A Retrospective Exploratory Analysis for Serum Extracellular Vesicles Reveals APRIL (TNFSF13), CXCL13, and VEGF-A as Prognostic Biomarkers for Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

Neoadjuvant chemotherapy (NAC) is widely used as a standard treatment for early-stage triple-negative breast cancer (TNBC). While patients who achieve pathologic complete response (pCR) have a highly favorable outcome, patients who do not achieve pCR have variable prognoses. It is important to identify patients who are most likely to have poor survival outcomes to identify candidates for more aggressive therapeutic approaches after NAC. Many studies have demonstrated that cytokines and growth factors packaged into extracellular vesicles (EVs) have an essential role in tumor progression and drug resistance. In this study, we examined the role of serum-derived EV-associated cytokines as prognostic biomarkers for long-term outcomes in patients who underwent anthracycline-taxane-based NAC. We isolated extracellular vesicles from the serum of 190 TNBC patients who underwent NAC between 2015 and 2018 at Samsung Medical Center. EV-associated cytokine concentrations were measured with ProcartaPlex Immune Monitoring 65-plex panels. The prognostic value of EV-associated cytokines was studied. We found that patients with high EV_APRIL, EV_CXCL13, and EV_VEGF-A levels had shorter overall survival (OS). We further evaluated the role of these selected biomarkers as prognostic factors in patients with residual disease (RD) after NAC. Even in patients with RD, high levels of EV_APRIL, EV_CXCL13, and EV_VEGF-A were correlated with poor OS. In all subgroup analyses, EV_CXCL13 overexpression was significantly associated with poor overall survival. Moreover, multivariate analysis indicated that a high level of EV_CXCL13 was an independent predictor of poor OS. Correlation analysis between biomarker levels in EVs and serum showed that EV_VEGF-A positively correlated with soluble VEGF-A but not CXCL13. An elevated level of soluble VEGF-A was also associated with poor OS. These findings suggest that EV_APRIL, EV_CXCL13, and EV_VEGF-A may be useful in identifying TNBC patients at risk of poor survival outcomes after NAC.

Genomic characteristics of triple negative apocrine carcinoma: a comparison to triple negative breast cancer.

Apocrine carcinoma is a rare breast cancer subtype. As such, the genomic characteristics of apocrine carcinoma with triple negative immunohistochemical results (TNAC), which has been treated as triple negative breast cancer (TNBC), have not been revealed. In this study, we evaluated the genomic characteristics of TNAC compared to TNBC with low Ki-67 (LK-TNBC). In the genetic analysis of 73 TNACs and 32 LK-TNBCs, the most frequently mutated driver gene in TNAC was TP53 (16/56, 28.6%), followed by PIK3CA (9/56, 16.1%), ZNF717 (8/56, 14.3%), and PIK3R1 (6/56, 10.71%). Mutational signature analysis showed enrichment of defective DNA mismatch repair (MMR)-related signatures (SBS6 and SBS21) and the SBS5 signature in TNAC, whereas an APOBEC activity-associated mutational signature (SBS13) was more prominent in LK-TNBC (Student's t test, p < 0.05). In intrinsic subtyping, 38.4% of TNACs were classified as luminal A, 27.4% as luminal B, 26.0% as HER2-enriched (HER2-E), 2.7% as basal, and 5.5% as normal-like. The basal subtype was the most dominant subtype (43.8%) in LK-TNBC (p < 0.001), followed by luminal B (21.9%), HER2-E (21.9%), and luminal A (12.5%). In the survival analysis, TNAC had a five-year disease-free survival (DFS) rate of 92.2% compared to 59.1% for LK-TNBC (P = 0.001) and a five-year overall survival (OS) rate of 95.3% compared to 74.6% for LK-TNBC (P = 0.0099). TNAC has different genetic characteristics and better survival outcomes than LK-TNBC. In particular, normal-like and luminal A subtypes in TNAC have much better DFS and OS than other intrinsic subtypes. Our findings are expected to impact medical practice for patients diagnosed with TNAC.

Elevated Level of Nerve Growth Factor (NGF) in Serum-Derived Exosomes Predicts Poor Survival in Patients with Breast Cancer Undergoing Neoadjuvant Chemotherapy.

