Taiwanese Breast Cancer Patients Research Articles

Analysis of the Abasic Sites in Breast Cancer Patients With 5 Year Postoperative Treatment Without Recurrence in Taiwan.

This prospective study aimed to investigate estrogen-induced carcinogenesis by assessing the background levels of abasic sites (apurinic/apyrimidinic sites, AP sites) in Taiwanese breast cancer patients following 5 years of postoperative treatment without recurrence (5-year survivors) (n = 70). The study also sought to compare the extent of these DNA lesions with those found in healthy controls and in breast cancer patients prior to treatment. Abasic sites were measured using an aldehyde reactive probe and quantified as the total number of abasic sites per total nucleotides. Characterization of the abasic sites in subjects recruited for this study was conducted using the abasic site cleavage assay using putrescine or T7 exonuclease (T7 Exo) and/or exonuclease III (Exo III). The number of abasic sites detected in 5year survivors (26.7 ± 10.2 per 106 total nucleotides, n = 70) was significantly reduced by 46.9% compared to those in breast cancer patients before treatment (50.3 ± 59.2 per 106 total nucleotides, P < 0.001), and was similar to the levels observed in healthy controls (23.3 ± 13.5 per 106 total nucleotides, P > 0.05). Further investigation into the specific types of abasic sites indicated that the number of abasic sites excisable by putrescine in controls, breast cancer patients, and 5-year survivors were 63.3%, 78.6%, and 67.7%, respectively. These findings suggest the involvement of oxidative stress rather than depurination/depyrimidination of DNA adducts in the formation of abasic sites. Further analyses were performed using exonuclease cleavage assay to characterize the specific types of abasic sites including 5'-cleaved, 3'-cleaved, intact, and residual abasic sites. Results demonstrated that the proportion of residual abasic sites detected in controls, breast cancer patients, and 5-year survivors were estimated to be 32.7%, 48.8%, and 34.0%, respectively. Overall, these findings suggest clear evidence of treatment-related effects on the reduction of levels of abasic sites as well as on the profile of abasic sites in 5year survivors.

Prevalence of BRCA1, BRCA2, and PALB2 genomic alterations among 924 Taiwanese breast cancer assays with tumor-only targeted sequencing: extended data analysis from the VGH-TAYLOR study

BackgroundThe homologous recombination (HR) repair pathway for DNA damage, particularly the BRCA1 and BRCA2 genes, has become a target for cancer therapy, with poly ADP-ribose polymerase (PARP) inhibitors showing significant outcomes in treating germline BRCA1/2 (gBRCA1/2) mutated breast cancer. Recent studies suggest that some patients with somatic BRCA1/2 (sBRCA1/2) mutation or mutations in HR-related genes other than BRCA1/2 may benefit from PARP inhibitors as well, particularly those with PALB2 mutations. The current analysis aims to evaluate the prevalence of genetic alterations specific to BRCA1, BRCA2, and PALB2 in a large cohort of Taiwanese breast cancer patients through tumor-targeted sequencing.MethodsA total of 924 consecutive assays from 879 Taiwanese breast cancer patients underwent tumor-targeted sequencing (Thermo Fisher Oncomine Comprehensive Assay v3). We evaluated BRCA1, BRCA2, and PALB2 mutational profiles, with variants annotated and curated by the ClinVAR, the Oncomine™ Knowledgebase Reporter, and the OncoKB™. We also conducted reflex germline testing using either whole exome sequencing (WES) or whole genome sequencing (WGS), which is ongoing.ResultsAmong the 879 patients analyzed (924 assays), 130 had positive mutations in BRCA1 (3.1%), BRCA2 (8.6%), and PALB2 (5.2%), with a total of 14.8% having genetic alterations. Co-occurrence was noted between BRCA1/BRCA2, BRCA1/PALB2, and BRCA2/PALB2 mutations. In BRCA1-mutated samples, only p.K654fs was observed in three patients, while other variants were observed no more than twice. For BRCA2, p.N372H was the most common (26 patients), followed by p.S2186fs, p.V2466A, and p.X159_splice (5 times each). For PALB2, p.I887fs was the most common mutation (30 patients). This study identified 176 amino acid changes; 60.2% (106) were not documented in either ClinVAR or the Oncomine™ Knowledgebase Reporter. Using the OncoKB™ for annotation, 171 (97.2%) were found to have clinical implications. For the result of reflex germline testing, three variants (BRCA1 c.1969_1970del, BRCA1 c.3629_3630del, BRCA2 c.8755-1G > C) were annotated as Pathogenic/Likely pathogenic (P/LP) variants by ClinVar and as likely loss-of-function or likely oncogenic by OncoKB; while one variant (PALB2 c.448C > T) was not found in ClinVar but was annotated as likely loss-of-function or likely oncogenic by OncoKB.ConclusionOur study depicted the mutational patterns of BRCA1, BRCA2, and PALB2 in Taiwanese breast cancer patients through tumor-only sequencing. This highlights the growing importance of BRCA1/2 and PALB2 alterations in breast cancer susceptibility risk and the treatment of index patients. We also emphasized the need to meticulously annotate variants in cancer-driver genes as well as actionable mutations across multiple databases.

