Taiwanese Breast Cancer Research Articles

Breast Cancer Screening: Comparison of Screening Modalities in Taiwan

Background: Screening for breast cancer is an important method for reducing the death rate in the Westernworld over the past 20-30 years. On this basis, since 2004 , the National Health Bureau of Taiwan has alsobegun providing free screening service. The purpose of the current research is to compare the efficacy of thethree-mammography modalities for breast cancer identification.Methods: Between July 2004 and December 2010, a total of 23,513 eligible women underwent screeningmammography. 6804 of these received screen-film mammography (SFM), 1610 received computedradiography (CR) mammography, and 15,099 received digital mammography (DM). The detection rate ofcarcinoma-in-situ, early detection rate of cancer, and the overall cancer detection rate of each modality wascalculated. To determine the statistical significance of performance differences between these modalitiesChi-square test was used.Results: SFM callback rate was 12.23%, cancer detection rate was 5.29%, Ductal carcinoma in situ (DCIS)detection rate was 1.76%. Computed radiography’s callback rate was 12.67%, with a cancer detection rateof 6.21% and DCIS detection rate of 1.24%. DM has the highest callback rate of 16.2%, but also the highestrate of cancer detection and DCIS detection rate of 9.21% and 3.58% respectively. DM was found to havebetter cancer detection rate and DCIS detection rate as compared to SFM and CR, it is only statisticallysignificant when compared to SFM. Although DM has better cancer and DCIS detection rates, it is alsoassociated with higher callback rate.Conclusions: In terms of image quality and greater contrast, optical mammography has many advantageswhen opposed to screen-film and computed radiography mammography. In the Asian population, wherewomen have higher breast densities, this is particularly vital. The incidence of Taiwanese breast cancer issteadily growing

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.

Residual risk stratification of Taiwanese breast cancers following curative therapies with the extended concurrent genes signature.

The aim of the study was to perform digital RNA counting to validate a gene expression signature for operable breast cancers initially treated with curative intention, and the risk of recurrence, distant metastasis, and mortality was predicted. Candidate genes were initially discovered from the coherent genomic and transcriptional alternations from microarrays, and the extended concurrent genes were used to build a risk stratification model from archived formalin-fixed paraffin-embedded (FFPE) tissues with the NanoString nCounter. The extended concurrent genes signature was prognostic in 144 Taiwanese breast cancers (5-year relapse-free survival: 89.8 and 69.4% for low- and high-risk group, log-rank test: P = 0.004). Cross-platform comparability was evidenced from significant and positive correlations for most genes as well as equal covariance matrix across 64 patients assayed for both microarray and digital RNA counting. Archived FFPE samples could be successfully assayed by the NanoString nCounter. The purposed signature was prognostic stratifying breast cancer patients into groups with distinct survival patterns, and clinical applicability of the residual risk model was proved.

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.

Interleukin-13 Promoter Genotypes and Taiwanese Breast Cancer Susceptibility.

The current study aimed at evaluating the contribution of IL-13 promoter rs1881457 and rs1800925 genotypes to the risk of breast cancer in Taiwan. A total of 1,232 breast cancer cases and 1,232 age-matched controls were genotyped by typical polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. As for IL-13 rs1881457, the rates of AA, AC and CC genotypes were 54.8, 37.9 and 7.3% among the cases, and 53.8, 38.7 and 7.5% among the healthy controls, respectively; there were no statistically significant differences between the two groups (p for trend=0.8889). Also, regarding IL-13 rs1800925, there were no statistically significant differences between the two groups either (p for trend=0.6803). Furthermore, the allelic frequencies for IL-13 rs1881457 and rs1800925 were not differentially distributed between the case and control groups (p=0.6515 and 0.8753, respectively). The rs1881457 and rs1800925 IL-13 promoter polymorphisms may not serve as breast cancer susceptibility determinants for Taiwanese.

