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Article ; Online: Comment on: "Hypoxia differently modulates the release of mitochondrial and nuclear DNA".

Wong, Boris K L / Zhang, Fan / Do, Hongdo / Testro, Adam / Muralidharan, Vijayaragavan / Dobrovic, Alexander / Cox, Daniel R A

British journal of cancer

2021 Volume 124, Issue 12, Page(s) 2035–2036

MeSH term(s)	DNA ; Humans ; Hypoxia ; Mitochondria/genetics
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2021-03-24
Publishing country	England
Document type	Letter ; Comment
ZDB-ID	80075-2
ISSN	1532-1827 ; 0007-0920
ISSN (online)	1532-1827
ISSN	0007-0920
DOI	10.1038/s41416-021-01287-z
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Article ; Online: In Reply to Leone.

Do, Hongdo / Arulananda, Surein / John, Thomas / Dobrovic, Alexander

Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

2018 Volume 13, Issue 2, Page(s) e22–e23

MeSH term(s)	Carcinoma, Non-Small-Cell Lung ; Developing Countries ; ErbB Receptors ; Humans ; Lung Neoplasms ; Mutation
Chemical Substances	EGFR protein, human (EC 2.7.10.1) ; ErbB Receptors (EC 2.7.10.1)
Language	English
Publishing date	2018-02-08
Publishing country	United States
Document type	Letter ; Comment
ZDB-ID	2432037-7
ISSN	1556-1380 ; 1556-0864
ISSN (online)	1556-1380
ISSN	1556-0864
DOI	10.1016/j.jtho.2017.11.119
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Article ; Online: A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification.

Goh, Su Kah / Cox, Daniel R A / Wong, Boris Ka Leong / Musafer, Ashan / Witkowski, Tom / Do, Hongdo / Muralidharan, Vijayaragavan / Dobrovic, Alexander

Clinical chemistry

2021 Volume 67, Issue 9, Page(s) 1201–1209

Abstract: Background: Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. ... ...

Abstract	Background: Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. Quantification of recovery efficiency after these steps can facilitate quality assurance and improve reliability when comparing serial samples. Methods: We developed an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulfite modification (CEREBIS) after cfDNA extraction and/or subsequent bisulfite modification from plasma. The strategic placement of cytosine bases in the 180 bp CEREBIS enabled PCR amplification of the construct by a single primer set both after plasma DNA extraction and following subsequent bisulfite modification. Results: Plasma samples derived from 8 organ transplant donors and 6 serial plasma samples derived from a liver transplant recipient were spiked with a known number of copies of CEREBIS. Recovery of CEREBIS after cfDNA extraction and bisulfite modification was quantified with high analytical accuracy by droplet digital PCR. The use of CEREBIS and quantification of its recovery was useful in identifying problematic extractions. Furthermore, its use was shown to be invaluable towards improving the reliability of the analysis of serial samples. Conclusions: CEREBIS can be used as a spike-in control to address the preanalytical variable of recovery efficiency both after cfDNA extraction from plasma and following bisulfite modification. Our approach can be readily implemented and its application may have significant benefits, especially in settings where longitudinal quantification of cfDNA for disease monitoring is necessary.
MeSH term(s)	Cell-Free Nucleic Acids/genetics ; DNA/genetics ; Humans ; Reproducibility of Results ; Sulfites
Chemical Substances	Cell-Free Nucleic Acids ; Sulfites ; DNA (9007-49-2) ; hydrogen sulfite (OJ9787WBLU)
Language	English
Publishing date	2021-06-19
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	80102-1
ISSN	1530-8561 ; 0009-9147
ISSN (online)	1530-8561
ISSN	0009-9147
DOI	10.1093/clinchem/hvab095
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Article ; Online: Adapting an Established Clinical Chemistry Quality Control Measure for Droplet Generation Performance in Digital PCR.

Goh, Su Kah / Wong, Boris Ka Leong / Muralidharan, Vijayaragavan / Christophi, Christopher / Do, Hongdo / Dobrovic, Alexander

Clinical chemistry

2018 Volume 64, Issue 8, Page(s) 1255–1257

MeSH term(s)	Chemistry, Clinical/methods ; Chemistry, Clinical/standards ; Polymerase Chain Reaction/methods ; Quality Control
Language	English
Publishing date	2018-06-11
Publishing country	England
Document type	Letter ; Research Support, Non-U.S. Gov't
ZDB-ID	80102-1
ISSN	1530-8561 ; 0009-9147
ISSN (online)	1530-8561
ISSN	0009-9147
DOI	10.1373/clinchem.2018.291120
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Article ; Online: Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization.