Abstract 2271: Characteristics of peripheral blood T cell receptor repertoire and correlation with response to chemotherapy in patients with breast cancer

Abstract Purpose: The success of immune checkpoint inhibitors therapy in triple negative breast cancer highlights the potential immunogenic characteristics of breast cancer. Increasing evidence have suggested that conventional chemotherapy can exert anti-cancer activity through various immune-based mechanisms. T cell receptor (TCR) diversity is crucial for immune responses and TCR repertoire has been reported acting as predictive and prognostic markers for cancer outcome. Here, we reported the TCR repertoire profiling in breast cancer and its association with chemotherapy and treatment response in breast cancer patients. Experimental design: This study is part of VGH-TAYLOR study, a comprehensive precision medicine study protocol on the heterogeneity of Taiwanese breast cancer patients (NCT04626440). A total of 856 female patients with luminal A, luminal B1, luminal B2, HER2-enriched, or triple-negative breast cancers were recruited. The enrolled subjects contains patients who receive surgery as the first-line treatment followed by adjuvant therapy and who receive neoadjuvant therapy as the first-line treatment followed by surgery. The blood samples from patients during chemotherapy were collected for TCR sequencing. The Oncomine TCR Beta-LR Assay was applied to determine the dynamic change in TCR repertoire. We examined TCR repertoire in terms of clonality, TCR richness/evenness and convergence. Results: Higher TCR clonality in breast cancer patients was associated with lower clonal richness. TCR clonality was positively correlated with age and stage but not tumor size. Patients with recurrence had higher TCR clonality and lower Shannon diversity. Patients with luminal B2 breast cancer showed lower TCR diversity compared to luminal A and triple-negative breast cancer. However, there were no significant differences in CDR3 length and the usage of variable and joining genes among breast cancer subtypes. Notably, both of adjuvant and neoadjuvant chemotherapies increased convergence and clonality while decreased Shannon diversity of TCR. In neoadjuvant settings, lower post-treatment blood TCR richness was associated with complete pathologic response. Conclusion: These data suggested that TCR repertoire is associated with clinicopathological characteristics including stage and recurrence. TCR clonal richness might provide prognostic value for patients receiving neoadjuvant chemotherapy. Citation Format: Chun-Yu Liu, Chi-Cheng Huang, Ji-Lin Chen, Yi-Fang Tsai, Ta-Chung Chao, Pei-Ju Lien, Yen-Shu Lin, Chin-Jung Feng, Yen-Jen Chen, Jiun-I Lai, Jen-Hwey Chiu, Chih-Yi Hsu, Ling-Ming Tseng. Characteristics of peripheral blood T cell receptor repertoire and correlation with response to chemotherapy in patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2271.

Abstract P2-23-08: Concordance of Targeted Sequencing from Circulating Tumor DNA and Paired Tumor Tissue

Abstract Purpose: In this study, we evaluated the concordance of targeted sequencing results between paired ctDNA and tumor samples from early breast cancers scheduled for curative surgery with or without adjuvant therapy. If high concordance was observed, pre-operative ctDNA testing could serve as a non-invasive surrogate for variants meant to be revealed after definite surgery. On the other hand, poor concordance may hamper wide clinical absorption of liquid biopsy early breast cancer. Materials and Methods: The study VGH-TAYLOR: Comprehensive precision medicine research on the heterogeneity of Taiwanese breast cancer patients, consisted of three years enrollment and approximately four years follow-up after enrollment. Individual subject was assigned into Group 1 [planned to received surgery as first-line treatment and followed by adjuvant therapy, Group 2 [planned to receive neoadjuvant therapy as the first-line treatment and followed by surgery], and Group 3 [diagnosed with de novo and treatment naïve stage IV breast cancer, or stage IV breast cancer with recurrence beyond three years after surgery]. Molecular profiling and potential biomarkers were determined using Oncomine Comprehensive Assay v3 from FFPE tissues and Oncomine Breast cfDNA Assay v2 from plasma. Oncomine Comprehensive Assay is a targeted sequencing of NGS experiments were performed with TMO comprehensive using FFPE tissues, included 161 cancer-releant genes and types of mutation detected such as frameshift, missense, synonymous, SNV, Indel, and CNV. Oncomine Breast cfDNA Assay detects breast-derived cfDNA including hotspot genes: AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, PIK3CA, SF3B1, TP53 (~152 hotspots), CNVs: CCND1, ERBB2, FGFR1, and full length TP53 (~80% coverage). Common genes interrogated by both ctDNA and TMO assay were identified, and concordance between paired targeted sequencing results from the same individuals were reported. Results: We reported the mutational landscape of initial 612 patients interrogated with the Oncomine Breast cfDNA assay; 239 out of 612 patients reported at least one mutation (39%). Among 246 patients assayed for both ctDNA and tumor tissue, cfDNA assay detected 73 (29.6%) and TMO comprehensive assay detected 201 (81.7%) breast cancers with at least one variant (c2 test, p=0.001). Sixty-seven (25.6%) were tested positive for both liquid and tissue assays, while cfDNA and TMO comprehensive assay detected additional 10 (4%) and 138 (56%) cases, which were not identified by the other platform. Table 1 details the distributions of called variants among common targeted genes. Conclusion: In current study, we evaluated the concordance of called variants between targeted sequencing from ctDNA and tumor tissue among an early breast cancer cohort in Taiwan. Only one-quarter of patients were positive for both cfDNA and TMO comprehensive assays from the same subject, indicating assay-specific sensitivity inevitably resulting in diagnostic discrepancy. Another plausible explanation came from the early stage nature of interrogated patients, which may inherit fewer ctDNA spillage from tumor and compromise detectability from liquid biopsy. The most prevalent mutant genes from both platforms were TP53 (68.3%) and KRAS (53.5%), both were well-known drivers in cancers. From breast cancer actionability, PIK3CA (39.4%) mutation is a biomarker for the FDA-approved PI3Ka inhibitor such as alpelisib, AKT1 (45.9%) mutation for agents such as capivasertib (AZD5363) and ipatasertib, and ERBB2 mutation for tyrosine kinase inhibitor such as neratinib. Our study showed that tumor should be the preferred source for targeted sequencing, and additional 4% of variants wound be revealed from liquid biopsy. Table 1. Distributions of variants from common targeted genes (n=6) from cfDNA and TMO comprehensive assay. Citation Format: Chi-Cheng Huang, Ling-Ming Tseng. Concordance of Targeted Sequencing from Circulating Tumor DNA and Paired Tumor Tissue [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-08.

