Apicella, Valerio; Vitale, Antonella; Testi, Anna Maria; Giudice, Ilaria Del; Chiaretti, Sabina; Foà, Robin; Guarini, Anna

doi:10.1111/bjh.14082

Cited by 60 publications

(46 citation statements)

References 38 publications

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“…MRD detection by dPCR is still under investigation. From current studies, the sensitivity and accuracy of dPCR appear to be at least comparable to those of ASO RQ‐PCR (Drandi et al , ; Della Starza et al , ). Also, dPCR allows absolute quantification of the number of copies of the target DNA molecules, without a need for a standard curve constructed by serial dilutions of diagnostic DNA or plasmid DNA.…”

Section: Digital Pcrsupporting

confidence: 57%

Molecular detection of minimal residual disease in multiple myeloma

2017

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Despite the significantly higher complete remission rates and improved survival achieved in the last decade, multiple myeloma (MM) patients continue to relapse due to persistence of minimal residual disease (MRD). Generally, MRD refers to persistence of low levels of disease in the order of one tumour cell in ≥10 normal cells. Currently, molecular and immunophenotypic techniques are employed for MRD detection. This review focuses on MRD detection by molecular techniques, including allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), next-generation sequencing (NGS) and digital PCR (dPCR), in addition to a brief description of, and comparison with, multiparameter flow cytometry. The basic principles, technical advantages and limitations, and the clinical impact of all three molecular techniques are reviewed and compared. They all have a sensitivity of at least 10 , among which ASO real-time quantitative PCR is the most well-standardized, and NGS carries the highest sensitivity and applicability, while dPCR is still under investigation. Furthermore, molecular MRD negativity is a favourable prognostic factor for survival of patients with MM. However, several challenges inherent to molecular detection of MRD still remain to be overcome, particularly false negativity and failure to detect extramedullary disease. Finally, detection of MRD from peripheral blood remains challenging.

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Section: Digital Pcrsupporting

confidence: 57%

Molecular detection of minimal residual disease in multiple myeloma

2017

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“…ddPCR reaches a sensitivity of less than 0.1%, as long as enough DNA input can be guaranteed, which is higher than the sensitivity of qPCR (around 0.5%) . Two studies have compared ddPCR to qPCR for minimal residual disease detection in peripheral blood and bone marrow aspirates of lymphomas and have shown comparable results with an advantage of ddPCR with regard to sensitivity …”

Section: Discussionmentioning

confidence: 99%

“…In this study, we present the newest generation of quantitative PCR, ie, droplet digital PCR (ddPCR). This technique is known for its superior sensitivity (<0.1%) compared with the currently used PCR strategies (0.5%–5%) …”

Section: Introductionmentioning

confidence: 99%

The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid

Hiemcke-Jiwa

Minnema

Loon

et al. 2017

Hematological Oncology

100

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The gold standard for diagnosis of central nervous system lymphomas still regards a stereotactic brain biopsy, with the risk of major complications for the patient. As tumor cells can be detected in cerebrospinal fluid (CSF), CSF analysis can be used as an alternative. In this respect, mutation analysis in CSF can be of added value to other diagnostic parameters such a cytomorphology and clonality analysis. A well-known example of targeted mutation analysis entails MYD88 p.(L265P) detection, which is present in the majority of Bing Neel syndrome and primary central nervous system lymphoma (PCNSL) patients. Unfortunately, tumor yield in CSF can be very low. Therefore, use of the highly sensitive droplet digital PCR (ddPCR) might be a suitable analysis strategy for targeted mutation detection. We analyzed 26 formalin fixed paraffin embedded (FFPE) samples (8 positive and 18 negative for MYD88 p.(L265P) mutation) by ddPCR, of which the results were compared with next generation sequencing (NGS). Subsequently, 32 CSF samples were analyzed by ddPCR. ddPCR and NGS results on FFPE material showed 100% concordance. Among the 32 CSF samples, 9 belonged to patients with lymphoplasmacytic lymphoma (LPL) and clinical suspicion of Bing Neel syndrome, and 3 belonged to patients with PCNSL. Nine of these samples tested positive for MYD88 p.(L265P) (8 LPL and 1 PCNSL). This study shows that sensitive MYD88 mutation analysis by ddPCR in CSF is highly reliable and can be applied even when DNA input is low. Therefore, ddPCR is of added value to current diagnostic parameters, especially when the available amount of DNA is limited.