Do, Hongdo / Dobrovic, Alexander

Clinical chemistry

2014 Volume 61, Issue 1, Page(s) 64–71

Abstract: Background: Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalin-fixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented ... ...

Abstract	Background: Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalin-fixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented and also contains DNA lesions that are the sources of sequence artifacts. Sequence artifacts can be difficult to distinguish from true mutations, especially in the context of tumor heterogeneity, and are an increasing interpretive problem in this era of massively parallel sequencing. Understanding of the sources of sequence artifacts in FFPE tissues and implementation of preventative strategies are critical to improve the accurate detection of actionable mutations. Content: This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis. In particular, fragmentation, base modification (including uracil and thymine deriving from cytosine deamination), and abasic sites are discussed as indirect or direct sources of sequence artifacts. We discuss strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts. These strategies are applicable for the detection of actionable mutations in both single amplicon and massively parallel amplicon sequencing approaches. Summary: Because FFPE tissues are usually the only available material for DNA analysis, it is important to maximize the accurate informational content from FFPE DNA. Careful consideration of each step in the work flow is needed to minimize sequence artifacts. In addition, validation of actionable mutations either by appropriate experimental design or by orthogonal methods should be considered.
MeSH term(s)	Artifacts ; DNA/genetics ; DNA Damage ; False Negative Reactions ; False Positive Reactions ; Formaldehyde ; Humans ; Neoplasms/genetics ; Paraffin Embedding ; Reproducibility of Results ; Sequence Analysis, DNA ; Templates, Genetic ; Tissue Fixation
Chemical Substances	Formaldehyde (1HG84L3525) ; DNA (9007-49-2)
Language	English
Publishing date	2014-11-24
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	80102-1
ISSN	1530-8561 ; 0009-9147
ISSN (online)	1530-8561
ISSN	0009-9147
DOI	10.1373/clinchem.2014.223040
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Article: Standard dose osimertinib for erlotinib refractory T790M-negative

Arulananda, Surein / Do, Hongdo / Rivalland, Gareth / Loh, Zoe / Musafer, Ashan / Lau, Eddie / Mitchell, Paul / Dobrovic, Alexander / John, Thomas

Journal of thoracic disease

2019 Volume 11, Issue 5, Page(s) 1756–1764

Abstract: Background: Leptomeningeal spread in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (: Methods: Eight patients on EGFR TKIs who progressed with cytology-proven leptomeningeal disease at our institution were studied. ...

Abstract	Background: Leptomeningeal spread in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor ( Methods: Eight patients on EGFR TKIs who progressed with cytology-proven leptomeningeal disease at our institution were studied. Results: None of the four patients who developed leptomeningeal disease while receiving 1st generation EGFR TKIs developed the Conclusions: Standard-dose osimertinib resulted in a clinically meaningful response in a patient with
Language	English
Publishing date	2019-06-28
Publishing country	China
Document type	Journal Article
ZDB-ID	2573571-8
ISSN	2077-6624 ; 2072-1439
ISSN (online)	2077-6624
ISSN	2072-1439
DOI	10.21037/jtd.2019.05.41
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Article ; Online: Reducing Artifactual

Do, Hongdo / Molania, Ramyar / Mitchell, Paul L / Vaiskunaite, Rita / Murdoch, John D / Dobrovic, Alexander

Clinical chemistry

2017 Volume 63, Issue 9, Page(s) 1506–1514

Abstract: Background: False-positive : Methods: Formalin-fixed normal lung tissues and lung squamous cell carcinomas were tested to measure the frequency of false-positive : Results: Artifactual : Conclusions: Both U:G and T:G lesions in formalin-fixed ... ...

Abstract	Background: False-positive Methods: Formalin-fixed normal lung tissues and lung squamous cell carcinomas were tested to measure the frequency of false-positive Results: Artifactual Conclusions: Both U:G and T:G lesions in formalin-fixed tissue are sources of false-positive
MeSH term(s)	Cell Line, Tumor ; DNA Glycosylases/metabolism ; Diagnostic Errors/prevention & control ; False Positive Reactions ; Genes, erbB-1/genetics ; Humans ; Molecular Diagnostic Techniques/methods ; Molecular Diagnostic Techniques/standards ; Mutation/genetics ; Neoplasms/diagnosis ; Neoplasms/genetics ; Paraffin Embedding ; Thymine/chemistry
Chemical Substances	DNA Glycosylases (EC 3.2.2.-) ; Thymine (QR26YLT7LT)
Language	English
Publishing date	2017-07-18
Publishing country	England
Document type	Journal Article
ZDB-ID	80102-1
ISSN	1530-8561 ; 0009-9147
ISSN (online)	1530-8561
ISSN	0009-9147
DOI	10.1373/clinchem.2017.271932
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Article ; Online: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil- DNA glycosylase.

