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GeneXpert® breast cancer STRAT4 assay demonstrates high concordance of ESR1, PgR, HER2, and Ki67 with central IHC and FISH testing in FFPE breast tumor tissues
15th St.Gallen International Breast Cancer Conference / The Breast 32S1 (2017) S22–S77
in similar or higher kappa values compared to dichotomous scoring of other histopathological features. This study demonstrates the robustness of dichotomous assessment of both stromal architecture and stromal inflammation in DCIS. Disclosure of Interest: No significant relationships. P075 The expression of cyclin D1b is increased in tamoxifen-resistant breast cancer cell line H.S. Won*, Y.H. Ko, D.S. Sun. Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, South Korea Aims: Cyclin D1 is a well-known regulator of cell-cycle progression, and a target gene of the estrogen receptor (ER). Cyclin D1 exists in two isoforms: conventional cyclin D1a and cyclin D1b. The cyclin D1b variant arises as a consequence of alternative splicing of the CCND1 transcript. Although it seems that cyclin D1b is a highly oncogenic variant of cyclin D1, little is known about the behavior of cyclin D1b. We aimed to investigate the expression of cyclin D1b and its related signaling pathway in tamoxifen-resistant breast cancer cells. Methods: According to previous studies, we have established a MCF7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with gradually increasing 4-hydroxytamoxifen concentration till 3 μM. The levels of protein expression and mRNA transcripts were determined using western blot analysis and realtime quantitative PCR. Cell cycle of control and TamR cells was analyzed by flow cytometry. Results: TamR cells exhibited a relatively decreased expression of ER and increased expression of human epidermal growth factor receptor 2 and the epidermal growth factor receptor. The viability of control cells decreased by 65.2% after 3 µM tamoxifen treatment, whereas the viability of TamR cells showed no change. TamR cells exhibited Ecadherin loss and increased expression of Snail and slug. TamR cells had an acceleration of the G1 to S phase transition compared to control cells. The cyclin D1b level was increased in TamR cells compared to control cells, instead of cyclin D1a. The phosphorylated signal transducers and activators of transcription 3 ( pSTAT3) was also increased in TamR cells compared to control cells. The expression of cyclin D1b and pSTAT3 were inhibited by small-molecule inhibitor of STAT3, Stattic. Conclusions: These results suggest that STAT3/cyclin D1b plays a role in tamoxifen-resistant breast cancer, and the inhibition of this pathway may be a potential target in tamoxifen-resistant breast cancer. Disclosure of Interest: No significant relationships. P076 The prognostic value of SOCS2 expression in breast cancer patient according to molecular subtype S.U. Woo*, S.H. Lee, W.Y. Kim, J.B. Lee. Korea University, Seoul, South Korea Objective: Suppressors of cytokine signaling (SOCS) has been known as one of the prognostic factors in breast cancer patients. The aims of this study were to verify the significant prognostic role of SCOS 2 being depended on the status of axillary lymphatic metastases and to evaluate the relationship between SOCS2 and molecular markers in breast cancer patients. Methods: We analyzed the tissue microarray which was contained 386 tissue samples from breast cancer patients who undertook proper treatment between Jan 1990 and Dec 2006. We measured the expression of SOCS2 by immunohistochemistry staining. Results: SOCS2 immunohistochemistry staining was shown high expressions of SOCS2 in 206 (53.4%) cases. The expression of SOCS2 decreased with higher tumor grade (P < 0.05). SOCS2 high expressions were related with estrogen receptor positivity. There was a
S49
significant local recurrence in a group of low SOCS2 expression (P = 0.04) A statistical difference of overall survival was shown in patients with axillary lymphatic metastases comparing low with high expression of SOCS2. Conclusions: SOCS2 expression closely related to estrogen receptor status. High SOCS2 expression related to low local recurrence. We suggest that SOCS2 might be useful disease free survival predictor for the breast patients. Disclosure of Interest: No significant relationships. P077 GeneXpert® breast cancer STRAT4 assay demonstrates high concordance of ESR1, PgR, HER2, and Ki67 with central IHC and FISH testing in FFPE breast tumor tissues E. Wong1 *, N. Wu1, B. Acca1, H. Dias1, Cepheid Oncology BC STRAT4 Team Cepheid OncologyBC STRAT4 Team1, N. Quigley2, S.S. Beqaj3, K. David4, S. Davenport5, M. Press5. 