Interlaboratory Diagnostic Validation of Conformation-Sensitive Capillary Electrophoresis for Mutation Scanning

Abstract Background: Indirect alternatives to sequencing as a method for mutation scanning are of interest to diagnostic laboratories because they have the potential for considerable savings in both time and costs. Ideally, such methods should be simple, rapid, and highly sensitive, and they should be validated formally to a very high standard. Currently, most reported methods lack one or more of these characteristics. We describe the optimization and validation of conformation-sensitive capillary electrophoresis (CSCE) for diagnostic mutation scanning. Methods: We initially optimized the performance of CSCE with a systematic panel of plasmid-based controls. We then compared manual analysis by visual inspection with automated analysis by BioNumerics software (Applied Maths) in a blinded interlaboratory validation with 402 BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 1, early onset) variants previously characterized by Sanger sequencing. Results: With automated analysis, we demonstrated a sensitivity of >99% (95% CI), which is indistinguishable from the sensitivity for conventional sequencing by capillary electrophoresis. The 95% CI for specificity was 90%–93%; thus, CSCE greatly reduces the number of fragments that need to be sequenced to fully characterize variants. By manual analysis, the 95% CIs for sensitivity and specificity were 98.3%–99.4% and 93.1%–95.5%, respectively. Conclusions: CSCE is amenable to a high degree of automation, and analyses can be multiplexed to increase both capacity and throughput. We conclude that once it is optimized, CSCE combined with analysis with BioNumerics software is a highly sensitive and cost-effective mutation-scanning technique suitable for routine genetic diagnostic analysis of heterozygous nucleotide substitutions, small insertions, and deletions.

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Multiclinician involvement in protocol-driven management of early breast cancer patients in Australia: High standard and excellent outcomes can be maintained

European Journal of Cancer ◽

10.1016/s0959-8049(02)80510-x ◽

2002 ◽

Vol 38 (11) ◽

pp. S152

Author(s):

K Taylor

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Early Breast Cancer ◽

High Standard ◽

Breast Cancer Patients

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Oral Contraceptive Use and Risk of Early-Onset Breast Cancer in Carriers and Noncarriers of BRCA1 and BRCA2 Mutations

Yearbook of Obstetrics Gynecology and Women s Health ◽

10.1016/s1090-798x(08)70471-5 ◽

2006 ◽

Vol 2006 ◽

pp. 269-270

Author(s):

L.P. Shulman

Keyword(s):

Breast Cancer ◽

Oral Contraceptive ◽

Early Onset ◽

Contraceptive Use ◽

Early Onset Breast Cancer ◽

Brca1 And Brca2 ◽

Oral Contraceptive Use ◽

Brca2 Mutations ◽

Brca1 And Brca2 Mutations

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Early onset isolated ostial left-main-stem stenosis after breast cancer radiation report of a case and review of literature

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-0031-1297851 ◽

2012 ◽

Vol 60 (S 01) ◽

Author(s):

M Wilbring ◽

SM Tugtekin ◽

S Schön ◽

D Joskowiak ◽

K Matschke ◽

...

Keyword(s):

Breast Cancer ◽

Early Onset ◽

Main Stem ◽

Review Of Literature ◽

Left Main ◽

Left Main Stem

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Faculty Opinions recommendation of Germline mutations in BRCA2: shared genetic susceptibility to breast cancer, early onset leukemia, and Fanconi anemia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718311724.793492279 ◽

2014 ◽

Author(s):

Wade Clapp ◽

Grzegorz Nalepa

Keyword(s):

Breast Cancer ◽

Genetic Susceptibility ◽

Fanconi Anemia ◽

Early Onset ◽

Germline Mutations ◽

Shared Genetic

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Pressure-assisted electrokinetic injection stacking for citalopram drug to achieve highly sensitive detection and enantioseparation by capillary electrophoresis

Chinese Journal of Chromatography ◽

10.3724/sp.j.1123.2015.03038 ◽

2015 ◽

Vol 33 (9) ◽

pp. 988 ◽

Cited By ~ 2

Author(s):

Zhongqi XU ◽

Feng YE ◽

Yongle WANG ◽

Aimei LI

Keyword(s):

Capillary Electrophoresis ◽

Sensitive Detection ◽

Electrokinetic Injection ◽

Highly Sensitive ◽

Highly Sensitive Detection

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PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method

Journal of Molecular Diagnostics ◽

10.2353/jmoldx.2010.100008 ◽

2010 ◽

Vol 12 (5) ◽

pp. 697-704 ◽

Cited By ~ 22

Author(s):

Panagiotis A. Vorkas ◽

Nikoleta Poumpouridou ◽

Sophia Agelaki ◽

Christos Kroupis ◽

Vassilis Georgoulias ◽

...