The effect of the preoperational psychoeducation program for Taiwanese breast cancer patients: A three-month follow-up study

Studies suggest that psychosocial interventions might be more effective during highly stressful periods, such as before surgery. This study aimed to explore the effectiveness of the Preoperational Health Psychology Education program among Taiwanese breast cancer patients. A total of 137 adult women (1) diagnosed with breast cancer; and (2) admitted to the ward for surgery were recruited to join the program one day before surgery. Emotional distress, worries, and cancer self-efficacy were evaluated at admission, post-program, and 3-month post-surgery. Patients were grouped into high/low distress groups, and mixed-design ANOVA was used to examine the program's effectiveness. The results showed significant interactions of Group × Time in emotional distress ( F = 16.15, p = .000) and worry ( F = 5.81, p = .005), but not in self-efficacy ( F = 2.97, p = .068). The post-hoc tests revealed significant decreases in emotional distress and worry in the high distress group. The program was found helpful in reducing emotional distress and worries. The effect lasting for three-month for those with higher preoperational emotional distresses. This psycho-education program with a relatively rare one-session design, targeted at a less-studied pre-surgery period, is helpful to a less-studied population, Asian cancer patients.

Promoter hypomethylation and overexpression of TSTD1 mediate poor treatment response in breast cancer.

Epigenetic alterations play a pivotal role in cancer treatment outcomes. Using the methylation array data and The Cancer Genome Atlas (TCGA) dataset, we observed the hypomethylation and upregulation of thiosulfate sulfurtransferase-like domain containing 1 (TSTD1) in patients with breast cancer. We examined paired tissues from Taiwanese patients and observed that 65.09% and 68.25% of patients exhibited TSTD1 hypomethylation and overexpression, respectively. A significant correlation was found between TSTD1 hypomethylation and overexpression in Taiwanese (74.2%, p = 0.040) and Western (88.0%, p < 0.001) cohorts. High expression of TSTD1 protein was observed in 68.8% of Taiwanese and Korean breast cancer patients. Overexpression of TSTD1 in tumors of breast cancer patients was significantly associated with poor 5-year overall survival (p = 0.021) and poor chemotherapy response (p = 0.008). T47D cells treated with TSTD1 siRNA exhibited lower proliferation than the control group, and transfection of TSTD1 in MDA-MB-231 induced the growth of MDA-MB-231 cells compared to the vector control. Additionally, overexpression of TSTD1 in MCF7 cells mediated a poor response to chemotherapy by epirubicin (p < 0.001) and docetaxel (p < 0.001) and hormone therapy by tamoxifen (p =0.025). Circulating cell-free hypomethylated TSTD1 was detected in plasma of Taiwanese breast cancer patients with disease progression and poor chemotherapy efficacy. Our results indicate that promoter hypomethylation and overexpression of TSTD1 in patients with breast cancer are potential biomarkers for poor 5-year overall survival and poor treatment response.

Association of Pathway Mutations With Survival in Taiwanese Breast Cancers.

Breast cancer is the most common invasive cancer in women worldwide. Next-generation sequencing (NGS) provides a high-resolution profile of cancer genome. Our study ultimately gives the insight for genetic screening to identify the minority of patients with breast cancer with a poor prognosis, who might benefit from the most intensive possible treatment. The detection of mutations can polish the traditional method to detect high-risk patients who experience poor prognosis, recurrence and death early. In total, 147 breast cancer tumors were sequenced with targeted sequencing using a RainDance Cancer Hotspot Panel. The average age of all 147 breast cancer patients in the study was 51.7 years, with a range of 21–77 years. The average sequencing depth was 5,222x (range 2,900x-8,633x), and the coverage was approximately 100%. A total of 235 variants in 43 genes were detected in 147 patients by high-depth Illumina sequencing. A total of 219 single nucleotide variations were found in 42 genes from 147 patients, and 16 indel mutations were found in 13 genes from 84 patients. After filtering with the 1000 Genomes database and for synonymous SNPs, we focused on 54 somatic functional point mutations. The functional point mutations contained 54 missense mutations in 22 genes. Additionally, mutation of genes within the RET, PTEN, CDH1, MAP2K4, NF1, ERBB2, RUNX1, PIK3CA, FGFR3, KIT, KDR, APC, SMO, NOTCH1, and FBXW7 in breast cancer patients were with poor prognosis. Moreover, TP53 and APC mutations were enriched in triple-negative breast cancer. APC mutations were associated with a poor prognosis in human breast cancer (log-rank P<0.001). Our study identified tumor mutation hotspot profiles in Taiwanese breast cancer patients, revealing new targetable gene mutations in Asian breast cancer patients.

