Multiplexed Droplet Digital PCR Assays for the Simultaneous Screening of Major Genetic Alterations in Tumors of the Central Nervous System

Appay, Romain; Fina, Frédéric; Barets, Doriane; Gallardo, Catherine; Nanni-Metellus, Isabelle; Scavarda, Didier; Henaff, Daniel; Vincent, Juline; Grewis, Lise; Pourquier, Philippe; Colin, Carole; Figarella‐Branger, Dominique

doi:10.3389/fonc.2020.579762

Cited by 23 publications

(26 citation statements)

References 24 publications

(35 reference statements)

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“…Despite the increased cost, longer turnaround time, requirement for moderate, or even high amount of biological material, these techniques offer the benefit of delineating the complex genomic and epigenomic landscape of tumors, which may provide prognostic as well as therapeutic information. A more detailed discussion of the advantages and disadvantages of each technique have been highlighted previously (18).…”

Section: Discussionmentioning

confidence: 99%

“…We have successfully used this technique for the detection of recurrent genome copy number variations (CNV) including duplications in CNS tumors, using, in the same DNA test sample, a standard dPCR design with two different colored probes directed against a target sequence and a reference sequence (invariable on the same chromosome, same arm or another chromosome or SNP) (18)(19)(20). However, the duplicated region of the BCOR-ITD is very small, hindering its segregation into different droplets and its CNV evaluation by standard designs.…”

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confidence: 99%

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Specific and Sensitive Diagnosis of BCOR-ITD in Various Cancers by Digital PCR

et al. 2021

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BCOR is an epigenetic regulator altered by various mechanisms including BCOR-internal tandem duplication (BCOR-ITD) in a wide range of cancers. Six different BCOR-ITD in the 3’-part of the coding sequence of exon 15 have been reported ranging from 89 to 114 bp in length. BCOR-ITD is a common genetic alteration found in clear cell sarcoma of the kidney and primitive myxoid mesenchymal tumor of infancy (PMMTI) and it characterizes a new type of central nervous system tumor: “CNS tumor with BCOR-ITD”. It can also be detected in undifferentiated round cell sarcoma (URCS) and in high-grade endometrial stromal sarcoma (HGESS). Therefore, it is of utmost importance to search for this genetic alteration in these cancers with the most frequent technique being RNA-sequencing. Here, we developed a new droplet PCR assay (dPCR) to detect the six sequences characterizing BCOR-ITD. To achieve this goal, we used a single colored probe to detect both the duplicated region and the normal sequence that acts as a reference. We first generated seven synthetic DNA sequences: ITD0 (the normal sequence) and ITD1 to ITD6 (the duplicated sequences described in the literature) and then we set up the optima dPCR conditions. We validated our assay on 19 samples from a representative panel of human tumors (9 HGNET-BCOR, 5 URCS, 3 HGESS, and 2 PMMTI) in which BCOR-ITD status was known using at least one other method including RNA sequencing, RT-PCR or DNA-methylation profiling for CNS tumors. Our results showed that our technique was 100% sensitive and specific. DPCR detected BCOR-ITD in 13/19 of the cases; in the remaining 6 cases additional RNA-sequencing revealed BCOR gene fusions. To conclude, in the era of histomolecular classification of human tumors, our modified dPCR assay is of particular interest to detect BCOR-ITD since it is a robust and less expensive test that can be applied to a broad spectrum of cancers that share this alteration.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Specific and Sensitive Diagnosis of BCOR-ITD in Various Cancers by Digital PCR

et al. 2021

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“…BRAF V600E mutation, BRAF exon 14 duplication, FGFR1 N546K/ K656E mutations and FGFR1 tyrosine kinase domain (TKD) duplication were assessed for every patient by previously described multiplexed digital PCR (mdPCR) assays [27][28][29]. Fractional abundance and copy number variation (CNV) were calculated with the cut-off values and detection thresholds defined by Appay et al [29].…”

Section: Multiplexed Droplet Digital Pcrmentioning

confidence: 99%

Low‐grade epilepsy‐associated neuroepithelial tumours with a prominent oligodendroglioma‐like component: The diagnostic challenges

