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A 30-kb deletion that eliminates the coding region of APOBEC3B (A3B) is >5 times more common in women of Asian compared to European descent. This polymorphism creates a chimera with the APOBEC3A (A3A) coding region and A3B 3'UTR, and is associated with an increased risk for breast cancer in Asian women. Here, we explored the relationship between the A3B deletion polymorphism with tumour characteristics in Asian women. Using whole exome and whole transcriptome sequencing data of 527 breast tumours, we report that germline A3B deletion polymorphism leads to expression of the A3A-B hybrid isoform and increased APOBEC-associated somatic hypermutation. Hypermutated tumours, regardless of A3B germline status, were associated with the Her2 molecular subtype and PIK3CA mutations. Compared to non-hypermutated tumours, hypermutated tumours also had higher neoantigen burden, tumour heterogeneity and immune activation. Taken together, our results suggest that the germline A3B deletion polymorphism, via the A3A-B hybrid isoform, contributes to APOBEC-mutagenesis in a significant proportion of Asian breast cancers. In addition, APOBEC somatic hypermutation, regardless of A3B background, may be an important clinical biomarker for Asian breast cancers. This article is protected by copyright. All rights reserved.

Molecular profiling of breast cancer has enabled the development of more robust molecular prognostic signatures and therapeutic options for breast cancer patients. However, non-Caucasian populations remain understudied. Here, we present the mutational, transcriptional, and copy number profiles of 560 Malaysian breast tumours and a comparative analysis of breast cancers arising in Asian and Caucasian women. Compared to breast tumours in Caucasian women, we show an increased prevalence of HER2-enriched molecular subtypes and higher prevalence of TP53 somatic mutations in ER+ Asian breast tumours. We also observe elevated immune scores in Asian breast tumours, suggesting potential clinical response to immune checkpoint inhibitors. Whilst HER2-subtype and enriched immune score are associated with improved survival, presence of TP53 somatic mutations is associated with poorer survival in ER+ tumours. Taken together, these population differences unveil opportunities to improve the understanding of this disease and lay the foundation for precision medicine in different populations., Molecular profiling of breast cancer in non-Caucasian populations remains underexplored. Here the authors report a high prevalence of HER2-subtypes and enriched immune score with improved survival and higher rates of TP53 somatic mutations with poorer survival in ER+ tumours in a Malaysian cohort.

A 30-kb deletion that eliminates the coding region of APOBEC3B (A3B) is5 times more common in women of Asian compared to European descent. This polymorphism creates a chimera with the APOBEC3A (A3A) coding region and A3B 3'UTR, and is associated with an increased risk for breast cancer in Asian women. Here, we explored the relationship between the A3B deletion polymorphism with tumour characteristics in Asian women. Using whole exome and whole transcriptome sequencing data of 527 breast tumours, we report that germline A3B deletion polymorphism leads to expression of the A3A-B hybrid isoform and increased APOBEC-associated somatic hypermutation. Hypermutated tumours, regardless of A3B germline status, were associated with the Her2 molecular subtype and PIK3CA mutations. Compared to non-hypermutated tumours, hypermutated tumours also had higher neoantigen burden, tumour heterogeneity and immune activation. Taken together, our results suggest that the germline A3B deletion polymorphism, via the A3A-B hybrid isoform, contributes to APOBEC-mutagenesis in a significant proportion of Asian breast cancers. In addition, APOBEC somatic hypermutation, regardless of A3B background, may be an important clinical biomarker for Asian breast cancers. This article is protected by copyright. All rights reserved.

A 30-kb deletion that eliminates the coding region ofAPOBEC3B(A3B) is >5 times more common in women of Asian compared to European descent. This polymorphism creates a chimera with theAPOBEC3A(A3A) coding region andA3B3’UTR, and is associated with an increased risk for breast cancer in Asian women. Here, we explored the relationship between theA3Bdeletion polymorphism with tumour characteristics in Asian women. Using whole exome and whole transcriptome sequencing data of 527 breast tumours, we report that germlineA3Bdeletion polymorphism leads to expression of theA3A-Bhybrid isoform and increased APOBEC-associated somatic hypermutation. Hypermutated tumours, regardless ofA3Bgermline status, were associated with the Her2 molecular subtype andPIK3CAmutations. Compared to non-hypermutated tumours, hypermutated tumours also had higher neoantigen burden, tumour heterogeneity and immune activation. Taken together, our results suggest that the germlineA3Bdeletion polymorphism, via theA3A-Bhybrid isoform, contributes to APOBEC-mutagenesis in a significant proportion of Asian breast cancers. In addition, APOBEC somatic hypermutation, regardless ofA3Bbackground, may be an important clinical biomarker for Asian breast cancers.Graphical Abstract