Hypermethylation of TMEM240 predicts poor hormone therapy response and disease progression in breast cancer

BackgroundApproximately 25% of patients with early-stage breast cancer experience cancer progression throughout the disease course. Alterations in TMEM240 in breast cancer were identified and investigated to monitor treatment response and disease progression.MethodsCirculating methylated TMEM240 in the plasma of breast cancer patients was used to monitor treatment response and disease progression. The Cancer Genome Atlas (TCGA) data in Western countries and Illumina methylation arrays in Taiwanese breast cancer patients were used to identify novel hypermethylated CpG sites and genes related to poor hormone therapy response. Quantitative methylation-specific PCR (QMSP), real-time reverse transcription PCR, and immunohistochemical analyses were performed to measure DNA methylation and mRNA and protein expression levels in 394 samples from Taiwanese and Korean breast cancer patients. TMEM240 gene manipulation, viability, migration assays, RNA-seq, and MetaCore were performed to determine its biological functions and relationship to hormone drug treatment response in breast cancer cells.ResultsAberrant methylated TMEM240 was identified in breast cancer patients with poor hormone therapy response using genome-wide methylation analysis in the Taiwan and TCGA breast cancer cohorts. A cell model showed that TMEM240, which is localized to the cell membrane and cytoplasm, represses breast cancer cell proliferation and migration and regulates the expression levels of enzymes involved in estrone and estradiol metabolism. TMEM240 protein expression was observed in normal breast tissues but was not detected in 88.2% (67/76) of breast tumors and in 90.0% (9/10) of metastatic tumors from breast cancer patients. QMSP revealed that in 54.5% (55/101) of Taiwanese breast cancer patients, the methylation level of TMEM240 was at least twofold higher in tumor tissues than in matched normal breast tissues. Patients with hypermethylation of TMEM240 had poor 10-year overall survival (p = 0.003) and poor treatment response, especially hormone therapy response (p < 0.001). Circulating methylated TMEM240 dramatically and gradually decreased and then diminished in patients without disease progression, whereas it returned and its levels in plasma rose again in patients with disease progression. Prediction of disease progression based on circulating methylated TMEM240 was found to have 87.5% sensitivity, 93.1% specificity, and 90.2% accuracy.ConclusionsHypermethylation of TMEM240 is a potential biomarker for treatment response and disease progression monitoring in breast cancer.

VGH-TAYLOR: Comprehensive precision medicine study protocol on the heterogeneity of Taiwanese breast cancer patients.

Heterogeneity in breast cancer leads to diverse morphological features and different clinical outcomes. There are inherent differences in breast cancer between the populations in Asia and in western countries. The use of immune-based treatment in breast cancer is currently in the developmental stage. The VGH-TAYLOR study is designed to understand the genetic profiling of different subtypes of breast cancer in Taiwan and define the molecular risk factors for breast cancer recurrence. The T-cell receptor repertoire and the potential effects of immunotherapy in breast cancer subjects isevaluated. The favorable biomarkers for early detection of tumor recurrence, diagnosis and prognosis may provide clues for the selection of individualized treatment regimens and improvement in breast cancer therapy.

Differential whole-genome doubling and homologous recombination deficiencies across breast cancer subtypes from the Taiwanese population

Whole-genome doubling (WGD) is an early macro-evolutionary event in tumorigenesis, involving the doubling of an entire chromosome complement. However, its impact on breast cancer subtypes remains unclear. Here, we performed a comprehensive and quantitative analysis of WGD and its influence on breast cancer subtypes in patients from Taiwan and consequently highlight the genomic association between WGD and homologous recombination deficiency (HRD). A higher manifestation of WGD was reported in triple-negative breast cancer, conferring high chromosomal instability (CIN), while HER2 + tumors exhibited early WGD events, with widely varied CIN levels, compared to luminal-type tumors. An association of higher activity of de novo indel signature 2 with WGD and HRD in Taiwanese breast cancer patients was reported. A control test between WGD and pseudo non-WGD samples was further employed to support this finding. The study provides a better comprehension of tumorigenesis in breast cancer subtypes, thus assisting in personalized treatment.

Comprehensive molecular profiling of Taiwanese breast cancers revealed potential therapeutic targets: prevalence of actionable mutations among 380 targeted sequencing analyses

BackgroundBreast cancer is one of the leading causes of cancer-related deaths in women, and there is a demand in developing an Asian-based genetic profiling database for breast cancer in improving the treatment response. This study aimed to determine molecular alternations and identify potential therapeutic targets by analyzing the genetic profiling from a cohort of Taiwanese breast cancers using a commercialized next-generation sequencing (NGS) targeted panel.MethodsThe study population comprised a broad spectrum of breast cancer patients in Taiwan, including Group 1: planned to receive first-line surgery and followed by adjuvant therapy, or early relapse within three years, Group 2: planned to receive first-line neoadjuvant therapy and followed by surgery, and Group 3: de novo stage IV, or stage IV with recurrence beyond three years. Molecular profiles were determined using Thermo Fisher™ Oncomine™ Comprehensive Assay version 3 (TMO comprehensive assay) from Formalin-Fixed Paraffin-Embedded (FFPE) tissues. Level of actionability was evaluated with the ESMO Scale of clinical actionability of molecular targets (ESCAT).ResultsA total of 380 TMO comprehensive assays were conducted on 372 patients, and we presented targeted sequencing analyses of Tier I: alteration-drug match associated with improved outcome in clinical trials including ERBB2 amplification, BRCA1/2 germline mutation, PIK3CA mutation, and NTRK translocation, and Tier II: antitumor activity associated with the matched alteration-drug but lack of prospective outcome data including PTEN loss, ESR1 mutation, AKT1 mutation, and ERBB2 mutation, and Tier III: matched drug-alteration that led to clinical benefit in another tumor type including MDM2 amplification, and ERBB3 mutation. Among them, 249 (66%) showed at least one actionable alternation based on the ESCAT criteria. The most frequent impacted genes (all variants combined within each sample) were PIK3CA (38%), followed by ERBB2 (23%), ESR1 (10%), AKT1 (6%), and BRCA2 (5%), and the remaining rare variants (less than 5% of assayed cohort) were BRCA1 (3%), MDM2 (2.2%), and ERBB3 (1.1%).ConclusionTargeted sequencing of actionable genes is believed to provide clinical applicability and substantial benefits for Taiwanese breast cancer patients. A valid scale of clinical actionability should be adopted for precision medicine practice under multidisciplinary molecular tumor board.

