TY - JOUR
T1 - Frequency, impact and a preclinical study of novel ERBB gene family mutations in HER2-positive breast cancer
AU - Elster, Naomi
AU - Toomey, Sinead
AU - Fan, Yue
AU - Cremona, Mattia
AU - Morgan, Clare
AU - Weiner Gorzel, Karolina
AU - Bhreathnach, Una
AU - Milewska, Malgorzata
AU - Murphy, Madeline
AU - Madden, Stephen
AU - Naidoo, Jarushka
AU - Fay, Joanna
AU - Kay, Elaine
AU - Carr, Aoife
AU - Kennedy, Sean
AU - Furney, Simon
AU - Mezynski, Janusz
AU - Breathhnach, Oscar
AU - Morris, Patrick
AU - Grogan, Liam
AU - Hill, Arnold
AU - Kennedy, Susan
AU - Crown, John
AU - Gallagher, William
AU - Hennessy, Bryan
AU - Eustace, Alex
N1 - Funding Information:
This work was supported by the Irish Cancer Society Collaborative Cancer Research Centre, BREAST-PREDICT (CCRC13GAL) (http:// www.breastpredict.com), an Irish Cancer Society Research Scholarship (CRS11ELS), the Health Research Board (HRA/POR2012/054), NECRET, the North-Eastern Cancer Research and Education Trust, and a Breast Cancer Now Catalyst Grant (2016NovPCC002).
Funding Information:
We thank Dr Scott Wilhelm and Bayer Pharmaceuticals for providing us with copanlisib. We thank Padraig Doolan for his helpful comments on the manuscript. We also acknowledge the following for their contributions to this manuscript: NE performed Chi-square and Fisher’s exact tests on the clinical data, performed the site-directed mutagenesis and performed all in vitro experiments with the stably transfected cell lines excluding the kinase assay, analyzed those results, and drafted this manuscript. ST designed the Agena Panel and performed the Agena analysis. YF performed bioinformatics analysis. MC and CM completed the RPPA experiments (subsequent RPPA analysis by NE and AJE, with advice from MC). KW-M, UB and MM (Murphy) stably transfected the construct cell lines used in this study. MM (Milewska) assisted in the design of the creation of the construct cell lines, and extracted protein from FFPE samples for use in the RPPA experiment. SM and SJF provided statistical expertise and guidance to the clinical study, including the generation of KM curves. JF and EWK performed H&E staining and quality control on all clinical samples used in this study. EWK also contributed to the revision of this manuscript. AC extracted DNA from FFPE clinical samples for use in Agena experiments. SK (RCSI) performed the kinase assays. JN, JM, OSB, PGM, LG, ADH, SK (SVUH) and JC facilitated this study through the provision of clinical samples and data, and assistance in the analysis of the data. PGM also contributed to the revision of this manuscript. WMG provided bioinformatics and NGS supervision and expertise to the project. BTH and AJE conceived of the need for this study, designed and supervised all stages of the project, and were major contributors to the revision and preparation of the final manuscript. All authors read and approved the final manuscript This work was supported by the Irish Cancer Society Collaborative Cancer Research Centre, BREAST-PREDICT (CCRC13GAL) (http://www.breastpredict.com), an Irish Cancer Society Research Scholarship (CRS11ELS), the Health Research Board (HRA/POR2012/054), NECRET, the North-Eastern Cancer Research and Education Trust, and a Breast Cancer Now Catalyst Grant (2016NovPCC002).
Publisher Copyright:
© The Author(s), 2018.
