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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: 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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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: PDCD1

Parakh, Sagun / Musafer, Ashan / Paessler, Sabrina / Witkowski, Tom / Suen, Connie S N Li Wai / Tutuka, Candani S A / Carlino, Matteo S / Menzies, Alexander M / Scolyer, Richard A / Cebon, Jonathan / Dobrovic, Alexander / Long, Georgina V / Klein, Oliver / Behren, Andreas

Frontiers in immunology

2021 Volume 12, Page(s) 672521

Abstract: A significant number of patients (pts) with metastatic melanoma do not respond to anti-programmed cell death 1 (PD1) therapies. Identifying predictive biomarkers therefore remains an urgent need. We retrospectively analyzed plasma DNA of pts with ... ...

Abstract	A significant number of patients (pts) with metastatic melanoma do not respond to anti-programmed cell death 1 (PD1) therapies. Identifying predictive biomarkers therefore remains an urgent need. We retrospectively analyzed plasma DNA of pts with advanced melanoma treated with PD-1 antibodies, nivolumab or pembrolizumab, for five PD-1 genotype single nucleotide polymorphisms (SNPs): PD1.1 (rs36084323, G>A), PD1.3 (rs11568821, G>A), PD1.5 (rs2227981, C>T) PD1.6 (rs10204225, G>A) and PD1.9 (rs2227982, C>T). Clinico-pathological and treatment parameters were collected, and presence of SNPs correlated with response, progression free survival (PFS) and overall survival (OS). 115 patients were identified with a median follow up of 18.7 months (range 0.26 - 52.0 months). All were Caucasian; 27% BRAF V600 mutation positive. At PD-1 antibody commencement, 36% were treatment-naïve and 52% had prior ipilimumab. The overall response rate was 43%, 19% achieving a complete response. Overall median PFS was 11.0 months (95% CI 5.4 - 17.3) and median OS was 31.1 months (95% CI 23.2 - NA). Patients with the G/G genotype had more complete responses than with A/G genotype (16.5%
MeSH term(s)	Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor/genetics ; Drug Resistance, Neoplasm/genetics ; Female ; Genotype ; Humans ; Immune Checkpoint Inhibitors/therapeutic use ; Male ; Melanoma/drug therapy ; Melanoma/genetics ; Melanoma/mortality ; Middle Aged ; Polymorphism, Single Nucleotide ; Programmed Cell Death 1 Receptor/genetics ; Progression-Free Survival ; Retrospective Studies
Chemical Substances	Biomarkers, Tumor ; Immune Checkpoint Inhibitors ; PDCD1 protein, human ; Programmed Cell Death 1 Receptor
Language	English
Publishing date	2021-06-09
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2021.672521
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Article ; Online: Comparison of 3 Methodologies for Genotyping of Small Deletion and Insertion Polymorphisms.

Goh, Su Kah / Musafer, Ashan / Witkowski, Tom / Muralidharan, Vijayaragavan / Christophi, Christopher / Do, Hongdo / Dobrovic, Alexander

Clinical chemistry

2016 Volume 62, Issue 7, Page(s) 1012–1019

Abstract: Background: The quantification of genomic chimerism is increasingly recognized for its clinical significance after transplantation. Before the measurement of chimerism, accurate genotyping of genetic polymorphisms for informative alleles that can ... ...