Pre-treatment high body mass index is associated with poor survival in Asian premenopausal women with localized breast cancer.

Background: Obesity is associated with poor prognosis in breast cancer patients. This study aimed to evaluate the effect of obesity measured by body mass index (BMI) on survival of Taiwanese breast cancer patients in a single institution.Methods: We observed 5000 patients who were diagnosed with stage I-III breast cancer between 1990 and 2005. Information on BMI at diagnosis, and clinical follow-up for disease recurrence and death, up to 20 years post-diagnosis were available. BMI (in kg/m2) categories included normal weight (BMI<24), overweight (24≤BMI<27), and obesity (BMI≥27), according to recommendations from the Bureau of Health Promotion of Taiwan. The role of BMI and other known prognostic factors for patient survival were evaluated in this patient cohort.Results: Obesity was associated with advanced stage, higher nuclear grade, and higher percentages of estrogen receptor (ER) positive. The median age of patients with a higher BMI was greater than the median age of patients with a lower BMI. Obesity was an independent prognostic factor of overall survival (OS) (P<0.001), but not disease-free survival (DFS) (P=0.067). We subsequently analyzed the impact of age-stratified BMI (age<50 and age≥50 years) to ameliorate the impact of age bias. Following subset analyses, obesity correlated with shorter DFS (P=0.004) and OS (P=0.009) only in women<50 years of age. Multivariate analysis revealed that BMI was an independent prognostic factor for both DFS and OS in this group of patients. Subset analysis revealed that in women <50 years old, the impact of BMI on survival was associated with higher stage, ER negativity.Conclusion: BMI is an independent prognostic factor of OS and DFS in breast cancer patients aged<50 years. Although the cause-effect relationship between obesity and survival is unclear, we recommend that weight control measures in young breast cancer survivors should be considered.

Abstract 2503: Molecular profiling of Taiwanese breast cancers reveals potential therapeutic targets: Preliminary results of 160 subjects

Abstract Introduction: Breast cancer is one of the leading causes of cancer-related death in women, and the annual incidence of breast cancer has continuously increased throughout the past two decades in Taiwan. There is a demand in developing an Asian-based genetic profiling database for breast cancer in improving the treatment response and recurrence rate. This study aimed to determine the difference of molecular alternations and identify the potential biomarkers regarding therapeutic target by analyzing the genetic profiling from a large cohort of breast cancer subjects using the method of next-generation sequencing (NGS). Materials and methods: The protocol consisted of three years enrollment and approximately four years follow-up after enrollment. Individual subject was assigned into Group 1 [planned to received surgery as first-line treatment and followed by adjuvant therapy, Group 2 [planned to receive neoadjuvant therapy as the first-line treatment and followed by surgery], and Group 3 [diagnosed with de novo and treatment naïve stage IV breast cancer, or stage IV breast cancer with recurrence beyond three years after surgery]. Molecular profiling and potential biomarkers were determined using Thermo FisherTM OncomineTM Comprehensive Assay version 3 (TMO comprehensive assay) from Formalin-Fixed Paraffin-Embedded (FFPE) tissues. Results: We presented the initial 160 subjects as preliminary results. The TMO comprehensive assay is a targeted, NGS assay that enables the detection of relevant SNVs, CNVs, gene fusions, and indels from 161 unique genes to help inform drug discovery research and clinical trial research programs such as the NCI-MATCH trial. Level of actionability of sequenced targets were evaluated with ESMO Scale of clinical actionability of molecular targets (ESCAT). Of note, Tier I [alteration-drug match associated with improved outcome in clinical trials], Tier II [antitumor activity associated with the matched alteration-drug but lack of prospective outcome data], and Tier III [matched drug-alteration that led to clinical benefit in another tumor type] matched targets including ERBB2 amplification, BRCA1/2 germline mutation, PIK3CA mutation, NTRK translocation (Tier I), PTEN loss, ESR1 mutation, AKT1 mutation, ERBB2 mutation (Tier II), and BRCA1/2 somatic mutation, MDM2 amplification, and ERBB3 mutation (Tier III) were retrieved and analyzed. 108 out of 160 sequenced subjects (67.5%) reported as least one variant (average 1.7 per case, ranged from 1 to 10). Two group 3 heavily pre-treated patients were identified to have all actionable genes varied. Ipatasertib, alpelisib, and talazoparib, for instance, had been advocated for the corresponding PTEN p.(Q219*), PIK3CA p.(G118S), and BRCA2 p.(Q1107*) mutations. Conclusion: Targeted sequencing of actionable genes is believed to provide clinical applicability and substantial benefits for Taiwanese breast cancer patients. A valid scale of clinical actionability should be adopted for precision medicine practice under multidisciplinary molecular tumor board. Further details will be updated when more samples are ready for sequencing and actionability analyses in coming years. Citation Format: Chi-Cheng Huang, Chun-Yu Liu, Ling-Ming Tseng. Molecular profiling of Taiwanese breast cancers reveals potential therapeutic targets: Preliminary results of 160 subjects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2503.

