Droplet digital PCR for large genomic rearrangements detection: A promising strategy in tissue BRCA1 testing

Paolis, Elisa De; Bonis, Maria De; Concolino, Paola; Piermattei, Alessia; Fagotti, Anna; Urbani, Andrea; Scambia, Giovanni; Minucci, Angelo; Capoluongo, Ettore

doi:10.1016/j.cca.2020.12.001

Cited by 20 publications

(26 citation statements)

References 36 publications

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“…1-6,8,10,12,19, 33 For this reason, it is important to use a method with sufficient sensitivity to detect ultra low-level mosaicism; and our study shows that ddPCR provides an optimal approach for this purpose. 12,14,15 When considering sample availability, it is very imperative to select the relevant tissue to efficiently identify mutations.…”

Section: Discussionmentioning

confidence: 99%

“…11 Therefore, ddPCR has been increasingly used as a clinical diagnostic method to efficiently and reliably detect and quantify common cancerassociated variants (such as the common PIK3CA hotspot mutations -p.C420R, p.E542K, p.E545K, p.H1047L, and p.H1047R -for PIK3CA-related overgrowth disorders (PROS), and the BRAF V600E mutation for brain and thyroid tumors, among others), and has also been recently used in other pediatric overgrowth, vascular and lymphatic malformation phentypes. [12][13][14][15] The PI3K-AKT-MTOR-related developmental brain disorders, including most notably FCD and HMEG, have a narrow mutational spectrum, with a few "hotspot" mutations or variants representing a sizable portion of molecularly-solved cases. 4,6,9,10 Therefore, we tested these common mutations as a first tier molecular method to complement the neuroimaging and neuropathology diagnosis, in a large cohort of affected individuals to efficiently identify mutations and studying the relationship between levels of mosaicism and key neuroimaging and neuropathological features.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of common PI3K-AKT-MTOR mutations in pediatric surgical epilepsy by droplet digital PCR reveals novel clinical and molecular insights

Pirozzi¹,

Berkseth²,

Shear³

et al. 2021

Preprint

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Focal malformations of cortical development (FMCD) including focal cortical dysplasia (FCD), hemimegalencephaly (HMEG) and megalencephaly (MEG), constitute a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism, and intellectual disability. Importantly, FCD is the most common cause of intractable pediatric focal epilepsy. Gain and loss of function mutations in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we aimed to assess droplet digital Polymerase Chain Reaction (ddPCR) as a first-tier molecular diagnostic method, as well as define genotype-phenotype relationships among the most common PI3K-AKT-MTOR pathway mutations in FMCD. A total of 144 specimens, including 113 brain samples, were collected from 58 individuals with intractable focal epilepsy phenotypes including FCD, MEG, HMEG and other types of developmental cortical lesions. We designed an ultra-deep and highly sensitive molecular diagnostic panel using ddPCR for six of the most common mutations in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key phenotype, neuroimaging and neuropathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions (VAF) ranged from 0.1% to 22.67% across all positive samples. Our data shows that MTOR mutations are mostly associated with FCD, whereas PIK3CA mutations are more frequent in the HMEG-DMEG spectrum. The presence of one of these common PI3K-AKT-MTOR-mutations correlated with earlier onset of seizures. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or neuropathological examination. Interestingly, we could not identify the six most common pathogenic variants in other types of cortical lesions (e.g., polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the FCD-HMEG-MEG spectrum. Finally, our data suggest that ultra-deep targeted molecular analysis for the most common PI3K-AKT-MTOR mutations via ddPCR is an effective molecular diagnostic approach for FMCD phenotypes with a good diagnostic yield when paired with neuroimaging and neuropathology evaluations. The high sensitivity and low DNA input requirements suggests that ddPCR is an effective molecular diagnostic tool for disorders caused by somatic mutations with a narrow mutational spectrum, including specific subtypes of pediatric epilepsy surgical phenotypes such as FCD and HMEG.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Analysis of common PI3K-AKT-MTOR mutations in pediatric surgical epilepsy by droplet digital PCR reveals novel clinical and molecular insights

