T-K Yoo

Abstract P2-08-26: Tumor-associated macrophages and platelet-derived growth factor C as a prognostic marker for breast cancer patients

Introduction: Tumor related inflammation plays an important role in breast cancer progression, tumor-associated macrophages (TAMs) being a crucial part of this microenvironment. Platelet-derived growth factor-C (PDGF-C) is abundant in the breast cancer microenvironment having an anti-apoptotic effect on macrophages. Previous reports suggest that tumor cell derived PDGF-C promotes TAM survival, enhancing tumor progression. In this study we analyzed the prognostic value of PDGF-C density associated with TAM infiltration in human breast cancer. Materials and Methods: TAM and PDGF-C density was evaluated by immunohistochemistry of CD163+, CD68+ and PDGF-C myeloid cells in tumor stroma. Tissue microarrays from 140 invasive breast cancer cases were used. Survival analysis to evaluate the impact of TAM and PDGF-C density on disease free survival (DFS) was done using Kaplan-Meier and Cox regression analysis. Results: Infiltration of CD163+ and CD68+ macrophages into tumor stroma had a tendency, but not significant, for reduced DFS (p=0.204, p=0.314 respectively). Whereas PDGF-C strong density was significantly associated with worse DFS (p=0.024). This inverse correlation with DFS was demonstrated stronger when PDGF-C density was combined with CD163+ and CD68+ macrophage infiltration (p=0.008, p=0.018, respectively). But, CD163+ or CD68+ infiltration with strong PDGF-C density did not demonstrate as an independent prognostic factor when adjusting for tumor size, lymph node metastasis, hormone receptor and histologic grade. This result is probably due to small number of patients having both TAM infiltration and strong PDGF-C density. Conclusion: Our results show that PDGF-C infiltration adversely affects DFS and also enhances the inverse correlation between tumor-associated macrophages and DFS. To support these results, a study with larger numbers is on progress. Citation Format: Yoo T-K, Lee K-M, Jung H, Na YR, Seok SH, Han W. Tumor-associated macrophages and platelet-derived growth factor C as a prognostic marker for breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-26.

Abstract P6-04-02: Identification of ESR1 splice variants associated with prognosis in estrogen receptor positive breast cancer

Background: Splice variants play a major role in carcinogenesis and disease progression. It is well known that androgen receptor splice variants are associated with resistance to prostate cancer treatment. Estrogen receptor (ER)-positive breast cancers constitute about 70% of all breast cancers and have better prognosis compared to ER-negative cancers. However, there are ER-positive breast cancers that acquire resistance to anti-estrogen therapy, and 12-55% of those tumors were shown to possess ESR1 mutations. The aim of this study was to identify common splice variants in the ESR1 gene and investigate their association with disease outcome. Methods: Whole transcriptome sequencing was performed on breast cancer specimens from 120 invasive breast cancer patients who underwent operation at Seoul National University Hospital (SNUH) and data from SNUH, GEO, and The Cancer Genome Atlas (TCGA) was used for normal breast tissue sequencing. Exon-exon junctions were identified on aligned RNA sequencing data and was used to construct exon graphs. Splice variant candidates were selected from exon graphs and were merged according to variant subtypes of samples. Subtypes were accessed differentially in relation to how frequent the junctions appear in tumor samples and common exon skipping types with frequent junctions were identified. TCGA RNA sequencing data was then used to search for the common exon skipping subtypes detected from SNUH RNA sequencing data. Results: Of the 120 tumor samples, 50 were clinically ER-positive by immunohistochemistry. Among exon paths logically possible, 125 paths were not observed in normal breast tissues. Exon 4-5 junction was the most commonly observed junction in the tumor samples. In a search for exon skipping type that results in missing ligand-binding domain of ER, three exon skipping types were identified. Exon skipping with exon 5-10 junction (type 1), exon 9-12 junction (type 2), and exon 10-12 (type 3) was seen in 4 (8%), 4 (8%), and 10 (20%) ER-positive samples, respectively. Retrospective medical chart review of the 18 patients showed recurrence in 4 (100%), 2 (50%), and 4 (40%) patients with type 1, 2, and 3 exon skipping, respectively. Evaluation of TCGA RNA sequencing data of 872 ER-positive samples suggested exon 4-5 junction as the most common junction. A search for exon skipping types in TCGA revealed 1 (0.1%), 9 (1.0%), and 454 (52.1%) samples with type 1, 2, and 3 exon skipping, respectively. However, none of the patients with type 1 or 2 had metastasis or had expired. Of the 454 patients with type 3 exon skipping, 54 patients had died, constituting 61.4% of 88 mortalities in the whole ER-positive population. Conclusion: Certain splice variants of ESR1 gene yields exon skipping subtypes commonly observed in the ER-positive breast cancer. Estrogen receptor-positive breast cancer with these exon skipping types resulting in a missing ligand-binding domain of ER may be associated with poorer disease outcome. Further investigation is warranted to validate the role of ESR1 exon skipping subtypes in the disease progression of breast cancer. Citation Format: Lee H-B, Han W, Ko S, Kim M-S, Lim S, Lee K-M, Kang YJ, Han JH, Kim Y, Yoo T-K, Moon H-G, Noh D-Y, Kim S, Han W. Identification of ESR1 splice variants associated with prognosis in estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-02.