Background: Oestrogen receptor positive (ER+) breast cancer patients can acquire endocrine resistance and 10-20% tumours have lost ER at relapse. While growth factor pathway hyperactivity and ER promoter methylation contribute to de novo ER negativity, ER loss in acquired resistance is largely unexplored. We have recently developed 11 lines from MCF7, T47D, BT474 & MDAMB361 cells to model acquired resistance emerging with prolonged (3 year) endocrine treatment both in ER+/HER2- and ER+/HER2+ disease. Here we establish prevalence of acquired ER loss in the panel, determine any association with aggressiveness, and explore ER loss mechanisms in acquired endocrine resistance. Methods: Authenticated acquired resistant models derived from endocrine responsive lines cultured for 3 years with 10-7M tamoxifen (TamR), 10-7M fulvestrant (FasR) or oestrogen deprivation (5% charcoal-stripped foetal calf serum SFCSR) were profiled for ER & PR by PCR, immunocytochemistry and Western blotting (+/- 1-2wk antihormone withdrawal), for 2nd-line endocrine responsiveness and for migration using Boyden chamber assays vs. time-matched controls. Src kinase, EGFR, HER2, MAPK & AKT activity were examined and whether their respective inhibition using saracatinib or gefitinib (1μM), trastuzumab (100nM), U0126 or LY294002 (5μM) for 1wk restored ER. ESR1 promoter methylation was examined by bisulfite modification & MethyLight PCR. Results: Substantial ER mRNA & protein loss occurred in 7/11 long-term acquired endocrine resistant lines. This was irreversible by antihormone withdrawal and paralleled by complete PR loss and endocrine growth-insensitivity. While seen in all fulvestrant resistant lines, ER loss was less frequent with tamoxifen (in MCF7TamR & MDATamR) and only seen in oestrogen deprived resistant T47DSFCSR cells. Increased migration accompanied acquired ER loss in ER+/HER2- derived MCF7TamR, MCF7FasR & T47DSFCSR cells and was saracatinib-sensitive. Src and further growth factor pathway activity increased in several acquired resistant models, and ER loss associated with increased EGFR/HER2 in the MCF7- & MDA-derived cells and with increased MAPK activity in all lines. Weak ER recovery was seen in antioestrogen resistant models treated with saracatinib (MDATamR, MCF7FasR), gefitinib (MDATamR, BT474FasR, MCF7FasR/TamR) or trastuzumab (MCF7TamR). ESR1 DNA methylation was only prominent in MDATamR and MCF7TamR cells. No inhibitor restored ER in the T47D-derived cells, including T47DSFCSR which also lacked ESR1 methylation. Conclusions: Although ER loss is very prominent in this acquired resistant cell panel, it demonstrates there is capacity of prolonged antihormones, chiefly antioestrogens, to promote receptor loss independent of initial HER2 status. Acquired ER loss clinically would be expected to confer endocrine insensitivity and poorer prognosis given the panel findings. Where ER loss emerged with antioestrogens there was some mechanistic-overlap with de novo ER negativity, including ER promoter methylation for acquired tamoxifen resistance. Our future studies will use the panel to address if targeting these mechanisms can be optimized for ER recovery or if further mechanisms also drive ER loss in acquired resistance, notably for prolonged oestrogen deprivation. Citation Format: Julia M Gee, Mei Yee Meng, Richard A McClelland, Huw J Mottram, Susan R Kyme, Pauline Finlay, Lindy Goddard, Carol M Dutkowski, Denise Barrow, Walther Parson, Heidi Fiegl. A new cell panel to study oestrogen receptor loss in acquired endocrine resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-19.