Multi-gene signature of microcalcification and risk prediction among Taiwanese breast cancer

Microcalcification is one of the most common radiological and pathological features of breast ductal carcinoma in situ (DCIS), and to a lesser extent, invasive ductal carcinoma. We evaluated messenger RNA (mRNA) transcriptional profiles associated with ectopic mammary mineralization. A total of 109 breast cancers were assayed with oligonucleotide microarrays. The associations of mRNA abundance with microcalcifications and relevant clinical features were evaluated. Microcalcifications were present in 86 (79%) patients by pathological examination, and 81 (94%) were with coexistent DCIS, while only 13 (57%) of 23 patients without microcalcification, the invasive diseases were accompanied with DCIS (χ2-test, P < 0.001). There were 69 genes with differential mRNA abundance between breast cancers with and without microcalcifications, and 11 were associated with high-grade (comedo) type DCIS. Enriched Gene Ontology categories included glycosaminoglycan and aminoglycan metabolic processes and protein ubiquitination, indicating an active secretory process. The intersection (18 genes) of microcalcificaion-associated and DCIS-associated genes provided the best predictive accuracy of 82% with Bayesian compound covariate predictor. Ten genes were further selected for prognostic index score construction, and five-year relapse free survival was 91% for low-risk and 83% for high-risk group (log-rank test, P = 0.10). Our study suggested that microcalcification is not only the earliest detectable radiological sign for mammography screening but the phenomenon itself may reflect the underling events during mammary carcinogenesis. Future studies to evaluate the prognostic significance of microcalcifications are warranted.

Abstract 2481: Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer

Abstract Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer Introduction Breast cancer is molecularly heterogeneous and at least five molecular subtypes have been defined according to intrinsic genes transcription. Currently microarrays, RT-PCR, and next generation RNA sequencing have been adopted for subtyping in addition to the original nanoString nCounter assay. This study aimed to evaluate the consistency in subtyping Taiwanese breast cancers. Methods A total of 109 Taiwanese breast cancers (24 with adjacent normal breast) were assayed with Affymetrix Human Genome U133 plus 2.0 arrays and 96 were assayed with the customized 145-gene nanoString Codeset BCeCSig, of which the academic PAM50 was investigated. Of note, 64 patients were assayed for both platforms. Subtyping with the nearest centroid (single sample prediction) was called for each patient; Spearman's rank correlation coefficients were used, and samples were unclassified if correlation coefficients to all 5 centroids were less than 0.1. Results Table 1 showed the distributions of molecular subtypes with clinical IHC results. For 24 matched normal breast tissues, 21 were classified as normal breast-like. For 64 breast cancers (one unclassified) tested for both platforms, 41 (65%) were classified with identical subtype, resulting in a fair Kappa statistic of 0.55. Taking nCounter as gold standard, classification accuracy was 50% (3/6), 72% (13/18), 53% (10/19), 87% (13/15), and 40% (2/5) for basal-like, HER2-enriched, luminal-A, luminal-B, and normal breast-like subtype using Affymetrix microarray. ER-/HER2- phenotype and basal-like subtype experienced worse relapse free survival at 8-year follow-up (55.6% and 63.6% censored). Table 1.Molecular subtypes and IHC phenotypes (two unclassified and one missing IHC result).Subtype IHCBasal-likeHER2-enrichedLuminal-ALuminal-BNormal breast-likeHR+/HER2+276174HR+/HER2-6442429HR-/HER2+625209HR-/HER2-144003 Conclusions: Using public domain microarrays as a handy shortcut for breast cancer molecular subtyping is luring in scientific communities. Our experiments showed that fundamental discrepancy existed between distinct approaches, and more cation should be taken without too much extrapolation. On the other hand, adjacent normal breast tissue did display normal breast-like character, and this subtype should be viewed as a continuum of surrounding stroma. Citation Format: Chi-Cheng Huang, Chao-Chiang Tu, Mong-Hsun Tsai, Liang-chuan Lai, Ching-Shui Huang. Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer [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 2481.

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.

35P The role of interplay between microRNA-133a and Mre11 in breast cancer

In Taiwan, breast cancer has been rapidly jumped to the first place in the incidence of women-specific malignancies. It will be urgent to discover some critical biomarkers predicting patient prognosis and outcome in Taiwan. Because Taiwanese breast cancer has a relatively lower incidence and earlier onset than Western population, the subset of clinical biomarkers and treatments applied in Western population may not be suitable for all Taiwanese population.

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.

Association of Interleukin-4 Polymorphisms With Breast Cancer in Taiwan.

The present study aimed at evaluating the contribution of IL-4 promoter T-1099G (rs2243248), C-589T (rs2243250), C-33T (rs2070874) genotypes to the risk of breast cancer in Taiwanese. A total of 1232 breast cancer patients and 1232 age-matched controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Genotypic frequencies of IL-4 rs2243248, rs2243250 and rs2070874 were not differentially distributed between case and control groups. Consistently, there was no difference in the distribution of allelic frequencies among patients and controls. IL-4 rs2243248, rs2243250 and rs2070874 do not confer breast cancer susceptibility in Taiwanese.