Abstract P4-07-12: Residual risk stratification of Taiwanese breast cancer following curative therapies with digital RNA counting and actionable mutations sequencing

Abstract Introduction: Breast cancer is the most common female malignancy in Taiwan and treatment outcomes have improved enormously with early detection and advancements in adjuvant therapy. Unfortunately, a subset of early stage breast cancer still suffers from recurrence, metastasis, or even cancer-specific death despite treatment improvement, and conventional pathological factors fail to provide sufficient explanatory power accounting for the observed prognostic discrepancy. The aim of the project is to take advantage of digital RNA counting for gene expression and targeted sequencing of actionable mutations to develop a predictive model for operable breast cancers initially treated with curative intention, and the risk of recurrence, distant metastasis, as well as breast cancer-specific mortality will be speculated. Methods: Two approaches are applied for candidate genes yield; we have identified concurrent genes from the coherent patterns between genomic and transcriptional profiles, and the synthesized signatures were prognostic. In addition, concordant genes from leading edge analysis of published gene expression signatures were retrieved, and the consensus of leading edge subsets could stratify breast cancer patients into groups with distinct survival patterns. With bioinformatics tools, the harvested genes will be used to build a risk stratification model, and the residual risk after surgery and adjuvant therapy will be evaluated from archived formalin-fixed paraffin embedded (FFPE) cancer tissues, augmenting the clinical significance of the proposed signature. And finally, actionable mutations will be identified for relapsing patients through targeted sequencing. Results: There were 1584 extended concurrent genes with the threshold MAD (medium absolute deviation) of 0.9, constituting 15.89% of common genes across copy number variation and gene expression arrays. We also constructed breast cancer risk prediction using concordance of genes from leading edge analysis of various prognostic signatures; enrolled signatures were split into either the up-regulated or down-regulated gene sets within each study, and leading edge analysis of filtered up-/down-regulated gene sets from all training data sets were performed, followed by the PLS (partial least square) risk predictive model composed of consensus of leading edge subsets among all assayed subjects. The 145-gene BCeC Sig platform designated for the current study has been established, and was prognostic in 96 Taiwanese breast cancers (log-rank test: P&amp;lt;0.01). Among 37 breast cancers categorized into the high-risk group, there were 383 SNPs, 23 deletes, and 11 insertions. Variations per breast cancer were between 91 and 162 (average: 120). When collapsed into genes, the most altered genes were: BRCA1/2 (30 variants), FANCA(27), NOTCH(21), ALK, AR (20), ATM, BSG(14), FGFR, MET(12), JAK2, PDFGR, RET(11), CSF1R, DAB2I, and TP53(10). Discussion and conclusions: Archived pathological specimens provide an invaluable source to validate the residual risk prediction model for breast cancer patients managed with contemporary multi-modality treatment including surgery, chemo-, endocrine, and targeted therapy. Degraded nucleic acid from FFPE samples could be tackled by the latest NanoString digital RNA counting. This breast cancer residual risk model, composed of concurrent genes and consensual leading edge subset of published gene expression signatures, is believed to provide clinical applicability and substantial benefits for Taiwanese breast cancer patients in terms of personalized medicine Citation Format: Chi-Cheng Huang, Ching-Shui Huang. Residual risk stratification of Taiwanese breast cancer following curative therapies with digital RNA counting and actionable mutations sequencing [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-07-12.

Long-Term Use of Tamoxifen Is Associated With a Decreased Subsequent Meningioma Risk in Patients With Breast Cancer: A Nationwide Population-Based Cohort Study

Background: Earlier studies have indicated a relatively higher risk of occurring meningioma among female breast cancer survivors and have suggested that tamoxifen might decrease this risk. The present study evaluated whether tamoxifen use in breast cancer patients can reduce the risk of meningioma. Methods: We designed a population-based cohort study by using data from the National Health Insurance system of Taiwan to assess this issue. Between January 1, 2000, and December 31, 2008, women with breast cancer and of age ≥20 years were included. They were divided into two groups: those who had not received tamoxifen therapy and those who had. The Cox’s proportion hazard regression analysis was conducted to estimate the effects of tamoxifen treatment and the subsequent meningioma risk. Results: We identified a total of 50,442 tamoxifen users and 30,929 non-tamoxifen users. Tamoxifen users had a borderline significantly lower overall risk of meningioma than non-tamoxifen users [adjusted hazard ratio (aHR) = 0.64, 95% confidence interval (95% CI) = 0.40–1.02]. A statistically significant difference was found in those patients with tamoxifen treatment duration longer than 1,500 days (aHR = 0.42, 95% CI = 0.19–0.91) or with cumulative dosage exceeding 26,320 mg (aHR = 0.44, 95% CI = 0.22–0.88). Furthermore, no statistically significant joint effect of aromatase inhibitors and tamoxifen on the occurrence of meningioma among breast cancer patients was seen. Conclusion: Tamoxifen users had a non-significantly (36%) lower risk of developing meningioma than did tamoxifen non-users; however, our data indicated that tamoxifen therapy is associated with a reduced meningioma risk for Taiwanese breast cancer patients receiving long duration or high cumulative dosage treatment with tamoxifen.