Neoadjuvant chemotherapy (NAC) is a standard treatment strategy for patients with locally advanced breast cancer (LABC). However, there are no established predictors of chemosensitivity and survival in LABC patients who undergo NAC. Many studies have demonstrated that exosomes and cytokines are important players in intercellular communication between tumors and their environments, and are involved in chemotherapy resistance. Recently, it was reported that cytokines can be packaged into exosomes, but whether exosomal cytokines serve as biomarkers in breast cancer patients is still unclear. In this study, we examined the roles of cytokines in both serum and exosomes as prognostic biomarkers for long-term outcomes in patients with breast cancer who undergo NAC. We isolated exosomes from the blood of 129 patients with early breast cancer who were receiving neoadjuvant chemotherapy between 2008 and 2011 at Samsung Medical Center. The levels of cytokines and growth factors in serum and exosomes were measured with ProcartaPlex immune-related panels. We investigated correlations between clinic-pathologic variables and patient survival, and Cox proportional hazards regression analysis was performed for prognostic evaluation. We detected significant differences in expression patterns between serum cytokines and exosomal cytokines. In both serum and exosomes, many cytokines were positively correlated with age. In univariate analysis, patients with high serum IP-10, serum MMP-1, and exosomal NGF had shorter overall survival. Exosomal NGF showed significantly poorer overall survival in multivariate analysis. These findings suggest that exosomal NGF is useful for identifying patients with poor survival outcomes.

Cytokine profiling in serum-derived exosomes isolated by different methods.

Exosomes in blood play an important role in cell-to-cell signaling and are a novel source of biomarkers for the diagnosis and prognosis of diseases. Recently, evidence has accumulated that cytokines are released from encapsulated exosomes and are capable of eliciting biological effects upon contact with sensitive cells. However, there is currently limited information on exosome isolation methods for cytokine research. In this study, we evaluated three exosome isolation methods for their usability, yield, purity, and effectiveness in subsequent cytokine profiling. We found that ultracentrifugation (UC) and Exoquick (EQ), but not exoEasy, yielded appropriate exosome sizes, and EQ had higher exosome extraction efficiency than the other two methods. Although UC generated markedly fewer particles than EQ, it yielded a relatively high purity. Next, we performed a multiplex assay with the ProcartaPlex Immune Monitoring 65-Plex Panel to determine the feasibility of these methods for cytokine profiling. The results indicated significant differences among isolation methods when analyzing exosomal cytokine profiles. We further investigated the changes of exosomal cytokines according to breast cancer progression in triple-negative breast cancer. We found significantly decreased concentrations of MIP-3 alpha, IL-23, M-CSF, Eotaxin-3, BLC, SDF-1 alpha, IL-2R, MDC, FGF-2, IL-22, and IL-31 in exosomes from metastatic breast cancer (MBC) patients.

Prognostication of a 13-immune-related-gene signature in patients with early triple-negative breast cancer.

PURPOSE: We investigated the expression profiles of immune genes in patients with triple-negative breast cancer (TNBC) to identify the prognostic value of immune genes and their clinical implications. METHODS: NanoString nCounter Analysis of 770 immune-related genes was used to measure immune gene expression in patients with TNBC who underwent curative surgery followed by adjuvant chemotherapy at Samsung Medical Center between 2000 and 2004. Statistical analyses were conducted to identify the associations between gene expression and distant recurrence-free survival (DRFS). RESULTS: Of 1189 patients who underwent curative BC surgery, 200 TNBC patients were included and stage was the only clinical factor predictive of DRFS. In terms of immune genes, 155 of 770 genes were associated with DRFS (p < 0.01). Further multivariate analysis revealed that 13 genes, CD1B, CD53, CT45A1, GTF3C1, IL11RA, IL1RN, LRRN3, MAPK1, NEFL, PRKCE, PTPRC, SPACA3 and TNFSF11, were associated with patient prognosis (p < 0.05). The prognostic value of stage and expression levels of 13 immune genes was analyzed and the area under the receiver operating characteristic curve (AUC) was 0.923. Based on the AUC, we divided patients into three genetic risk groups and DRFS rate was significantly different according to genetic risk groups, even in the same stage (p < 0.001). CONCLUSIONS: In this study, a 13-gene expression profile in combination with stage precisely predicted distant recurrence of early TNBC. Therefore, this 13-immune-gene signature could help predict TNBC prognosis and provide guidance for treatment as well as the opportunity to develop new targets for immunotherapy in TNBC patients.