The Performance of Different Artificial Intelligence Models in Predicting Breast Cancer among Individuals Having Type 2 Diabetes Mellitus.

Objective: Early reports indicate that individuals with type 2 diabetes mellitus (T2DM) may have a greater incidence of breast malignancy than patients without T2DM. The aim of this study was to investigate the effectiveness of three different models for predicting risk of breast cancer in patients with T2DM of different characteristics. Study design and methodology: From 2000 to 2012, data on 636,111 newly diagnosed female T2DM patients were available in the Taiwan’s National Health Insurance Research Database. By applying their data, a risk prediction model of breast cancer in patients with T2DM was created. We also collected data on potential predictors of breast cancer so that adjustments for their effect could be made in the analysis. Synthetic Minority Oversampling Technology (SMOTE) was utilized to increase data for small population samples. Each datum was randomly assigned based on a ratio of about 39:1 into the training and test sets. Logistic Regression (LR), Artificial Neural Network (ANN) and Random Forest (RF) models were determined using recall, accuracy, F1 score and area under the receiver operating characteristic curve (AUC). Results: The AUC of the LR (0.834), ANN (0.865), and RF (0.959) models were found. The largest AUC among the three models was seen in the RF model. Conclusions: Although the LR, ANN, and RF models all showed high accuracy predicting the risk of breast cancer in Taiwanese with T2DM, the RF model performed best.

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.

A qualitative exploration of the fear of recurrence among Taiwanese breast cancer survivors.

Fear of cancer recurrence (FCR) is the most commonly reported problem and unmet need among breast cancer survivors. The aim of this qualitative study was to explore the experience of recurrence fears among Taiwanese breast cancer survivors. Three focus groups were included in this study. A purposive sampling technique was used to recruit 11 recurrence-free women at least 20years of age from southern Taiwan who were diagnosed with breast cancer within the previous 2years. Data were analysed and interpreted using content analysis. Three themes reflecting the experience of FCR were extracted from the transcript analyses: "Trapped in insecurity," "Suffering in silence," and "Pretending as if nothing happened." Participants endured a complex set of circumstances akin to silently walking the survival tightrope. Breast cancer survivors were still insecure and suffering, rather than celebrating survival, after the completion of treatment. Fear of cancer recurrence silently dominated their lives. An avoidance mindset and an unspoken FCR leave survivors insecure, resulting in a silent survival journey. These difficult thoughts and feelings that may impact the reconstruction of post-cancer life should be acknowledged. The development of appropriate survivorship care programs and emotional resilience regarding recurrence is needed for Taiwanese breast cancer survivors.

Abstract P4-04-09: Comparing tumor mutation burden detection between whole exome and target enrichment sequencing among Taiwanese breast cancers

Abstract Purpose: The ability to detect tumor mutation burden (TMB) is increasingly demanding and has become a pre-requisite for breast cancer personalized therapies, especially for those interrogating immune-modulating agents. The optimized quantification method determining TMB, however, remains inconclusive, and the current study compared tumor mutation yields among Taiwanese breast cancers, either with whole exome or target enrichment sequencing. Methods: Sporadic breast cancers were prospectively recruited. Target-enrichment sequencing was performed with Illumina SolexaTM technology with read length of 150 paired ends and was analyzed with Agilent SureCallTM. There were 56 targets comprising 990 regions with a total regional size of 173.999 kbp. For whole exome sequencing, SureSelectTM Human All Exome V6r2 was adopted. The minimum coverage was set to 20 and the minimum alternative reads was set to 10 to enhance sequencing reliability. Samtools version 1.2 was utilized as variant caller for both whole exome and targeted sequencing. Novel and synonymous variants, as well as those located outside coding regions were removed. TMB was calculated as the number of somatic mutations per megabase (mb). Results: A total of 61 and 52 Taiwanese breast cancers underwent target enrichment and whole exome sequencing, respectively. The number of somatic mutations ranged from 36 to 292 (median: 205), equivalent to 207~1678 (median: 1178) mutations/mb for targeted sequencing. On the other hand, there were 9947 to 10981 somatic mutations (median: 10727) when the whole exome was sequenced, resulting in TMB between 333 and 366 (median: 358). TMB for ER+/HER-, ER+/HER2+, ER-/HER2+, and ER-/HER2- was 1322 (n=31), 713 (n=11), 793 (n=10), and 1230 (n=9) for targeted sequencing and was 358 (n=29), 358 (n=12), 354 (n=5), and 357 (n=6) for whole exome sequencing. The most frequent nonsense mutations were NOTCH1 (S255*), RET (Q87*), KRAS (G57*), JAK3 (Y267*), and SMO (Y61*), while MAP2K1 (G148C), FNACA (G175V), BSG (H254P), ABL1 (T57P), and NOTCH1 (H416P) were the most common missense mutations. Discussion and conclusions: High-throughput parallel massive sequencing can identify large numbers of variants, dependent both on the size of the sequenced regions and the variant caller algorithm utilized. Although Samtools identified more variants against reference than other methods, there should be no differential between whole exome and targeted sequencing. The number of somatic mutations, as well as the variability in TMB, was much higher in target enrichment approach than whole exome sequencing, highlighting that different TMB threshold should be established before wide clinical applications. Uneven distributions of “hotspot” mutation regions across genome as well as hundred-fold of sequencing length of whole exome versus targeted sequencing sequencing may contribute to such discrepancy. Citation Format: Huang C-C, Huang C-S. Comparing tumor mutation burden detection between whole exome and target enrichment sequencing among Taiwanese breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-04-09.