PY - 2018/1/1
Y1 - 2018/1/1
N2 - Background: Somatic mutations in the ERBB genes (epidermal growth factor receptor: EGFR, ERBB2, ERBB3, ERBB4) promote oncogenesis and lapatinib resistance in metastatic HER2+ (human epidermal growth factor-like receptor 2) breast cancer in vitro. Our study aimed to determine the frequency of mutations in four genes: EGFR, ERBB2, ERBB3 and ERBB4 and to investigate whether these mutations affect cellular behaviour and therapy response in vitro and outcomes after adjuvant trastuzumab-based therapy in clinical samples. Methods: We performed Agena MassArray analysis of 227 HER2+ breast cancer samples to identify the type and frequency of ERBB family mutations. Of these, two mutations, the somatic mutations ERBB4-V721I and ERBB4-S303F, were stably transfected into HCC1954 (PIK3CA mutant), HCC1569 (PIK3CA wildtype) and BT474 (PIK3CA mutant, ER positive) HER2+ breast cancer cell lines for functional in vitro experiments. Results: A total of 12 somatic, likely deleterious mutations in the kinase and furin-like domains of the ERBB genes (3 EGFR, 1 ERBB2, 3 ERBB3, 5 ERBB4) were identified in 7% of HER2+ breast cancers, with ERBB4 the most frequently mutated gene. The ERBB4-V721I kinase domain mutation significantly increased 3D-colony formation in 3/3 cell lines, whereas ERBB4-S303F did not increase growth rate or 3D colony formation in vitro. ERBB4-V721I sensitized HCC1569 cells (PIK3CA wildtype) to the pan class I PI3K inhibitor copanlisib but increased resistance to the pan-HER family inhibitor afatinib. The combinations of copanlisib with trastuzumab, lapatinib, or afatinib remained synergistic regardless of ERBB4-V721I or ERBB4-S303F mutation status. Conclusions: ERBB gene family mutations, which are present in 7% of our HER2+ breast cancer cohort, may have the potential to alter cellular behaviour and the efficacy of HER- and PI3K-inhibition.
AB - Background: Somatic mutations in the ERBB genes (epidermal growth factor receptor: EGFR, ERBB2, ERBB3, ERBB4) promote oncogenesis and lapatinib resistance in metastatic HER2+ (human epidermal growth factor-like receptor 2) breast cancer in vitro. Our study aimed to determine the frequency of mutations in four genes: EGFR, ERBB2, ERBB3 and ERBB4 and to investigate whether these mutations affect cellular behaviour and therapy response in vitro and outcomes after adjuvant trastuzumab-based therapy in clinical samples. Methods: We performed Agena MassArray analysis of 227 HER2+ breast cancer samples to identify the type and frequency of ERBB family mutations. Of these, two mutations, the somatic mutations ERBB4-V721I and ERBB4-S303F, were stably transfected into HCC1954 (PIK3CA mutant), HCC1569 (PIK3CA wildtype) and BT474 (PIK3CA mutant, ER positive) HER2+ breast cancer cell lines for functional in vitro experiments. Results: A total of 12 somatic, likely deleterious mutations in the kinase and furin-like domains of the ERBB genes (3 EGFR, 1 ERBB2, 3 ERBB3, 5 ERBB4) were identified in 7% of HER2+ breast cancers, with ERBB4 the most frequently mutated gene. The ERBB4-V721I kinase domain mutation significantly increased 3D-colony formation in 3/3 cell lines, whereas ERBB4-S303F did not increase growth rate or 3D colony formation in vitro. ERBB4-V721I sensitized HCC1569 cells (PIK3CA wildtype) to the pan class I PI3K inhibitor copanlisib but increased resistance to the pan-HER family inhibitor afatinib. The combinations of copanlisib with trastuzumab, lapatinib, or afatinib remained synergistic regardless of ERBB4-V721I or ERBB4-S303F mutation status. Conclusions: ERBB gene family mutations, which are present in 7% of our HER2+ breast cancer cohort, may have the potential to alter cellular behaviour and the efficacy of HER- and PI3K-inhibition.
KW - HER2+ BC
KW - PI3K
KW - PI3K inhibition
KW - breast cancer
KW - somatic mutations
KW - trastuzumab resistance
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U2 - 10.1177/1758835918778297
DO - 10.1177/1758835918778297
M3 - Article
C2 - 30023006
AN - SCOPUS:85054570621
SN - 1758-8340
VL - 10
JO - Therapeutic Advances in Medical Oncology
JF - Therapeutic Advances in Medical Oncology
ER -