Molecular alterations and poziotinib efficacy, a pan-HER inhibitor, in human epidermal growth factor receptor 2 (HER2)-positive breast cancers: Combined exploratory biomarker analysis from a phase II clinical trial of poziotinib for refractory HER2-positive breast cancer patients.

We aimed to investigate the impact of genetic alterations on the efficacy of poziotinib in a phase II clinical trial of patients with heavily treated HER2-positive metastatic breast cancer (BC). We performed targeted ultra-deep sequencing with a customized cancer gene panel and RNA expression assay using BC specimens. Of 106 patients, biomarker data were available for 85. Copy number (CN) amplifications of HER2 were observed in 72 patients (85%), and CN >8 in 50 (59%). Single nucleotide variants (SNVs) of HER2 were found in 16 patients (19%). Genetic alterations of PIK3CA pathway were found in 40 patients (47%). Median progression free survival (PFS) of the biomarker analysis group was 3.61 months. In terms of PFS, HER2 with CN >8 prolonged (hazard ratio (HR) 0.61, 95% CI: 0.38, 0.97, p = 0.037) and alteration of PIK3CA pathway shortened the duration of survival (HR 2.25, 95% CI: 1.39, 3.63, p = 0.001). SNVs of HER2 increased survival duration, but the effect was not significant (HR: 0.58, 95% CI: 0.31, 1.08, p = 0.085). In addition, SNVs in the ERBB3 cytoplasmic domain decreased poziotinib response (HR: 4.58, 95% CI: 2.02, 10.37, p < 0.001). In multigene analysis, BC with HER2 CN >8 and intact PIK3CA pathway had significantly longer PFS compared to others (HR: 0.37, 95% CI: 0.21, 0.66, p = 0.001), while SNVs in the ERBB3 cytoplasmic domain predicted poor prognosis (HR: 4.28, 95% CI: 1.71, 10.71, p < 0.001). In conclusion, HER2 CN amplification, PIK3CA pathway alteration, and ERBB3 cytoplasmic mutation showed predictive roles on clinical outcomes of HER2-positive MBC treated with poziotinib.

Validation of the new AJCC eighth edition of the TNM classification for breast cancer with a single-center breast cancer cohort.

PURPOSE: The new eighth edition TNM classification by the AJCC for breast cancer (BC) incorporates biologic factors and gene expression prognostic panels, in addition to traditional anatomic factors. In this study, we evaluated the prognostic value of this new staging system compared to the previous AJCC 7th edition staging system. METHODS: We conducted a retrospective analysis of women with stage I, II, or III BC who underwent curative surgery with/without adjuvant systemic therapy at Samsung Medical Center between July 2004 and December 2008. RESULTS: Of 3,208 BCs, this study was analyzed using the information of 2,790 BC patients. Hormone receptor-positive (HR+) and human epidermal growth factor 2 (HER2)- BCs were observed in 62.9% of BCs, HR+/ HER2+ in 9.3%, HR-/HER2- in 17.0%, and HR-/HER2+ in 10.8%. In survival analysis, we observed 245 distant recurrences and 198 deaths caused by BC progression. The median follow-up duration was 116.2 months. 10-year disease-specific survival (DSS) rates according to the AJCC 7th edition criteria were 97.2% of stage IA, 100% of IB, 94.9% of IIA, 87.9% of IIB, 86.4% of IIIA, 95.7% of IIIB, and 65.7% of IIIC (p < 0.001). After applying 8th edition criteria, the 10-year DSS rates were 98.1% of stage IA, 97.7% of IB, 93.8% of IIA, 92.7% of IIB, 88.2% of IIIA, 80.8% of IIIB, and 70.3% of IIIC (p < 0.001). CONCLUSIONS: The AJCC 8th edition clinical staging system provides a good prognostic value and addresses the weakness of the AJCC 7th edition, which uses only anatomical pathologic staging.

Multi-omics profiling of younger Asian breast cancers reveals distinctive molecular signatures.