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“…Recent evidences, as well as our experience, showed that MRD by NGS appears more specific for relapse prediction than MRD by RQ, reducing the proportion of false‐positive results.…”

Section: Discussionmentioning

confidence: 73%

“…Next‐generation sequencing (NGS) has also been shown to represent an excellent tool for MRD detection in precursor and mature B‐cell tumors . One important advantage of NGS compared with RQ‐based MRD assessment is that its quantitative discrimination is always superimposable to the sensitivity, while the RQ quantitative range is usually inferior to the sensitivity threshold.…”

Section: Discussionmentioning

confidence: 99%

Minimal residual disease (MRD) in non‐Hodgkin lymphomas: Interlaboratory reproducibility on marrow samples with very low levels of disease within the FIL (Fondazione Italiana Linfomi) MRD Network

Starza

Cavalli

Novi

et al. 2019

Hematological Oncology

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In 2009, the four laboratories of the Fondazione Italiana Linfomi (FIL) minimal residual disease (MRD) Network started a collaborative effort to harmonize and standardize their methodologies at the national level, performing quality control (QC) rounds for follicular lymphoma (FL) and mantle cell lymphoma (MCL) MRD assessment. In 16 QC rounds between 2010 and 2017, the four laboratories received 208 bone marrow (BM) samples (126 FL; 82 MCL); 187 were analyzed, according to the EuroMRD Consortium guidelines, by both nested (NEST) polymerase chain reaction (PCR) and real‐time quantitative (RQ) PCR for BCL2/IGH MBR or IGHV rearrangements. Here, we aimed at analyzing the samples that challenged the interlaboratory reproducibility and data interpretation. Overall, 156/187 BM samples (83%) were concordantly classified as NEST+/RQ+ or NEST−/RQ− by all the four laboratories. The remaining 31 samples (17%) resulted alternatively positive and negative in the interlaboratory evaluations, independently of the method and the type of rearrangement, and were defined “borderline” (brd) samples: 12 proved NEST brd/RQ brd, 7 NEST−/RQ brd, 10 NEST brd/RQ positive not quantifiable (PNQ), and 2 NEST brd/RQ−. Results did not change even increasing the number of replicates/sample. In 6/31 brd samples, droplet digital PCR (ddPCR) was tested and showed no interlaboratory discordance. Despite the high interlaboratory reproducibility in the MRD analysis obtained and maintained by the QC round strategy, samples with the lowest MRD levels can still represent a challenge: 17% (31/187) of our samples showed discordant results in interlaboratory assessments, with 6.4% (12/187) remained brd even applying the two methods. Thus, although representing a minority, brd samples are still problematic, especially when a clinically oriented interpretation of MRD results is required. Alternative, novel methods such as ddPCR and next‐generation sequencing have the potential to overcome the current limitations.

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Comparative analysis between RQ‐PCR and digital‐droplet‐PCR of immunoglobulin/T‐cell receptor gene rearrangements to monitor minimal residual disease in acute lymphoblastic leukaemia

Cited by 60 publications

References 38 publications

Molecular detection of minimal residual disease in multiple myeloma

Molecular detection of minimal residual disease in multiple myeloma

The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid

Minimal residual disease (MRD) in non‐Hodgkin lymphomas: Interlaboratory reproducibility on marrow samples with very low levels of disease within the FIL (Fondazione Italiana Linfomi) MRD Network

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