Abstract	Background: The quantification of genomic chimerism is increasingly recognized for its clinical significance after transplantation. Before the measurement of chimerism, accurate genotyping of genetic polymorphisms for informative alleles that can distinguish donor DNA from recipient DNA is essential. The ease of allelic discrimination of small deletion and insertion polymorphisms (DIPs) makes DIPs attractive markers to track chimerism. Current methodologies for the genotyping of DIPs are largely based on "open-tube" approaches. "Closed-tube" approaches involving no or minimal post-PCR handling are preferred. We compared 3 distinct methodologies to determine an optimal platform for DIP genotyping. Methods: Genomic DNA from 19 normal individuals was genotyped for 6 small biallelic DIPs using high-resolution melting analysis (HRMA), probe-free droplet digital PCR (ddPCR), and microfluidic electrophoresis of PCR products. For HRMA, 3 different platforms were compared. Results: Our newly developed probe-free ddPCR approach allowed the genotype of each DIP to be determined by fluorescence intensity based on amplicon size. Microfluidic electrophoresis also allowed genotypes to be determined by amplicon size. HRMA assays allowed the genotype of each DIP to be determined by melting profile. Genotyping results were concordant between the 3 methodologies. HRMA was the most readily performed methodology and was robust across 3 separate HRMA-capable platforms. Conclusions: We demonstrated the effectiveness of probe-free ddPCR to accurately genotype small biallelic DIPs. Nevertheless, HRMA proved to be the optimal approach for genotyping small DIPs because closed-tube approaches are preferred owing to rapid and less laborious workflows and least risk of PCR contamination.
MeSH term(s)	Alleles ; DNA/genetics ; Genotype ; Genotyping Techniques/methods ; Humans ; Microfluidic Analytical Techniques ; Mutagenesis, Insertional/genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide/genetics ; Sequence Deletion/genetics
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2016-06-24
Publishing country	England
Document type	Comparative Study ; Journal Article
ZDB-ID	80102-1
ISSN	1530-8561 ; 0009-9147
ISSN (online)	1530-8561
ISSN	0009-9147
DOI	10.1373/clinchem.2016.256388
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Article ; Online: Molecular and clinical determinants of response and resistance to rucaparib for recurrent ovarian cancer treatment in ARIEL2 (Parts 1 and 2).

Swisher, Elizabeth M / Kwan, Tanya T / Oza, Amit M / Tinker, Anna V / Ray-Coquard, Isabelle / Oaknin, Ana / Coleman, Robert L / Aghajanian, Carol / Konecny, Gottfried E / O'Malley, David M / Leary, Alexandra / Provencher, Diane / Welch, Stephen / Chen, Lee-May / Wahner Hendrickson, Andrea E / Ma, Ling / Ghatage, Prafull / Kristeleit, Rebecca S / Dorigo, Oliver /

Musafer, Ashan / Kaufmann, Scott H / Elvin, Julia A / Lin, Douglas I / Chambers, Setsuko K / Dominy, Erin / Vo, Lan-Thanh / Goble, Sandra / Maloney, Lara / Giordano, Heidi / Harding, Thomas / Dobrovic, Alexander / Scott, Clare L / Lin, Kevin K / McNeish, Iain A

Nature communications

2021 Volume 12, Issue 1, Page(s) 2487

Abstract: ARIEL2 (NCT01891344) is a single-arm, open-label phase 2 study of the PARP inhibitor (PARPi) rucaparib in relapsed high-grade ovarian carcinoma. In this post hoc exploratory biomarker analysis of pre- and post-platinum ARIEL2 samples, RAD51C and RAD51D ... ...

Abstract	ARIEL2 (NCT01891344) is a single-arm, open-label phase 2 study of the PARP inhibitor (PARPi) rucaparib in relapsed high-grade ovarian carcinoma. In this post hoc exploratory biomarker analysis of pre- and post-platinum ARIEL2 samples, RAD51C and RAD51D mutations and high-level BRCA1 promoter methylation predict response to rucaparib, similar to BRCA1/BRCA2 mutations. BRCA1 methylation loss may be a major cross-resistance mechanism to platinum and PARPi. Genomic scars associated with homologous recombination deficiency are irreversible, persisting even as platinum resistance develops, and therefore are predictive of rucaparib response only in platinum-sensitive disease. The RAS, AKT, and cell cycle pathways may be additional modulators of PARPi sensitivity.
MeSH term(s)	Adult ; Aged ; Aged, 80 and over ; Antineoplastic Agents/adverse effects ; Antineoplastic Agents/therapeutic use ; BRCA1 Protein/genetics ; BRCA2 Protein/genetics ; Carcinoma, Ovarian Epithelial/drug therapy ; DNA Methylation/genetics ; DNA-Binding Proteins/genetics ; Female ; Humans ; Indoles/adverse effects ; Indoles/therapeutic use ; Middle Aged ; Neoplasm Recurrence, Local/drug therapy ; Ovarian Neoplasms/drug therapy ; Platinum/therapeutic use ; Poly(ADP-ribose) Polymerase Inhibitors/adverse effects ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use ; Promoter Regions, Genetic/genetics
Chemical Substances	Antineoplastic Agents ; BRCA1 Protein ; BRCA1 protein, human ; BRCA2 Protein ; BRCA2 protein, human ; DNA-Binding Proteins ; Indoles ; Poly(ADP-ribose) Polymerase Inhibitors ; RAD51C protein, human ; RAD51D protein, human ; Platinum (49DFR088MY) ; rucaparib (8237F3U7EH)
Language	English
Publishing date	2021-05-03
Publishing country	England
Document type	Clinical Trial, Phase II ; Journal Article ; Multicenter Study ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-021-22582-6
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Article ; Online: Acquired