Breast cancer (BC) in the Asia Pacific regions is enriched in younger patients and rapidly rising in incidence yet its molecular bases remain poorly characterized. Here we analyze the whole exomes and transcriptomes of 187 primary tumors from a Korean BC cohort (SMC) enriched in pre-menopausal patients and perform systematic comparison with a primarily Caucasian and post-menopausal BC cohort (TCGA). SMC harbors higher proportions of HER2+ and Luminal B subtypes, lower proportion of Luminal A with decreased ESR1 expression compared to TCGA. We also observe increased mutation prevalence affecting BRCA1, BRCA2, and TP53 in SMC with an enrichment of a mutation signature linked to homologous recombination repair deficiency in TNBC. Finally, virtual microdissection and multivariate analyses reveal that Korean BC status is independently associated with increased TIL and decreased TGF-ß signaling expression signatures, suggesting that younger Asian BCs harbor more immune-active microenvironment than western BCs.

Circulating tumor DNA shows variable clonal response of breast cancer during neoadjuvant chemotherapy.

Circulating tumor DNA (ctDNA) correlates with tumor burden and provides early detection of treatment response and tumor genetic alterations in breast cancer (BC). In this study, we aimed to identify genetic alterations during the process of tumor clonal evolution and examine if ctDNA level well indicated clinical response to neoadjuvant chemotherapy (NAC) and BC recurrence. We performed targeted ultra-deep sequencing of plasma DNAs, matched germline DNAs and tumor DNAs from locally advanced BC patients. Serial plasma DNAs were collected at diagnosis, after the 1st cycle of NAC and after curative surgery. For the target enrichment, we designed RNA baits covering a total of ∼202kb regions of the human genome including a total of 82 cancer-related genes. For ctDNA, 15 serial samples were collected and 87% of plasma SNVs were detected in 13 BC samples that had somatic alterations in tumor tissues. The TP53 mutation was most commonly detected in primary tumor tissues and plasma followed by BRCA1 and BRCA2. At BC diagnosis, the amount of plasma SNVs did not correlate with clinical stage at diagnosis. With respect to the therapeutic effects of NAC, we found two samples in which ctDNA disappeared after the 1st NAC cycle achieved a pathologic complete response (pCR). In addition, the amount of ctDNA correlated with residual cancer volume detected by breast MRI. This targeted ultra-deep sequencing for ctDNA analysis would be useful for monitoring tumor burden and drug resistance. Most of all, we suggest that ctDNA could be the earliest predictor of NAC response.

Immune signature of metastatic breast cancer: Identifying predictive markers of immunotherapy response.

In breast cancer (BC), up to 10-20% patients were known to have clinical benefit with immune checkpoint inhibitors, and biomarkers are needed for optimal use of this multi-potential therapeutic strategy. Accordingly, we conducted an experiment to identify expression of genes associated with immune checkpoints that represent potential targets of cancer immunotherapy. We performed whole-transcriptome sequencing and whole-exome sequencing using 37 refractory BC specimens. In the immune pathway gene set expression analysis, we found that HER2 expression and previous taxane treatment were positively correlated with high expression of immune gene set expression (p = 0.070 and 0.008, respectively). The nine genes associated with immune checkpoints - PDCD1(PD-1), CD274(PD-L1), CD276(B7-H3), CTLA-4, IDO1, LAG3, VTCN1, HAVCR2, and TNFRSF4(OX40) - interacted with each other. In addition, HER2 expression also affected the expression levels of these genes (p = 0.044). Lastly, expression of immune checkpoint genes and tissue-infiltrating lymphocytes were positively correlated in metastatic BCs (p < 0.001). In conclusion, we suggest that HER2 expression and previous taxane treatment are potential surrogate markers for high expression of immune checkpoint genes and immune pathway gene sets. Further study of the BC immune signature with large-scale, translational data sets is warranted.

Clinical implications of genomic profiles in metastatic breast cancer with a focus on TP53 and PIK3CA, the most frequently mutated genes.