Abstract P1-02-01: Genomic analysis of single cells isolated by a pulse laser retrieval system

Background: Isolating tumor cells of interest and harvesting histologically pure samples is important for genomic studies. Laser capture microdissection (LCM) is an established method to obtain such purified cell populations for various applications including DNA, gene expression, and single cell analyses. However, LCM possesses problems such as limited optical resolution, cell fragmentation from dissection, and adherence of adjacent tissue to the cells which interrupts with single cell isolation from tissue sections. To overcome these obstacles, we developed a high-throughput pulse laser retrieval system which uses a wavelength that minimizes damage to the cellular content and is processed with a sacrificial layer that provides applicable optical resolution. The aim of this study was to evaluate the performance of the pulse laser retrieval system to provide appropriate samples for genomic analysis using breast cancer tissue. Methods: An indium tin oxide (ITO) coated glass slide was prepared using fresh frozen breast cancer tissue sections of 4㎛ thickness and stained by hematoxylin and eosin. The slide was mounted on the cell isolation machine and imaging was performed with a charge-coupled device camera using a 20× lens. Following identification of the target cells by a pathologist, nano-second pulsed laser (wavelength= 1064nm) was irradiated on the target. Isolated cells were collected in a polymerase chain reaction tube and whole genome amplification (WGA) was carried out using Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Amplified genomic DNA was fragmented and Illumina sequencing libraries were constructed. Sequencing was carried out to generate data with 0.1∼0.2× depth throughout the whole genome for each sample. Copy number variation (CNV) was analyzed by the Variable binning algorithm. Results: Whole genome amplification was performed using bulk tissue and 10 captured single cells from the same specimen. No difference in amplification coverage was observed between the two samples. A CNV analysis of captured single cells revealed similar CNV profiles with those in a matched bulk tumor. Whole exome sequencing (WES) of captured single cells yielded a variant frequency of 15% at a read depth of 15× and 50M base coverage, compared to 0% at 100× and 50M for WES using bulk tumor and 0.5% at 1200× and 100K for targeted sequencing using bulk tumor. Laser capture was performed for DCIS and stromal cells from the same slide. CNV analysis of the two samples showed minimal CNV in normal stromal cells in contrast to DCIS where multiple CNVs were observed. Conclusions: Newly developed pulse laser retrieval system is suitable for capturing single cells for genomic analysis of breast cancer. WGA, WES, and CNV analysis was successfully carried out using the captured single cells and showed no difference in profile compared to those performed with bulk tissue. This method may have the potential to replace LCM for certain applications such as single cell analyses. Citation Format: Lee H-B, Kim S, Lee K-M, Jung Y, Lee AC, Kim J, Bae S, Ryu HS, Yoo T-K, Moon H-G, Noh D-Y, Kwon S, Han W. Genomic analysis of single cells isolated by a pulse laser retrieval system. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-02-01.