Do, Hongdo / Dobrovic, Alexander

Oncotarget

2012 Volume 3, Issue 5, Page(s) 546–558

Abstract: Non-reproducible sequence artefacts are frequently detected in DNA from formalinfixed and paraffin-embedded (FFPE) tissues. However, no rational strategy has been developed for reduction of sequence artefacts from FFPE DNA as the underlying causes of the ...

Abstract	Non-reproducible sequence artefacts are frequently detected in DNA from formalinfixed and paraffin-embedded (FFPE) tissues. However, no rational strategy has been developed for reduction of sequence artefacts from FFPE DNA as the underlying causes of the artefacts are poorly understood. As cytosine deamination to uracil is a common form of DNA damage in ancient DNA, we set out to examine whether treatment of FFPE DNA with uracil-DNA glycosylase (UDG) would lead to the reduction of C>T (and G>A) sequence artefacts. Heteroduplex formation in high resolution melting (HRM)-based assays was used for the detection of sequence variants in FFPE DNA samples. A set of samples that gave false positive HRM results for screening for the E17K mutation in exon 4 of the AKT1 gene were chosen for analysis. Sequencing of these samples showed multiple non-reproducible C:G>T:A artefacts. Treatment of the FFPE DNA with UDG prior to PCR amplification led to a very marked reduction of the sequence artefacts as indicated by both HRM and sequencing analysis, indicating that uracil lesions are the major cause of sequence artefacts. Similar results were shown for the BRAF V600 region in the same sample set and EGFR exon 19 in another sample set. UDG treatment specifically suppressed the formation of artefacts in FFPE DNA as it did not affect the detection of true KRAS codon 12 and true EGFR exon 19 and 20 mutations. We conclude that uracil in FFPE DNA leads to a significant proportion of sequence artefacts. These can be minimised by a simple UDG pretreatment which can be readily carried out, in the same tube, as the PCR immediately prior to commencing thermal cycling. HRM is a convenient way of monitoring both the degree of damage and the effectiveness of the UDG treatment. These findings have immediate and important implications for cancer diagnostics where FFPE DNA is used as the primary genetic material for mutational studies guiding personalised medicine strategies and where simple effective strategies to detect mutations are required.
MeSH term(s)	Artifacts ; Biopsy ; DNA Mutational Analysis/standards ; Diagnostic Errors/prevention & control ; Fixatives/adverse effects ; Fixatives/chemistry ; Formaldehyde/adverse effects ; Formaldehyde/chemistry ; Genes, erbB-1/genetics ; Humans ; Mutation/genetics ; Neoplasms/diagnosis ; Neoplasms/genetics ; Precision Medicine ; Proto-Oncogene Proteins/genetics ; Proto-Oncogene Proteins B-raf/genetics ; Proto-Oncogene Proteins c-akt/genetics ; Proto-Oncogene Proteins p21(ras) ; Sequence Analysis, DNA ; Substrate Specificity/genetics ; Tissue Fixation/methods ; Uracil-DNA Glycosidase/metabolism ; ras Proteins/genetics
Chemical Substances	Fixatives ; KRAS protein, human ; Proto-Oncogene Proteins ; Formaldehyde (1HG84L3525) ; BRAF protein, human (EC 2.7.11.1) ; Proto-Oncogene Proteins B-raf (EC 2.7.11.1) ; Proto-Oncogene Proteins c-akt (EC 2.7.11.1) ; Uracil-DNA Glycosidase (EC 3.2.2.-) ; Proto-Oncogene Proteins p21(ras) (EC 3.6.5.2) ; ras Proteins (EC 3.6.5.2)
Language	English
Publishing date	2012-05-30
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2560162-3
ISSN	1949-2553 ; 1949-2553
ISSN (online)	1949-2553
ISSN	1949-2553
DOI	10.18632/oncotarget.503
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Article ; Online: Probe-Free Digital PCR Quantitative Methodology to Measure Donor-Specific Cell-Free DNA after Solid-Organ Transplantation.

Goh, Su Kah / Muralidharan, Vijayaragavan / Christophi, Christopher / Do, Hongdo / Dobrovic, Alexander

Clinical chemistry

2017 Volume 63, Issue 3, Page(s) 742–750

Abstract: Background: Donor-specific cell-free DNA (dscfDNA) is increasingly being considered as a noninvasive biomarker to monitor graft health and diagnose graft rejection after solid-organ transplantation. However, current approaches used to measure dscfDNA ... ...