Breast cancer (BC) has been genetically profiled through large-scale genome analyses. However, the role and clinical implications of genetic alterations in metastatic BC (MBC) have not been evaluated. Therefore, we conducted whole-exome sequencing (WES) and RNA-Seq of 37 MBC samples and targeted deep sequencing of another 29 MBCs. We evaluated somatic mutations from WES and targeted sequencing and assessed gene expression and performed pathway analysis from RNA-Seq. In this analysis, PIK3CA was the most commonly mutated gene in estrogen receptor (ER)-positive BC, while in ER-negative BC, TP53 was the most commonly mutated gene (p = 0.018 and p < 0.001, respectively). TP53 stopgain/loss and frameshift mutation was related to low expression of TP53 in contrast nonsynonymous mutation was related to high expression. The impact of TP53 mutation on clinical outcome varied with regard to ER status. In ER-positive BCs, wild type TP53 had a better prognosis than mutated TP53 (median overall survival (OS) (wild type vs. mutated): 88.5 ± 54.4 vs. 32.6 ± 10.7 (months), p = 0.002). In contrast, mutated TP53 had a protective effect in ER-negative BCs (median OS: 0.10 vs. 32.6 ± 8.2, p = 0.026). However, PIK3CA mutation did not affect patient survival. In gene expression analysis, CALM1, a potential regulator of AKT, was highly expressed in PIK3CA-mutated BCs. In conclusion, mutation of TP53 was associated with expression status and affect clinical outcome according to ER status in MBC. Although mutation of PIK3CA was not related to survival in this study, mutation of PIK3CA altered the expression of other genes and pathways including CALM1 and may be a potential predictive marker of PI3K inhibitor effectiveness.

The effect of androgen receptor expression on clinical characterization of metastatic breast cancer.

In breast cancer (BC), androgen receptor (AR) expression is related to estrogen receptor (ER) and/or progesterone receptor (PgR) expression. AR expression is an indicator of good prognosis in breast cancer regardless of hormone receptor (HR) status. In this study, we evaluated the effect of AR-related gene expression on clinical characterization of metastatic BC. We performed RNA-Seq to evaluate gene expression using mRNA extracted from 37 patients with metastatic BC. Intrinsic subtype prediction, analysis of differential gene expression, and gene set enrichment pathway analysis were then performed. Metastatic BCs were categorized into three subgroups based on AR, ER, PgR, and HER2 expression. According to this subcategorization, 70 genes including AR, ER, and HER2 were differentially expressed among the three groups. In gene set enrichment pathway analysis, the low AR group was associated with the cell cycle pathway, whereas mammalian target of rapamycin (mTOR) pathways was prevalent in the high ER and AR group. In survival analysis, a higher level of AR expression correlated with prolonged overall survival in metastatic BC (high expression vs. low expression, median OS 53.1 vs. 27.2 months, p=.001). In conclusion, we propose that AR and AR-related gene expression could be utilized to predict the prognosis of metastatic BC and thus may be useful in treatment planning for refractory BC.

Statins affect ETS1-overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency.

We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 µM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4.

The relationship between nuclear factor (NF)-κB family gene expression and prognosis in triple-negative breast cancer (TNBC) patients receiving adjuvant doxorubicin treatment.

We investigated gene expression profiles of the NF-κB pathway in patients with triple-negative breast cancer (TNBC) receiving adjuvant chemotherapy to determine the prognostic value of NF-κB pathway genes according to chemotherapeutic regimen. We used the nCounter expression assay to measure expression of 11 genes (NFKB1, NFKB2, RELA, RELB, REL, TP53, FOXC1, TBP, SP1, STAT3 and IRF1 genes) belonging to the NF-κB pathway using mRNA extracted from paraffin-embedded tumor tissues from 203 patients diagnosed with TNBC. Of the 203 patients, 116 were treated with a chemotherapeutic regimen containing doxorubicin. As revealed by the expression profiles of the 11 genes, increased expression of SP1 was associated with poor prognosis in TNBC patients treated with adjuvant doxorubicin chemotherapy (5-year distant recurrence-free survival [5Y DRFS], low vs. high expression [cut-off: median]: 92.3% vs. 71.6%, P = 0.001). In a multivariate Cox regression model, SP1 expression was a useful marker for predicting long-term prognosis in TNBC patients receiving doxorubicin treatment, and we thus suggest that SP1 expression could serve as a prognostic marker in these patients.

Gene Expression Profiling of Breast Cancer Brain Metastasis.