Plasma endoxifen and 4-hydroxytamoxifen levels in CYP2D6(C100T) carrying breast cancer patients and association with serum cholesterol

Breast cancer patients with high cholesterol biosynthesis signature had poorer therapeutic outcome. Cytochrome P450 (CYP) 2D6 is crucial in the oxidation of tamoxifen to generate active metabolites, 4-hydroxytamoxifen and endoxifen. CYP2D6 variants with C100T substitution encode null or poor functional proteins. This study aims to examine the association of C100T genotypes and serum lipid levels with plasma drug levels in patients. Plasma tamoxifen concentration was positively associated with serum triglyceride concentration, adjusting for age and C100T genotype. Overweight (body mass index >24.0) patients with high serum cholesterol (≥200 mg/dL) had increased risks of ineffective endoxifen levels (<5.97 ng/mL). Compared to the low-cholesterol group, the high-cholesterol group had a lower 4-hydroxytamoxifen or endoxifen level in T/T carriers. In T/T carriers, the high-cholesterol group had an increased risk of an ineffective endoxifen level. Metastasis, hot flash/flushing, and high alanine transaminase did not relate to plasma 4-hydroxytamoxifen or endoxifen levels. Results indicate that C100T and high serum cholesterol are risk factors of ineffective endoxifen levels in Taiwanese breast cancer patients. These findings warrant further studies of a large hypercholesterolemic population to examine the outcome of increased doses of tamoxifen.

Hypermethylation of CCND2 in Lung and Breast Cancer Is a Potential Biomarker and Drug Target.

Lung and breast cancer are the leading causes of mortality in women worldwide. The discovery of molecular alterations that underlie these two cancers and corresponding drugs has contributed to precision medicine. We found that CCND2 is a common target in lung and breast cancer. Hypermethylation of the CCND2 gene was reported previously; however, no comprehensive study has investigated the clinical significance of CCND2 alterations and its applications and drug discovery. Genome-wide methylation and quantitative methylation-specific real-time polymerase chain reaction (PCR) showed CCND2 promoter hypermethylation in Taiwanese breast cancer patients. As compared with paired normal tissues and healthy individuals, CCND2 promoter hypermethylation was detected in 40.9% of breast tumors and 44.4% of plasma circulating cell-free DNA of patients. The western cohort of The Cancer Genome Atlas also demonstrated CCND2 promoter hypermethylation in female lung cancer, lung adenocarcinoma, and breast cancer patients and that CCND2 promoter hypermethylation is an independent poor prognostic factor. The cell model assay indicated that CCND2 expression inhibited cancer cell growth and migration ability. The demethylating agent antroquinonol D upregulated CCND2 expression, caused cell cycle arrest, and inhibited cancer cell growth and migration ability. In conclusion, hypermethylation of CCND2 is a potential diagnostic, prognostic marker and drug target, and it is induced by antroquinonol D.

Abstract 2587: Imbalances in the disposition of estrogen and naphthalene in Taiwanese breast cancer patients

Abstract The aims of this study were to measure the background level of quinone metabolites of 17β-estradiol (E2), including E2-2,3-Q and E2-3,4-Q, and naphthalene quinone (1,2-NPQ and 1,4-NPQ)-derived protein adducts in human albumin (Alb) derived from breast cancer patients (n=143) and healthy controls (n=122) in Taiwanese women and to explore the relationships between these adducts with risk factors of breast cancer. Results from the analysis indicated that Alb adducts of estrogen quinones, including E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb were detected in all subjects with mean levels at 400 and 676 pmole/g in cancer patients and 204 and 67 pmole/g in controls, respectively. In addition, the mean levels of Alb adducts of 1,2-NPQ and 1,4-NPQ on human serum albumin were estimated to be 209 and 73.6 pmole/g in cancer patients and 113 and 174 pmole/g in controls, respectively. Levels of E2-2,3-Q-4-S-Alb correlated significantly with those of E2-3,4-Q-2-S-Alb. Similarly, levels of 1,4-NPQ-Alb correlated significantly with those of 1,2-NPQ-Alb. Additionally, we noticed that E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb positively correlated with 1,4-NPQ-Alb (p&amp;lt;0.001) whereas levels of E2-2,3-Q-4-S-Alb associated with 1,2-NPQ-Alb (p&amp;lt;0.01). Furthermore, we observed that ratio of E2-3,4-Q-2-S-Alb to E2-2,3-Q-4-S-Alb in breast cancer patients were ~5 fold greater than those of healthy controls whereas ratio of 1,2-NPQ-Alb to 1,4-NPQ-Alb in cancer patients were ~4 fold greater than those of healthy controls. This evidence suggests that the cumulative body burden of estrogen quinones is a significant indicator of the risk of developing breast cancer and that breast cancer risk is associated with the imbalances in the disposition of estrogen and naphthalene in Taiwanese women. Citation Format: Po-Hsiung Lin, Dar-Ren Chen, Wei-Chung Hsieh, Mao-Huei Ko, Yi-Lun Liao. Imbalances in the disposition of estrogen and naphthalene in Taiwanese breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2587.