Abstract	Background: Donor-specific cell-free DNA (dscfDNA) is increasingly being considered as a noninvasive biomarker to monitor graft health and diagnose graft rejection after solid-organ transplantation. However, current approaches used to measure dscfDNA can be costly and/or laborious. A probe-free droplet digital PCR (ddPCR) methodology using small deletion/insertion polymorphisms (DIPs) was developed to circumvent these limitations without compromising the quantification of dscfDNA. This method was called PHABRE-PCR ( Methods: dscfDNA was serially measured in 3 liver transplant recipients. Donor and recipient genomic DNA was first genotyped against a panel of DIPs to identify donor-specific alleles. Alleles that differentiated donor-specific from recipient-specific DNA were then selected to quantify dscfDNA in the recipient plasma. Results: Lack of amplification of nontargeted alleles confirmed that PHABRE-PCR was highly specific. In recipients who underwent transplantation, dscfDNA was increased at day 3, but decreased and plateaued at a low concentration by 2 weeks in the 2 recipients who did not develop any complications. In the third transplant recipient, a marked increase of dscfDNA coincided with an episode of graft rejection. Conclusions: PHABRE-PCR was able to quantify dscfDNA with high analytical specificity and sensitivity. The implementation of a DIP-based approach permits surveillance of dscfDNA as a potential measure of graft health after solid-organ transplantation.
MeSH term(s)	DNA/genetics ; Humans ; Organ Transplantation ; Polymerase Chain Reaction ; Tissue Donors
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2017-01-18
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	80102-1
ISSN	1530-8561 ; 0009-9147
ISSN (online)	1530-8561
ISSN	0009-9147
DOI	10.1373/clinchem.2016.264838
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Article ; Online: Combination Osimertinib and Gefitinib in C797S and T790M EGFR-Mutated Non-Small Cell Lung Cancer.

Arulananda, Surein / Do, Hongdo / Musafer, Ashan / Mitchell, Paul / Dobrovic, Alexander / John, Thomas

Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

2017 Volume 12, Issue 11, Page(s) 1728–1732

Abstract: Introduction: Osimertinib, a third-generation EGFR tyrosine kinase inhibitor has demonstrated efficacy in tumors harboring the EGFR T790M resistance mutation. Inevitably, resistance to third-generation inhibitors results in disease progression, with the ...

Abstract	Introduction: Osimertinib, a third-generation EGFR tyrosine kinase inhibitor has demonstrated efficacy in tumors harboring the EGFR T790M resistance mutation. Inevitably, resistance to third-generation inhibitors results in disease progression, with the EGFR C797S mutation being one of several resistance pathways identified to date. On the basis of preclinical data, we report what is the first known case of a patient harboring the T790M and C797S mutations in trans treated with combination gefitinib and osimertinib. Methods: On development of progressive disease after multiple therapies, the patient's plasma was sequenced using the Oncomine Lung cfDNA Assay (Thermo Fisher Scientific, Waltham, MA). Subsequent monitoring of circulating tumor DNA in plasma was performed by droplet digital polymerase chain reaction. Results: Sequencing showed that the T790M and C797S mutations were in trans. Within 2 weeks of commencement of combination therapy, rapid clinical improvement occurred. Accompanying this, a rapid decline in the C797S mutation subclone in plasma was detected. However, the levels of the EGFR exon 19 deletion driver mutation and the T790M resistance mutation in the circulating tumor DNA continued to rise and the patient died from progressive disease 6 weeks after commencement of combination therapy. There were no adverse events seen with the combination therapy. Conclusion: This is, to the best of our knowledge, the first reported case of combination EGFR tyrosine kinase inhibitor therapy tailored to the allelic conformation of T790M and C797S mutation that resulted in brief clinical improvement without toxicity.
MeSH term(s)	Acrylamides ; Adult ; Aniline Compounds ; Carcinoma, Non-Small-Cell Lung/drug therapy ; Carcinoma, Non-Small-Cell Lung/pathology ; ErbB Receptors/genetics ; Gefitinib ; Humans ; Lung Neoplasms/drug therapy ; Lung Neoplasms/pathology ; Male ; Piperazines/pharmacology ; Piperazines/therapeutic use ; Quinazolines/pharmacology ; Quinazolines/therapeutic use
Chemical Substances	Acrylamides ; Aniline Compounds ; Piperazines ; Quinazolines ; osimertinib (3C06JJ0Z2O) ; ErbB Receptors (EC 2.7.10.1) ; Gefitinib (S65743JHBS)
Language	English
Publishing date	2017-08-24
Publishing country	United States
Document type	Case Reports ; Journal Article
ZDB-ID	2432037-7
ISSN	1556-1380 ; 1556-0864
ISSN (online)	1556-1380
ISSN	1556-0864
DOI	10.1016/j.jtho.2017.08.006
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