The biology of breast cancer brain metastasis (BCBM) is poorly understood. We aimed to explore genes that are implicated in the process of brain metastasis of primary breast cancer (BC). NanoString nCounter Analysis covering 252 target genes was used for comparison of gene expression levels between 20 primary BCs that relapsed to brain and 41 BCBM samples. PAM50-based intrinsic subtypes such as HER2-enriched and basal-like were clearly over-represented in BCBM. A panel of 22 genes was found to be significantly differentially expressed between primary BC and BCBM. Five of these genes, CXCL12, MMP2, MMP11, VCAM1, and MME, which have previously been associated with tumor progression, angiogenesis, and metastasis, clearly discriminated between primary BC and BCBM. Notably, the five genes were significantly upregulated in primary BC compared to BCBM. Conversely, SOX2 and OLIG2 genes were upregulated in BCBM. These genes may participate in metastatic colonization but not in primary tumor development. Among patient-matched paired samples (n = 17), a PAM50 molecular subtype conversion was observed in eight cases (47.1%), with a trend toward unfavorable subtypes in patients with the distinct gene expression. Our findings, although not conclusive, reveal differentially expressed genes that might mediate the brain metastasis process.

Association between Mutation and Expression of TP53 as a Potential Prognostic Marker of Triple-Negative Breast Cancer.

PURPOSE: TP53, the most frequently mutated gene in breast cancer, is more frequently altered in HER2-enriched and basal-like breast cancer. However, no studies have clarified the role of TP53 status as a prognostic and predictive marker of triple-negative breast cancer (TNBC). MATERIALS AND METHODS: We performed p53 immunohistochemistry (IHC), nCounter mRNA expression assay, and DNA sequencing to determine the relationship between TP53 alteration and clinical outcomes of TNBC patients. RESULTS: Seventy-seven of 174 TNBC patients were found to harbor a TP53 mutation. Patients with missense mutations showed high protein expression in contrast to patients with deletion mutations (positivity of IHC: wild type vs. missense vs. deletion mutation, 53.6% vs. 89.8% vs. 25.0%, respectively; p < 0.001). TP53 mRNA expression was influenced by mutation status (mRNA expression [median]: wild type vs. missense vs. deletion mutation, 207.36± 132.73 vs. 339.61±143.21 vs. 99.53±99.57, respectively; p < 0.001). According to survival analysis, neither class of mutation nor protein or mRNA expression status had any impact on patient prognosis. In subgroup analysis, low mRNA expression was associated with poor prognosis in patients with a TP53 missense mutation (5-year distant recurrence-free survival [5Y DRFS]: low vs. high, 50.0% vs. 87.8%; p=0.009), while high mRNA expression with a TP53 deletion mutation indicated poor prognosis (5Y DRFS: low vs. high, 91.7% vs. 75.0%; p=0.316). CONCLUSION: Association between TP53 mutation and expression indicates a potential prognostic marker of TNBC; hence both DNA sequencing and mRNA expression analysis may be required to predict the prognosis of TNBC patients.

Prognostic value of ERBB4 expression in patients with triple negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is known for aggressive biologic features and poor prognosis. Epidermal growth factor receptor (EGFR) overexpression in TNBC indicates poor prognosis. However, there is no previous study of the relationship between expression of the entire human epidermal growth factor receptor (HER) family genes and patient prognosis in TNBC. Accordingly, we investigated the expression profiles of HER family genes in patients with TNBC to determine the prognostic value and clinical implications of HER family expression. METHODS: We used the nCounter expression assay (NanoString®) to measure the expression of EGFR, erb-B2 receptor tyrosine kinase 2 (ERBB2), ERBB3, ERBB4, and estrogen receptor 1 (ESR1) genes using mRNA extracted from paraffin-embedded tumor tissues from 203 patients diagnosed with TNBC. Our data were validated using a separate cohort of 84 TNBC patients. RESULTS: A total of 203 TNBC patients who received adjuvant chemotherapy after curative surgery from 2000 to 2004 formed the training set. The 84 TNBC patients in the validation consort were selected from breast cancer patients who received curative surgery since 2005 to 2010. Analysis of the expression profiles of the HER family genes in TNBC tissue specimens revealed that increased expression of ERBB4 was associated with poor prognosis according to survival analysis (5-year distant relapse free survival [5Y DRFS], low vs. high expression [cut-off: median]: 90.1% vs. 80.2%; p = 0.022). This trend was also observed in the validation set of TNBC patients (5Y DRFS, low vs. high: 69.4% vs. 44.7%; p = 0.053). In a multivariate Cox regression model, ERBB4 expression was identified as a indicator of long-term prognosis in patients with TNBC. CONCLUSIONS: The expression profile of ERBB4, a member of the HER family, might serve as a prognostic marker in patients with TNBC.