Abstract P2-02-15: Discovery of putative circulating tumor cells through somatic mutation profile of epithelial cell adhesion molecule positive single cells from blood of metastatic breast cancer patients

Background: Circulating tumor cell (CTC) enumeration provides prognostic information for chemotherapy in metastatic breast cancer. However, due to its rarity and heterogeneity, it is difficult to distinguish true CTCs from normal blood cells and perform genomic analysis on them for use in therapeutic strategies. The main application of most currently available CTC detection systems consists of an enumeration of putative CTCs without further analysis. The aim of this study was to evaluate the feasibility of single cell picking and target sequencing of epithelial cell adhesion molecule (EpCAM)-positive cells for detecting CTCs. Methods: Whole blood sampled from metastatic breast cancer patients who were newly diagnosed with metastasis or who had disease progression during palliative treatment were used for this study. After applying IsoFlux Circulating Tumor Cell Enrichment Kit (Fluxion, South San Francisco, CA, USA), single CTC candidates were picked from a pool of EpCAM-positive cells. Genomic DNA from the picked cells was whole genome amplified and target sequencing was performed using Ion AmpliSeq™ Cancer Hotspot Panel (Life Technologies, Carlsbad, CA, USA). Target sequencing reads were mapped to human genome reference (hg19) using BWA-MEM (0.7.10). Single nucleotide variants (SNVs) were annotated using dbSNP, Variome Data 0.2, and COSMIC databases. Results: A total of 172 EpCAM-positive cells were selected according to size and EpCAM status from whole blood of 11 patients. The remaining cells were grouped into a pooled sample for each patient. The mean read depth of the target genes was 13455×. A mean 7.82 mutations as determined by SNVs listed in the COSMIC database but not in dbSNP and Variome Data 0.2 were detected in each patient. Cells with multiple mutated genes, or those with a mutated gene repeatedly observed in another cell from the same patient were judged to be putative CTCs. At least 2 putative CTCs were detected in 7 patients while no CTCs were detected in 2 patients. Mutated genes observed in the putative CTCs were ABL1, AKT1, APC, CDH1, CDKN2A, ERBB2, FGFR3, HRAS, IDH1, JAK2, KDR, NPM1, RB1, RET, SMARCB1, STK11, and TP53. Conclusions: Potential CTCs were successfully identified by single cell picking and target sequencing of EpCAM-positive cells from whole blood of metastatic breast cancer patients. Unique mutations not detected in other single cells and pooled samples can be used to distinguish putative CTCs from normal cells. Genomic profiling of corresponding primary tumor and metastatic site biopsy is warranted to verify the CTCs and investigate their role in disease progression. Citation Format: Lee H-B, Jeon S, Kim BC, Jho S, Kim J, Kang YJ, Yoo T-K, Han JH, Kim Y, Im S-A, Moon H-G, Noh D-Y, Han W. Discovery of putative circulating tumor cells through somatic mutation profile of epithelial cell adhesion molecule positive single cells from blood of metastatic breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-15.

Abstract P5-08-23: Ki-67 expression is not a valuable predictive prognostic factor when progesterone receptor expression is high in estrogen receptor-positive breast cancer

Background Immunohistochemistry markers are recognized as a predictive prognostic factor for women with breast cancer. Ki-67 and progesterone receptor (PgR) expression are reported to be independently associated with breast cancer prognosis. Some studies report high Ki-67 expression as a negative predictive marker. Whereas other studies report tendency of similar survival between high and low Ki67 cancers when PgR expression is high. In this study, we examined the prognostic significance of Ki-67 expression under PgR expression status. Methods The records of 2,366 patients were retrospectively reviewed. The patients underwent surgery for primary breast cancer from July 2009 to December 2012 at a single institution. We studied the prognostic significance of Ki-67 expression under PgR expression. We used 20% and 10% as the cut-off value for PgR and Ki-67, respectively. The end point was recurrence-free survival (RFS) evaluated by use of Kaplan-Meier analysis. Result Of the 2,366 analyzed patients, the median follow-up time was 43 months. During follow-up, 44 patients had recurrence, loco-regional recurrence developed in 23 patients and distant recurrence developed in 21 patients. In patients with low PgR expression, high Ki-67 expression group showed significantly worse prognosis compared to low Ki-67 expression group (p=0.005). On the other hand, no significant difference was shown between low and high Ki-67 expression group when PgR expression was high (p=0.637). Also multivariate analysis demonstrated that high Ki-67 expression was an independent prognostic factor only when PgR expression was low. (95% confidence interval [CI], 1.35-10.48; p=0.011) Conclusion This is the largest reported study that prognostic significance of Ki-67 expression is defined by PgR expression. Our study presents that high Ki-67 expression is inversely correlated with recurrence risk in early breast cancer patients only under low PgR expression. At high PgR expression, Ki-67 expression has no influence on breast cancer prognosis. Therefore, attention should be paid to correlation between PgR and Ki-67 expression. Citation Format: Han JH, Kang YJ, Han W, Lee H-B, Kim Y, Yoo T-K, Moon H-G, Noh D-Y. Ki-67 expression is not a valuable predictive prognostic factor when progesterone receptor expression is high in estrogen receptor-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-23.