Nesic, Ksenija / Kondrashova, Olga / Hurley, Rachel M / McGehee, Cordelia D / Vandenberg, Cassandra J / Ho, Gwo-Yaw / Lieschke, Elizabeth / Dall, Genevieve / Bound, Nirashaa / Shield-Artin, Kristy / Radke, Marc / Musafer, Ashan / Chai, Zi Qing / Ghamsari, Mohammad Reza Eftekhariyan / Harrell, Maria I / Kee, Damien / Olesen, Inger / McNally, Orla / Traficante, Nadia /

Australian Ovarian Cancer Study / DeFazio, Anna / Bowtell, David D L / Swisher, Elizabeth M / Weroha, S John / Nones, Katia / Waddell, Nicola / Kaufmann, Scott H / Dobrovic, Alexander / Wakefield, Matthew J / Scott, Clare L

Cancer research

2021 Volume 81, Issue 18, Page(s) 4709–4722

Abstract: In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair ... ...

Abstract	In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene
MeSH term(s)	Animals ; Antineoplastic Agents/pharmacology ; Antineoplastic Agents/therapeutic use ; Cell Line, Tumor ; Computational Biology ; Cystadenocarcinoma, Serous/drug therapy ; Cystadenocarcinoma, Serous/genetics ; Cystadenocarcinoma, Serous/metabolism ; Cystadenocarcinoma, Serous/pathology ; DNA Methylation ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Disease Models, Animal ; Drug Resistance, Neoplasm/genetics ; Female ; Gene Expression Profiling ; Gene Silencing ; Homozygote ; Humans ; Mice ; Neoplasm Grading ; Neoplasm Staging ; Ovarian Neoplasms/drug therapy ; Ovarian Neoplasms/genetics ; Ovarian Neoplasms/metabolism ; Ovarian Neoplasms/pathology ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Prognosis ; Promoter Regions, Genetic ; Xenograft Model Antitumor Assays
Chemical Substances	Antineoplastic Agents ; DNA-Binding Proteins ; Poly(ADP-ribose) Polymerase Inhibitors ; RAD51C protein, human
Language	English
Publishing date	2021-07-28
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1432-1
ISSN	1538-7445 ; 0008-5472
ISSN (online)	1538-7445
ISSN	0008-5472
DOI	10.1158/0008-5472.CAN-21-0774
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Article ; Online: A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification.

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Article ; Online: Combination Osimertinib and Gefitinib in C797S and T790M EGFR-Mutated Non-Small Cell Lung Cancer.

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Article: Standard dose osimertinib for erlotinib refractory T790M-negative

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Article ; Online: PDCD1

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Article ; Online: Comparison of 3 Methodologies for Genotyping of Small Deletion and Insertion Polymorphisms.

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Article ; Online: Molecular and clinical determinants of response and resistance to rucaparib for recurrent ovarian cancer treatment in ARIEL2 (Parts 1 and 2).

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Article ; Online: Acquired

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