Keyword(s):

High Resolution ◽

Analysis Method ◽

Mutation Scanning ◽

Highly Sensitive ◽

Melting Analysis ◽

Hotspot Mutation

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High likelihood of actionable pathogenic variant detection in breast cancer genes in women with very early onset breast cancer

Journal of Medical Genetics ◽

10.1136/jmedgenet-2020-107347 ◽

2021 ◽

pp. jmedgenet-2020-107347

Author(s):

D Gareth Evans ◽

Elke Maria van Veen ◽

Helen J Byers ◽

Sarah J Evans ◽

George J Burghel ◽

...

Keyword(s):

Breast Cancer ◽

Early Onset ◽

Ductal Carcinoma ◽

Cancer Genes ◽

Early Onset Breast Cancer ◽

Younger Women ◽

Pathogenic Variants ◽

Younger Age ◽

Predisposition Genes

BackgroundWhile the likelihood of identifying constitutional breast cancer-associated BRCA1, BRCA2 and TP53 pathogenic variants (PVs) increases with earlier diagnosis age, little is known about the correlation with age at diagnosis in other predisposition genes. Here, we assessed the contribution of known breast cancer-associated genes to very early onset disease.MethodsSequencing of BRCA1, BRCA2, TP53 and CHEK2 c.1100delC was undertaken in women with breast cancer diagnosed ≤30 years. Those testing negative were screened for PVs in a minimum of eight additional breast cancer-associated genes. Rates of PVs were compared with cases ≤30 years from the Prospective study of Outcomes in Sporadic vs Hereditary breast cancer (POSH) study.ResultsTesting 379 women with breast cancer aged ≤30 years identified 75 PVs (19.7%) in BRCA1, 35 (9.2%) in BRCA2, 22 (5.8%) in TP53 and 2 (0.5%) CHEK2 c.1100delC. Extended screening of 184 PV negative women only identified eight additional actionable PVs. BRCA1/2 PVs were more common in women aged 26–30 years than in younger women (p=0.0083) although the younger age group had rates more similar to those in the POSH cohort. Out of 26 women with ductal carcinoma in situ (DCIS) alone, most were high-grade and 11/26 (42.3%) had a PV (TP53=6, BRCA2=2, BRCA1=2, PALB2=1). This PV yield is similar to the 61 (48.8%) BRCA1/2 PVs identified in 125 women with triple-negative breast cancer. The POSH cohort specifically excluded pure DCIS which may explain lower TP53 PV rates in this group (1.7%).ConclusionThe rates of BRCA1, BRCA2 and TP53 PVs are high in very early onset breast cancer, with limited benefit from testing of additional breast cancer-associated genes.

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Automated analysis of 3D-echocardiography using spatially registered patient-specific CMR meshes

European Heart Journal - Cardiovascular Imaging ◽

10.1093/ehjci/jeaa356.425 ◽

2021 ◽

Vol 22 (Supplement_1) ◽

Author(s):

D Zhao ◽

E Ferdian ◽

GD Maso Talou ◽

GM Quill ◽

K Gilbert ◽

...

Keyword(s):