Métais

Appay

Pagès

et al. 2021

Neuropathology Appl Neurobio

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Aims: We searched for recurrent pathological features and molecular alterations in a retrospective series of 72 low-grade epilepsy-associated neuroepithelial tumours (LEATs) with a prominent oligodendroglioma-like component, in order to classify them according to the 2021 World Health Organization (WHO) classification of central nervous system (CNS) tumours. Methods: Centralised pathological examination was performed as well as targeted molecular analysis of v-Raf murine sarcoma viral oncogene homologue B (BRAF) and fibroblast growth factor receptor 1 (FGFR1) by multiplexed digital polymerase chain reaction (mdPCR). DNA methylation profiling was performed in cases with sufficient DNA. In cases with no genetic alteration by mdPCR and sufficient material, RNA sequencing was done. Results: We first reclassified our cohort into three groups: ganglioglioma (GG, n = 14), dysembryoplastic neuroepithelial tumours (DNTs, n = 19) and glioneuronal tumours/ paediatric-type low-grade glioma (LGG) not otherwise specified (GNT/PLGG NOS, n = 39). mdPCR found an alteration in 38/72 cases. Subsequent RNA sequencing revealed a fusion transcript involving BRAF, FGFR1/2/3 or neurotrophic tyrosine kinase receptor type 2 [NTRK2] in 9/25 cases. DNA methylation profiling found 12/46 cases with a calibrated score ≥0.9. Unsupervised hierarchical clustering revealed two clusters: Cluster 1 was enriched with cases classified as DNT at histology, belonging to the LGG-DNT methylation class (MC), with haematopoietic progenitor cell antigen (CD34) negativity and FGRF1 alterations; Cluster 2 was enriched with cases classified at histology as GG, belonging to the LGG-GG MC MC, with BRAF V600E mutation and CD34 positivity. The tumours reclassified as GNT/PLGG NOS were equally distributed across both clusters. Interestingly, all polymorphous low-grade neuroepithelial tumour of the young Romain Appay and Mélanie Pagès contributed equally to this study.

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“…FGFR1 clone selection for rare resistant has been also reported in lung and colorectal resistant tumors, thus revealing a change in variant allele frequency of FGFR1 somatic variants 55;56 . Of note, N546K mutation was also found in Ewing sarcoma and brain tumors [27][28][29][30][31] .…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, in addition to the already reported single relapsed NB case 15 , N546K mutation has also been recently reported in 6 patients 25 . Speci cally, N546K represents an activating mutation that alters FGFR1 auto-phosphorylation 26 , resulting in an increase of kinase activity and malignant transformation in Ewing sarcoma and brain tumors [27][28][29][30][31] .…”

Section: Introductionmentioning

confidence: 99%

FGFR1 is a Potential Therapeutic Target in Neuroblastoma

Cimmino

Montella

Tirelli

et al. 2022

Preprint

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Background: FGFR1 regulates cell-cell adhesion and extracellular matrix architecture and acts as oncogene in several cancers. Potential cancer driver mutations of FGFR1 occur in neuroblastoma NB, a neural crest-derived pediatric tumor arising in sympathetic nervous system but so far, they have not been studied experimentally. We investigated the driver-oncogene role of FGFR1 and the implication of N546K mutation in therapy-resistance in NB cells.Methods: Public datasets were used to predict the correlation of FGFR1 expression with NB clinical outcomes. Whole genome sequencing data of 19 paired diagnostic and relapse NB samples were used to find somatic mutations. In NB cell lines, silencing and transient overexpression of FGFR1 by short hairpin RNA were performed to evaluate the effect of the found mutation by cell growth, invasion and cologenicity assays. HEK293, SHSY5Y and SKNBE2 were selected to investigate subcellular wildtype and mutated protein localization. FGFR1 inhibitor (AZD4547), alone or in combination with PI3K inhibitor (GDC0941), was used to rescue malignant phenotypes induced by overexpression of FGFR1 wildtype and mutated protein.Results: High FGFR1 expression correlated with low relapse-free survival in two independent NB gene expression datasets. In addition, we found the somatic mutation N546K, the most recurrent point mutation of FGFR1 in all cancers and already reported in NB, in one out of 19 matched primary and recurrent tumors. Loss of FGFR1 function attenuated invasion and cologenicity in NB cells, whereas FGFR1 overexpression enhanced oncogenicity. The overexpression of FGFR1N546K protein showed a higher nuclear localization compared to wildtype protein and increased cellular invasion and cologenicity. Moreover, N546K mutation caused the failure in response to treatment with FGFR1 inhibitor by activation of ERK, STAT3 and AKT pathways. The combination of FGFR1 and PI3K pathway inhibitors was effective in reducing the invasive and colonigenic ability of cells overexpressing FGFR1 mutated protein.Conclusion: FGFR1 is an actionable driver oncogene in NB and a promising therapy may consist in targeting FGFR1 mutations in patients with therapy-resistant NB.

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Multiplexed Droplet Digital PCR Assays for the Simultaneous Screening of Major Genetic Alterations in Tumors of the Central Nervous System

Cited by 23 publications

References 24 publications

Specific and Sensitive Diagnosis of BCOR-ITD in Various Cancers by Digital PCR

Specific and Sensitive Diagnosis of BCOR-ITD in Various Cancers by Digital PCR

Low‐grade epilepsy‐associated neuroepithelial tumours with a prominent oligodendroglioma‐like component: The diagnostic challenges

FGFR1 is a Potential Therapeutic Target in Neuroblastoma

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