1 Cepheid, Sunnyvale, United States, 2Molecular Pathology Laboratory Network, Inc., Maryville, United States, 3Molecular Testing Labs, Vancouver, United States, 4Indivumed, Hamburg, Germany, 5University of Southern California, Los Angeles, United States The GeneXpert® Breast Cancer STRAT4 (STRAT4) assay is a semiquantitative, RT-qPCR assay for detection of ESR1, PGR, ERBB2 (HER2), and MKi67 mRNAs using FFPE specimens. The assay is fast, robust, and easy to perform. This multicenter clinical study sought to establish the performance characteristics for the STRAT4 assay mRNA measurements relative to IHC results for ESR1, PgR, HER2, Ki67 and to fluorescence in situ hybridization (FISH) for HER2 gene amplification. Additional analyses were conducted to evaluate concordance and indeterminate rate (ND) of mRNA measurements between standard (1x) and concentrated (4x) FFPE lysate preparation procedures. A total of 199 invasive primary tumor FFPE specimens were included in this study. For each FFPE specimen, 12 adjacent 4-µm sections on slides were prepared for STRAT4 and for ESR1, PgR, HER2, Ki67 IHC and for HER2 FISH. STRAT4 analysis was performed at 3 different sites (2 US and 1 EU). Each site prepared its own 1x FFPE lysate per specimen for testing with STRAT4. Each specimen was also processed using a 4x concentrated lysate procedure for STRAT4 testing at Cepheid. All IHC and FISH reference testing was performed in a central reference laboratory in the US. Overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) between STRAT4 and IHC (IHC and FISH for HER2) were determined for ESR1, PGR, HER2 and MKi67. Of the 199 samples, 197 (98.5%) yielded valid results for all four targets from STRAT4 with either the 1x or 4x concentrated lysate preparation protocol. OPA between STRAT4 with 1x lysate and IHC was 96.4% for ESR1, 89.2% for PGR, 91.8% for HER2 (IHC and FISH) (93% for HER2 IHC excluding IHC 2+), and 86.1% for MKi67. When comparing results between the 1x and 4x lysate preparation for STRAT4, the ND rate dropped from 10% to 1% with the 4x method for PGR and from 16.5% to 1.5% for MKi67. Concordance between the two lysate preparation methods was 97% for ESR1, 91% for PGR, 90% for HER2 and 80% for MKi67. STRAT4 assay measurements for ESR1, PGR, HER2 and MKi67 mRNA expression are highly concordant with IHC (IHC and FISH for HER2). Utilizing a concentrated lysate further improves assay validity rate. Further investigations using clinical outcomes from independent studies are in progress. Disclosure of Interest: the author mentions a conflict of interest which is not further specified.
in similar or higher kappa values compared to dichotomous scoring of other histopathological features. This study demonstrates the robustness of dichotomous assessment of both stromal architecture and stromal inflammation in DCIS. Disclosure of Interest: No significant relationships. P075 The expression of cyclin D1b is increased in tamoxifen-resistant breast cancer cell line H.S. Won*, Y.H. Ko, D.S. Sun. Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, South Korea Aims: Cyclin D1 is a well-known regulator of cell-cycle progression, and a target gene of the estrogen receptor (ER). Cyclin D1 exists in two isoforms: conventional cyclin D1a and cyclin D1b. The cyclin D1b variant arises as a consequence of alternative splicing of the CCND1 transcript. Although it seems that cyclin D1b is a highly oncogenic variant of cyclin D1, little is known about the behavior of cyclin D1b. We aimed to investigate the expression of cyclin D1b and its related signaling pathway in tamoxifen-resistant breast cancer cells. Methods: According to previous studies, we have established a MCF7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with gradually increasing 4-hydroxytamoxifen concentration till 3 μM. The levels of protein expression and mRNA transcripts were determined using western blot analysis and realtime quantitative PCR. Cell cycle of control and TamR cells was analyzed by flow cytometry. Results: TamR cells exhibited a relatively decreased expression of ER and increased expression of human epidermal growth factor receptor 2 and the epidermal growth factor receptor. The viability of control cells decreased by 65.2% after 3 µM tamoxifen treatment, whereas the viability of TamR cells showed no change. TamR cells exhibited Ecadherin loss and increased expression of Snail and slug. TamR cells had an acceleration of the G1 to S phase transition compared to control cells. The cyclin D1b level was increased in TamR cells compared to control cells, instead of cyclin D1a. The phosphorylated signal transducers and activators of transcription 3 ( pSTAT3) was also increased in TamR cells compared to control cells. The expression of cyclin D1b and pSTAT3 were inhibited by small-molecule inhibitor of