New Zealand ◽

Interobserver Variability ◽

Ground Truth ◽

Automated Analysis ◽

3D Echocardiography ◽

Training Data ◽

Patient Specific ◽

Manual Analysis ◽

Lv Mass ◽

3D Echo

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Heart Foundation (NHF) of New Zealand Health Research Council (HRC) of New Zealand Artificial intelligence shows considerable promise for automated analysis and interpretation of medical images, particularly in the domain of cardiovascular imaging. While application to cardiac magnetic resonance (CMR) has demonstrated excellent results, automated analysis of 3D echocardiography (3D-echo) remains challenging, due to the lower signal-to-noise ratio (SNR), signal dropout, and greater interobserver variability in manual annotations. As 3D-echo is becoming increasingly widespread, robust analysis methods will substantially benefit patient evaluation. We sought to leverage the high SNR of CMR to provide training data for a convolutional neural network (CNN) capable of analysing 3D-echo. We imaged 73 participants (53 healthy volunteers, 20 patients with non-ischaemic cardiac disease) under both CMR and 3D-echo (<1 hour between scans). 3D models of the left ventricle (LV) were independently constructed from CMR and 3D-echo, and used to spatially align the image volumes using least squares fitting to a cardiac template. The resultant transformation was used to map the CMR mesh to the 3D-echo image. Alignment of mesh and image was verified through volume slicing and visual inspection (Fig. 1) for 120 paired datasets (including 47 rescans) each at end-diastole and end-systole. 100 datasets (80 for training, 20 for validation) were used to train a shallow CNN for mesh extraction from 3D-echo, optimised with a composite loss function consisting of normalised Euclidian distance (for 290 mesh points) and volume. Data augmentation was applied in the form of rotations and tilts (<15 degrees) about the long axis. The network was tested on the remaining 20 datasets (different participants) of varying image quality (Tab. I). For comparison, corresponding LV measurements from conventional manual analysis of 3D-echo and associated interobserver variability (for two observers) were also estimated. Initial results indicate that the use of embedded CMR meshes as training data for 3D-echo analysis is a promising alternative to manual analysis, with improved accuracy and precision compared with conventional methods. Further optimisations and a larger dataset are expected to improve network performance. (n = 20) LV EDV (ml) LV ESV (ml) LV EF (%) LV mass (g) Ground truth CMR 150.5 ± 29.5 57.9 ± 12.7 61.5 ± 3.4 128.1 ± 29.8 Algorithm error -13.3 ± 15.7 -1.4 ± 7.6 -2.8 ± 5.5 0.1 ± 20.9 Manual error -30.1 ± 21.0 -15.1 ± 12.4 3.0 ± 5.0 Not available Interobserver error 19.1 ± 14.3 14.4 ± 7.6 -6.4 ± 4.8 Not available Tab. 1. LV mass and volume differences (means ± standard deviations) for 20 test cases. Algorithm: CNN – CMR (as ground truth). Abstract Figure. Fig 1. CMR mesh registered to 3D-echo.

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Prevalence of RECQL germline variants in Pakistani early-onset and familial breast cancer patients

Hereditary Cancer in Clinical Practice ◽

10.1186/s13053-020-00159-6 ◽

2020 ◽

Vol 18 (1) ◽

Cited By ~ 1

Author(s):

Muhammad Usman Rashid ◽

Noor Muhammad ◽

Faiz Ali Khan ◽

Umara Shehzad ◽

Humaira Naeemi ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Early Onset ◽

Familial Breast Cancer ◽

High Performance ◽

Breast Cancer Patients ◽

Germline Variants ◽

Variants Of Unknown Significance ◽

Breast Cancer Predisposition ◽

Novel Variants

Abstract Background The RecQ Like Helicase (RECQL) gene has previously been shown to predispose to breast cancer mainly in European populations, in particular to estrogen receptor (ER) and/or progesterone receptor (PR) positive tumor. Here, we investigated the contribution of pathogenic RECQL germline variants to hereditary breast cancer in early-onset and familial breast cancer patients from Pakistan. Methods Comprehensive RECQL variant analysis was performed in 302 BRCA1 and BRCA2 negative patients with ER and/or PR positive breast tumors using denaturing high-performance liquid chromatography followed by DNA sequencing. Novel variants were classified using Sherloc guidelines. Results One novel pathogenic protein-truncating variant (p.W75*) was identified in a 37-year-old familial breast cancer patient. The pathogenic variant frequencies were 0.3% (1/302) in early-onset and familial breast cancer patients and 0.8% (1/133) in familial patients. Further, three novel variants of unknown significance, p.I141F, p.S182S, and p.C475C, were identified in familial breast cancer patients at the age of 47, 68, and 47 respectively. All variants were absent in 250 controls. Conclusions Our data suggest that the RECQL gene plays a negligible role in breast cancer predisposition in Pakistan.

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