Pirozzi¹,

Berkseth²,

Shear³

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…Our MGDUnit routinely assesses the BRCA molecular status using the amplicon-based library preparation kit Devyser BRCA (Devyser, Stockholm, Sweden) on Illumina MiSeq NGS platform [7,8].…”

Section: Introductionmentioning

confidence: 99%

Tumor BRCA testing in ovarian cancer and EQA scheme: our experience of a critical evaluation

et al. 2021

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Next generation sequencing (NGS) is a widespread molecular biology method integrated into clinical practice to detect genetic variants, for diagnostic and prognostic purposes. The scheduled external quality assessments (EQA) is integral part of clinical molecular laboratory quality assurance. The EQA provides an efficient system to compare analytic test performances among different laboratories, which is essential to evaluate consistency of molecular test. EQA failures demands targeted corrective action plans. In this context, the complexity of the NGS techniques requires careful and continuous quality control procedures. We report a tumor BRCA1/2 (tBRCA) testing benchmark discrepancy provided by the European Molecular Genetics Quality Network in our laboratory during a round of EQA for somatic mutation testing of BRCA genes in relation to ovarian cancer. The critical analysis emerging from the tBRCA EQA is presented. We underline that harmonization processes are still required for the EQA in the molecular biology field, especially if applied to the evaluation of methods characterized by high complexity.

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“…As a consequence, the somatic bioinformatics pipeline must require computational algorithms developed ad hoc and specific characteristics of sequencing raw data (e.g. maximum amount, coverage uniformity and sufficient reads depth) 1 . Even if the majority of methods are optimized for somatic CNAs identification 6;8 , attention should be given in the comparison of blood and tissue tests results 13 .…”

mentioning

confidence: 99%

“…We argue that only harmonized guidelines encompassing the abovementioned methodological and postanalytical steps could solve the BRCAgermline and somatic testing bias. In our laboratory, BRCAgenetic testing is routinely performed on blood, FFT and FFPE samples 1 . In many cases, we routinely analyze matched blood and tissue samples belonging from the same patient, in order to perform an efficient BRCA test comprehensive of both germline and somatic evaluation.…”

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confidence: 99%

A commentary on the discrepancy between blood and tumor BRCA testing: an open question

Paolis

Marchetti

Concolino

et al. 2021

Preprint

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Molecular evaluation of BRCA1/2 (BRCA ) genes represents a well-known example of precision oncology. The availability of Poly ADP Ribose Polymerase inhibitors (PARPi) as target therapy option for several BRCA mutated cancers types (e.g. ovarian, breast, prostate, and pancreatic) 1 changed the course ofBRCA testing over the last years. In this context, an emerging path of molecular evaluation is represented by the BRCA testing performed directly on tumor tissue (tBRCA ): this increased the chance to identify more patients with higher likelihood of benefiting from PARPi treatment. This approach leads to the simultaneous identification of both constitutional and somatically acquired variants, with a lower turnaround time: the identification of BRCApathogenic variants (PVs) could lead to a secondary "reflex" germlineBRCA (gBRCA) testing in order to assess personal and familiar risks. In contrast, performing gBRCA as first molecular test causes the loss of a relevant proportion of patients with tissue acquired BRCA PVs, needing of a following tumor test 2;3 . However, challenges exist in tBRCA that may lead to inefficient germline variant call. A recently published paper by Kordes et al. reported a pancreatic adenocarcinoma patient with a germlinenovel BRCA2 c.516+4A>G variant classified as deleterious by the authors based on in silico and functional data 4 . Also the tumor tissue was sequenced in order to achieve the enrolment criteria for a clinical trial of Olaparib in

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Droplet digital PCR for large genomic rearrangements detection: A promising strategy in tissue BRCA1 testing

Cited by 20 publications

References 36 publications

Analysis of common PI3K-AKT-MTOR mutations in pediatric surgical epilepsy by droplet digital PCR reveals novel clinical and molecular insights

Analysis of common PI3K-AKT-MTOR mutations in pediatric surgical epilepsy by droplet digital PCR reveals novel clinical and molecular insights

Tumor BRCA testing in ovarian cancer and EQA scheme: our experience of a critical evaluation

A commentary on the discrepancy between blood and tumor BRCA testing: an open question

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