Abstract PD4-2: Whole exome and transcriptome sequencing of 120 primary breast cancer to discover novel therapeutic target

Introduction: Many somatic mutations, structural alterations, and gene expression changes are causally implicated in oncogenesis and tumor progression, and as a result, affect clinical outcome. Although majority of breast cancer patients have benefits from therapeutics targeting tumor biology, such as estrogen receptor and HER-2, still many patients suffer from disease recurrence and metastasis. More kind of specific target therapies are needed, especially for hormone-resistant tumor and triple-negative breast cancer. Materials and Method: To find novel therapeutic target in breast cancer, here we examine the both whole exome and whole transcriptome of fresh-frozen primary breast cancer tissues from 120 patients whose clinical, pathological, and survival data are available. Patients with Stage IV disease or who received neoadjuvant chemotherapy were excluded. 36 patients had distant metastasis within 5 years from surgery, and 84 patients were NED at least 5 years. RNA and DNA were extracted and qualities were assessed in all samples. Exome and transcriptome sequencing were done using NGS technology (Illumina HiSeq 2000). As a control, exome sequencing was done for 93 normal DNA from matched patients. Single nucleotide variations (SNV) identified in cancer samples on exonic region, nonsynonymous SNV or stop gain/loss, whose quality ≥20, and not found in 93 normal samples were included. SNVs registered in dbSNP135_common or 1000 genome allele frequency >0.001 were excluded.Results and Discussion: We identified 11,684 putative somatic mutations in 7,373 genes. Of them, 6,547 were deleterious or damaging mutation by Provean or SIFT analysis. Mutations were found in potential drug target genes, such as PIK3CA(25), PTEN(3), AKT1(3), ALK(3), ROS1(2), FGFR4(3), FGFR3(2), ERBB2(2), and IDH1(1) etc. In a pathway analysis, mutations in insulin signaling pathway were most dominant. We hypothesized that driver gene and therapeutic target has to have recurrent mutation and gene expression at least more than average expression. We calculated expression “Volume” according to the median normalized FPKM value of individual gene9s RNA-seq data. With a cut-off of 3 or more mutations in each gene, 1,116 genes were selected. After the filtering of Volume Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-2.

Abstract P3-14-20: Neoadjuvant chemotherapy in young age breast cancer: Survival benefit over adjuvant chemotherapy in clinically T2 node positive patients

Background: The downstaging of the primary tumor and the increase in breast conservation rates seems to be the only clinical benefit of Neoadjuvant systemic therapy(NST) in breast cancer treatment, given that several studies failed to demonstrate an improvement of overall survival compared with postoperative adjuvant chemotherapy. In Europe, S6 trial showed better early outcome in survival in favour of the neoadjuvant chemotherapy group compared to adjuvant chemotherapy group in premenopausal patients without significantly modifying long-term event rates. The aim of this study was to assess a potential advantage in survival by neoadjuvant as compared to adjuvant chemotherapy in young age breast cancer patients. Methods: Between January 2001 and December 2008, 1169 consecutive patients with breast cancer aged under 40 underwent adjuvant chemotherapy before or after surgery. Prospectively collected medical records for all patients were reviewed retrospectively. For the comparison of survival between neoadjuvant versus adjuvant chemotherapy group, cinically T2 and node positive patients were retrieved. Survival curves were derived from Kaplan-Meier estimates and compared by log-rank test. Results: Of the 1169 patients, 203(17.3%) patients were treated with neoadjuvant chemotherapy, and they were grouped as ‘NST’ and ‘non-NST’ according to initial treatment. About 47% patients in each group were clinically T2 patients. (99(47.8%) in NST group, 453(46.9%) in non-NST group) Among them, clinically T2 and node positive patients were 188, 97 patients in NST group, 91 patients in non-NST group each. The median age was 35.11±3.9 years old and HER2 amplification was observed as 23.5%, and they were not different between two groups.(p = 0.146 and 0.941 each) Significant lower hormone receptor expression rate and higher Ki-67 level were observed in NST group(p = 0.03 and Conclusion: A potential advantage of primary over adjuvant chemotherapy in young age breast cancer patients’ survival might be proposed by this results. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-14-20.