Mutational profiling of brain metastasis from breast cancer: matched pair analysis of targeted sequencing between brain metastasis and primary breast cancer.

Although breast cancer is the second most common cause of brain metastasis with a notable increase of incidence, genes that mediate breast cancer brain metastasis (BCBM) are not fully understood. To study the molecular nature of brain metastasis, we performed gene expression profiling of brain metastasis and matched primary breast cancer (BC). We used the Ion AmpliSeq Cancer Panel v2 covering 2,855 mutations from 50 cancer genes to analyze 18 primary BC and 42 BCBM including 15 matched pairs. The most common BCBM subtypes were triple-negative (42.9%) and basal-like (36.6%). In a total of 42 BCBM samples, 32 (76.2%) harbored at least one mutation (median 1, range 0-7 mutations). Frequently detected somatic mutations included TP53 (59.5%), MLH1 (14.3%), PIK3CA (14.3%), and KIT (7.1%). We compared BCBM with patient-matched primary BC specimens. There were no significant differences in mutation profiles between the two groups. Notably, gene expression in BCBM such as TP53, PIK3CA, KIT, MLH1, and RB1 also seemed to be present in primary breast cancers. The TP53 mutation frequency was higher in BCBM than in primary BC (59.5% vs 38.9%, respectively). In conclusion, we found actionable gene alterations in BCBM that were maintained in primary BC. Further studies with functional testing and a delineation of the role of these genes in specific steps of the metastatic process should lead to a better understanding of the biology of metastasis and its susceptibility to treatment.

Role of HER2 mutations in refractory metastatic breast cancers: targeted sequencing results in patients with refractory breast cancer.

In women with metastatic breast cancer (MBC), introduction of the anti-HER2 (human epidermal growth factor receptor-2) directed therapies including trastuzumab, pertuzumab, lapatinib, and/or trastuzumab-DM1 has markedly improved overall survival. However, not all cases of HER2-positive breast tumours derive similar benefit from HER2-directed therapy, and a significant number of patients experience disease progression because of primary or acquired resistance to anti-HER2-directed therapies. We integrated genomic and clinicopathological analyses in a cohort of patients with refractory breast cancer to anti-HER2 therapies to identify the molecular basis for clinical heterogeneity. To study the molecular basis underlying refractory MBC, we obtained 36 MBC tumours tissues and used next-generation sequencing to investigate the mutational and transcriptional profiles of 83 genes. We focused on HER2 mutational sites and HER2 pathways to identify the roles of HER2 mutations and the HER2 pathway in the refractoriness to anti-HER2 therapies. Analysis using massively parallel sequencing platform, CancerSCAN™, revealed that HER2 mutations were found in six of 36 patients (16.7%). One patient was ER (estrogen receptor)-positive and HER2-negative and the other five HER2 mutated patients were HER2-positive and HR (hormone receptor)-negative. Most importantly, four of these five patients did not show any durable clinical response to HER2-directed therapies. The HER2 pathway score obtained through transcriptional analyses identified that Growth Receptor Biding protein 2 (GRB2) was the most significantly down regulated gene in the HER2 mutated samples. Detection of HER2 mutations using higher deep DNA sequencing may identify a predictive biomarker of resistance to HER2-directed therapy. Functional validation is warranted.

Molecular characterization of patients with pathologic complete response or early failure after neoadjuvant chemotherapy for locally advanced breast cancer using next generation sequencing and nCounter assay.

Neoadjuvant chemotherapy (NAC) has the added advantage of increasing breast conservation rates with equivalent survival outcomes compared with adjuvant chemotherapy. A subset of breast cancer patients who received NAC experienced early failure (EF) during the course of therapy or within a short period after curative breast surgery. In contrast, patients with pathological complete response (pCR) were reported to have markedly favorable outcomes. This study was performed to identify actionable mutation(s) and to explain refractoriness and responsiveness to NAC. Included in this analysis were 76 patients among 397 with locally advanced breast cancer for whom a preoperative fresh-frozen paraffin-embedded tumor block was available for next-generation sequencing using AmpliSeq. The incidence of missense mutations in KRAS was much higher in patients with EF than in other groups (p < 0.01). In contrast, polymorphisms of the cMET gene were found in patients with pCR exclusively (p < 0.01).