Abstract 5370: Targeted sequencing of actionable mutations to enhance the prognostication of concurrent genes-defined high-risk breast cancers

Abstract Purpose: The aim of the study is to perform targeted sequencing to enhance the prognostic power of breast cancer concurrent genes. Somatic mutations with clinical significance will be identified. Materials and Methods: Our previous study had identified the concurrent genes signature from the coherent patterns between genomic and transcriptional profiles among Taiwanese breast cancers, and those predicted into the high-risk group were furthered assayed by targeted sequencing. Target-enrichment sequencing was performed with Illumina SolexaTM technology with read length of 150 bp and was analyzed with Agilent SureCallTM software. Results: In total, 109 breast cancer samples were collected during surgery and analyzed with AffymetrixTM U133 plus 2.0 microarrays. Breast cancer risk prediction was performed with the published concurrent genes signature. Targeted sequencing of 61 high-risk breast cancers revealed 1061 variants, including 76 pathogenic and 545 likely pathogenic variants based on ACMG classification. The most impacted genes were NOTCH, BRCA1, AR, ERBB2, FANCA, ATM, and BRCA2, harboring 57, 36, 30, 27, 27, 26, and 26 variants. Table 1 showed that the most frequent pathogenic deletions were FGFR1 , ATM, and WT1 (47, 47, and 37 patients) while BRCA1 (rs1799965, non-sense mutation), FGFR2 (missense mutation), and BRCA1 (rs1799949, non-sense mutation) were the most frequent pathogenic SNPs (44, 35, and 11 breast cancers, respectively). The distributions of deletions and pathogenic SNPs were similar across ER+, HER2+, and triple-negative breast cancers. Conclusion: This two-stage predictive model, concurrent genes signature risk stratification followed by targeted sequencing of actionable genes, is believed to provide clinical applicability and substantial benefits for Taiwanese breast cancer patients. Table 1. Pathogenic mutations affecting more than 10 high-risk breast cancers.GeneMutation typeEffectAffected subjectsPathwayPotential therapyFGFR1DeletionFRAME_SHIFT|NM_00117406347PI3K, FGF signalingDovitinibATMDeletionFRAME_SHIFT|NM_00005147DNA damage responseATR inhibitor, PARP inhibitor, Chk 1/2 inhibitorWT1DeletionFRAME_SHIFT|NM_00037837Mutated in Wilm's tumorPDGFRADeletionFRAME_SHIFT|NM_00620618PDGF signalingSorafenib, imatinib, sunitinibPIK3RDeletionFRAME_SHIFT|NM_18152316PI3K signalingBuparlisibRUNX1DeletionFRAME_SHIFT|NM_00100189015Transcription factor, mutated in AMLBRCA1 (rs1799965)SNPnonsense44DNA damage responsePARP inhibitor, cisplatinFGFR2 (chr10:123298158:T:C )SNPmissense35PI3K, FGF signalingBrivanib, nintedanib, dovitinibBRCA1 (rs1799949)SNPnonsense11DNA damage responsePARP inhibitor, cisplatin Citation Format: Chi-Cheng Huang, Chao-Chiang Tu, Ching-Shui Huang. Targeted sequencing of actionable mutations to enhance the prognostication of concurrent genes-defined high-risk breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5370.

Abstract P1-04-10: Using the allele specific copy number profile to predict the poor prognosis breast cancer patients of Taiwanese

Abstract Introduction:Strategies are needed for the identification of a poor response to treatment and determination of appropriate strategies for breast cancer patients. As an integral component in the tumor's genetic profile, knowledge of somatic copy number aberrations can lead to insights into the tumor's genetic history and may allow for more accurate prognosis and most appropriate treatment for the patient. By quantifying the number of copies of each allele at each variant loci rather than the total number of chromosome copies, allele-specific copy number (AsCN) estimation is an important step to the characterization of tumor genomes. In this study, AsCN profile was generated and used to predict the poor prognosis breast cancer patients. Methods: The blood DNA samples from 56 Taiwanese breast cancer patients were collected from Sep 2005 to May 2015. There are 24 patients were administrated with chemotherapy (42.85%), and 9 patients were recurrent (16.07%), 5 patients were metastasis (8.92%). Affymetrix Human SNP 6.0 microarray chip was utilized to detect the alleles and copy number (CN) variations. The state of CN was distinguished from the intensity of the CN probe on the array. These states of CN were created from unpaired analysis with reference samples (HAPMAP Asian 44 samples), and utilized BRLMM-P+ algorithm from Affymetrix. The AsCN profile of patients were established with HMM algorithm, and analyzed by unpaired analysis in Partek Genomic Suite 6.6 (Partek Inc., St. Louis, MO, USA). The final result was reported after combined the AsCN result with the segmentation result of copy number variation (CNV) analysis. Two segmentation algorithms were utilized to determine the CNV region. First is circular segmentation algorithm, with minimum 10 genomic markers in each segment and P-value threshold &amp;lt; 0.0001. Second algorithm is hidden Markov model segmentation (HMM), with the parameter sigma equal to 0.99 and likelihood equal to 0.985. Results: Four allele-specific imbalance regions which located on gene ABCB11 in 2q31.1, PREX2 in 8q13.2, GPHN in 14q23.3, and mir8062 in 20p 12.3 were detected and highly related to the poor prognosis of chemotherapy. The mean of CN state of these regions was lower than 1.2. The most significant region is the located on gene mir8062 in 20p12.3 (hsa-mir-8062), 22 of 54 patients were detected to bring this imbalance region on genome. The function of this miRNA is not clear now, and needs more investigated. Conclusions: We have demonstrated the feasibility of finding the association of allele-specific imbalance regions on genome with the poor prognosis in breast cancer by generating the AsCN profile from patients. This preliminary result shows these alleles-specific imbalance regions of the genome have the possibility of playing the important role in the recurrent and metastasis of breast cancer. Citation Format: Shia W, Chen D. Using the allele specific copy number profile to predict the poor prognosis breast cancer patients of Taiwanese [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-10.