Multiplex Droplet Digital PCR Quantification of Recurrent Somatic Mutations in Diffuse Large B-Cell and Follicular Lymphoma

Alcaide, Miguel; Yu, Stephen; Bushell, Kevin; Fornika, Daniel; Nielsen, Julie S.; Nelson, Brad H.; Mann, Koren K.; Assouline, Sarit; Johnson, Nathalie A.; Morin, Ryan D.

doi:10.1373/clinchem.2016.255315

Cited by 45 publications

(37 citation statements)

References 39 publications

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“…To investigate the feasibility of ddPCR for MYD88 p.(L265P) detection, it was first analyzed whether results of ddPCR on FFPE material were comparable to those of former NGS analysis. As shown in Table , the results of ddPCR and NGS matched perfectly, which corresponds to previous studies that investigated correlation of NGS and ddPCR for MYD88 p.(L265P) detection or proved applicability of ddPCR for MYD88 p.(L265P) detection for several non‐Hodgkin lymphomas on FFPE material, as well as on fresh tumor tissue and in liquid biopsies (blood) …”

Section: Discussionmentioning

confidence: 99%

“…Considering the complication risk of a brain biopsy, mutation detection in CSF is preferred. 20,21 In addition, we have shown that ddPCR is a highly reliable and sensitive method for detection of MYD88 p.(L265P) in CSF ( Notwithstanding the previously mentioned advantages of ddPCR, one must realize that NGS is becoming more and more standard procedure for analyzing lymphomas. Also, NGS is evolving rapidly and is increasingly able to analyze small DNA concentrations.…”

Section: Discussionmentioning

confidence: 99%

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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

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

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

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“…There are other recent studies that evaluated plasma cfDNA levels in DLBCL (Kurtz et al, 2018), addressing whether it can predict or monitor therapy response or if it can predict tumor burden or prognosis of FL cases (Delfau-Larue et al, 2018). Using droplet digital PCR, Alcaide et al detected previously known mutations (e.g., EZH2 Y641 and STAT6 D419) in circulating tumor DNA in major B cell NHL subtypes (Alcaide et al, 2016).…”

Section: Noninvasive Clinical Applications Of Genomic Alterations Of mentioning

confidence: 99%

Genetic alterations in B cell lymphoma subtypes as potential biomarkers for non-invasive diagnosis, prognosis, therapy, and disease monitoring

Sönmez

Küçük

2020

Turk J Biol

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Neoplastic transformation of germinal center B (GCB) cells may give rise to a variety of different B cell lymphoma subtypes, most of which show substantial heterogeneity in terms of genetic alterations and clinical features. The mutations observed in cancerrelated genes in GCB cells are related to abnormalities in the immunogenetic mechanisms associated with germinal center reaction. Recent studies have rapidly identified genomic alterations in B cell lymphomas that may be useful for better subclassification, noninvasive diagnosis, and prediction of response to therapy. The WHO recognizes different lymphoma subsets classified within 2 major categories of B cell lymphoma: Hodgkin's lymphoma (HL) and B cell non-Hodgkin's lymphoma (NHL), each with distinct genetic aberrations, including chromosomal translocations, copy number abnormalities, or point mutations. Next-generation sequencing-based technologies have allowed cancer researchers to identify somatic mutations and gene expression signatures at a rapid pace so that novel diagnostic or prognostic biomarkers, as well as therapeutic targets, can be discovered much faster than before. Indeed, deep sequencing studies have recently revealed that lymphoma-specific somatic mutations may be detected in cell-free circulating DNA obtained from the peripheral blood of B cell lymphoma patients, suggesting the possibility of minimally invasive diagnosis, monitoring, and predicting response to therapy of B cell lymphoma patients. In this study, the current status of the recurrent genetic aberrations observed during diagnosis and/ or relapse in HL and the major subtypes of B cell NHL (i.e. diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, and Burkitt lymphoma) are discussed to shed light on their potential use as noninvasive diagnostic or prognostic biomarkers and to reveal their role in lymphomagenesis as a target in therapy for newly diagnosed and chemotherapy-resistant cases.

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“…Recently, the ddPCR assay has been widely used to detect genetic and epigenetic alterations . ddPCR can detect very rare sequences with high precision and sensitivity by partitioning individual target molecules within distinct compartments and, therefore, reduces the limitations of conventional methods such as classic real‐time quantitative PCR (qPCR) or the amplification refractory mutation system qPCR (ARMS qPCR) .…”

Section: Discussionmentioning

confidence: 99%

“…Although the sensitivity of NGS is superior to that of direct Recently, the ddPCR assay has been widely used to detect genetic and epigenetic alterations. [18][19][20][21] ddPCR can detect very rare sequences with high precision and sensitivity by partitioning individual target molecules within distinct compartments and, therefore, reduces the limitations of conventional methods such as classic real-time quantitative PCR (qPCR) or the amplification refractory mutation system qPCR (ARMS qPCR). [22][23][24] However, the PNA-LNA clamp method is a molecular probe-based PCR method; its unique design and allele-specific approach allow exceptionally high specificity and sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Droplet digital polymerase chain reaction assay and peptide nucleic acid‐locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T‐cell lymphoma

et al. 2018

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Angioimmunoblastic T‐cell lymphoma (AITL) is a subtype of nodal peripheral T‐cell lymphoma (PTCL). Somatic RHOA mutations, most frequently found at the hotspot site c.50G > T, p.Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL. Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL. However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next‐generation sequencing (NGS), droplet digital PCR (ddPCR) and peptide nucleic acid‐locked nucleic acid (PNA‐LNA) clamp method for detecting the G17V RHOA mutation. G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS. ddPCR and PNA‐LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with ddPCR and those with NGS showed high concordance (P < .001). Three other RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in PTCL198; c.[50G > T;51A > C], p.Gly17Val in PTCL216; and c.50G > A, p.Gly17Glu in PTCL223) were detected using NGS. These sequence changes could not appropriately be detected using the ddPCR assay and the PNA‐LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of ddPCR/PNA‐LNA clamp methods and NGS are best method to assist the diagnosis of AITL by detecting RHOA mutations at the p.Gly17 position.

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Multiplex Droplet Digital PCR Quantification of Recurrent Somatic Mutations in Diffuse Large B-Cell and Follicular Lymphoma

Cited by 45 publications

References 39 publications

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

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

Genetic alterations in B cell lymphoma subtypes as potential biomarkers for non-invasive diagnosis, prognosis, therapy, and disease monitoring

Droplet digital polymerase chain reaction assay and peptide nucleic acid‐locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T‐cell lymphoma

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