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 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 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 P5-07-04: Nuclear miR-133 expression is associated with breast cancer progression and survival

Abstract Background:MiRNAs (microRNAs) can function as the tumor suppressor gene or oncogene to affect carcinogenesis, therapy response and progression in human cancer. The prevailing view is that miRNAs function to regulate mRNA stability and translation in the cytoplasm. However, multiple studies have detected miRNAs in the nuclear compartment. Nuclear miRNAs have been proven to regulate the functions of nuclear protein and synthesis of nuclear miRNA. We have found circulating muscle-enriched miR-133a can serve as prognostic biomarkers for breast cancer. Herein, we aim to explore the subcellular distribution, prognostic significance and treatment response of miR-133a in Taiwanese breast cancer. Methods: Circulating miR-133a levels were conducted with the serum from 188 patients prior to surgery and from 82 healthy controls. The subcellular distribution patterns of miR-133a were analyzed by Chromogenic In Situ Hybridization (CISH) in paraffin embedded tissue sections from 224 cases. Cell viability and cell cycle were determined by XTT assay and flowcytometry. Protein and mRNA levels were examined by western blotting and RT-qPCR (TaqMan) assay. Results: Our results showed that circulating miR-133a levels of breast cancer patient are significantly higher than those of health subjects, suggesting that increased miR-133a secretion from unknown tissues or cells may promote carcinogens in Taiwanese breast cancer. Using the receiver operating characteristic (ROC) curve to examine the diagnostic value of circulating miR-133a in breast cancer (AUC=0.834), we found that increased miR-133a levels could serve as a diagnostic biomarker in accessing Taiwanese breast cancer risk. Nevertheless, CISH analysis revealed that nuclear miR-133a frequency was inversely correlated with tumor grade, stage and lymph node metastasis (p=0.029, 0.012, and 0.0008), and cytoplasmic miR-133a intensity was positively correlated with recurrence (p=0.010). Furthermore, high frequency of nuclear miR-133a was correlated with a better overall survival rate (p=0.039) and improved the survival rate of patients who received hormone therapy (p=0.009). To further investigate the role of nuclear miR-133a, an additional miR-133a, denoted as Nu-miR-133a, ending in nuclear localization element was constructed and found to be highly enriched in the nucleus. Ectopic expression of wild-type and Nu-miR-133 inhibited proliferation of breast cancer cells. Both wild type miR-133a and Nu-miR-133a increased S phase arrest as well as the levels and foci of γ-H2AX. Unexpectedly, we found that wild type miR-133a decreased ATM expression but activated p38 MAPK. Notably, H2AX can be also phosphorylated by p38 MAPK kinase in addition to ATM and ATR. The underlying mechanism between nuclear miR-133a and potentially p38-mediated DNA damage response (DDR, γ-H2AX and S phase arrest) will be needed further elucidation. Conclusions: In conclusion, we provide the first evidence that nuclear miR-133a may play a crucial role in the development and treatment response of breast cancer potentially through its mediated DDR. The interplay among nuclear miR-133a, p38 and DDR require further investigations. Citation Format: Hou M-F, Chen F-M, Yang S-F, Dai H-Y, Yeh Y-T. Nuclear miR-133 expression is associated with breast cancer progression and survival [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-07-04.