STAT3, Stattic. Conclusions: These results suggest that STAT3/cyclin D1b plays a role in tamoxifen-resistant breast cancer, and the inhibition of this pathway may be a potential target in tamoxifen-resistant breast cancer. Disclosure of Interest: No significant relationships. P076 The prognostic value of SOCS2 expression in breast cancer patient according to molecular subtype S.U. Woo*, S.H. Lee, W.Y. Kim, J.B. Lee. Korea University, Seoul, South Korea Objective: Suppressors of cytokine signaling (SOCS) has been known as one of the prognostic factors in breast cancer patients. The aims of this study were to verify the significant prognostic role of SCOS 2 being depended on the status of axillary lymphatic metastases and to evaluate the relationship between SOCS2 and molecular markers in breast cancer patients. Methods: We analyzed the tissue microarray which was contained 386 tissue samples from breast cancer patients who undertook proper treatment between Jan 1990 and Dec 2006. We measured the expression of SOCS2 by immunohistochemistry staining. Results: SOCS2 immunohistochemistry staining was shown high expressions of SOCS2 in 206 (53.4%) cases. The expression of SOCS2 decreased with higher tumor grade (P < 0.05). SOCS2 high expressions were related with estrogen receptor positivity. There was a
S49
significant local recurrence in a group of low SOCS2 expression (P = 0.04) A statistical difference of overall survival was shown in patients with axillary lymphatic metastases comparing low with high expression of SOCS2. Conclusions: SOCS2 expression closely related to estrogen receptor status. High SOCS2 expression related to low local recurrence. We suggest that SOCS2 might be useful disease free survival predictor for the breast patients. Disclosure of Interest: No significant relationships. P077 GeneXpert® breast cancer STRAT4 assay demonstrates high concordance of ESR1, PgR, HER2, and Ki67 with central IHC and FISH testing in FFPE breast tumor tissues E. Wong1 *, N. Wu1, B. Acca1, H. Dias1, Cepheid Oncology BC STRAT4 Team Cepheid OncologyBC STRAT4 Team1, N. Quigley2, S.S. Beqaj3, K. David4, S. Davenport5, M. Press5. 1 Cepheid, Sunnyvale, United States, 2Molecular Pathology Laboratory Network, Inc., Maryville, United States, 3Molecular Testing Labs, Vancouver, United States, 4Indivumed, Hamburg, Germany, 5University of Southern California, Los Angeles, United States The GeneXpert® Breast Cancer STRAT4 (STRAT4) assay is a semiquantitative, RT-qPCR assay for detection of ESR1, PGR, ERBB2 (HER2), and MKi67 mRNAs using FFPE specimens. The assay is fast, robust, and easy to perform. This multicenter clinical study sought to establish the performance characteristics for the STRAT4 assay mRNA measurements relative to IHC results for ESR1, PgR, HER2, Ki67 and to fluorescence in situ hybridization (FISH) for HER2 gene amplification. Additional analyses were conducted to evaluate concordance and indeterminate rate (ND) of mRNA measurements between standard (1x) and concentrated (4x) FFPE lysate preparation procedures. A total of 199 invasive primary tumor FFPE specimens were included in this study. For each FFPE specimen, 12 adjacent 4-µm sections on slides were prepared for STRAT4 and for ESR1, PgR, HER2, Ki67 IHC and for HER2 FISH. STRAT4 analysis was performed at 3 different sites (2 US and 1 EU). Each site prepared its own 1x FFPE lysate per specimen for testing with STRAT4. Each specimen was also processed using a 4x concentrated lysate procedure for STRAT4 testing at Cepheid. All IHC and FISH reference testing was performed in a central reference laboratory in the US. Overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) between STRAT4 and IHC (IHC and FISH for HER2) were determined for ESR1, PGR, HER2 and MKi67. Of the 199 samples, 197 (98.5%) yielded valid results for all four targets from STRAT4 with either the 1x or 4x concentrated lysate preparation protocol. OPA between STRAT4 with 1x lysate and IHC was 96.4% for ESR1, 89.2% for PGR, 91.8% for HER2 (IHC and FISH) (93% for HER2 IHC excluding IHC 2+), and 86.1% for MKi67. When comparing results between the 1x and 4x lysate preparation for STRAT4, the ND rate dropped from 10% to 1% with the 4x method for PGR and from 16.5% to 1.5% for MKi67. Concordance between the two lysate preparation methods was 97% for ESR1, 91% for PGR, 90% for HER2 and 80% for MKi67. STRAT4 assay measurements for ESR1, PGR, HER2 and MKi67 mRNA expression are highly concordant with IHC (IHC and FISH for HER2). Utilizing a concentrated lysate further improves assay validity rate. Further investigations using clinical outcomes from independent studies are in progress. Disclosure of Interest: the author mentions a conflict of interest which is not further specified.