Breast Cancer Biopsies Research Articles

Sentinel Lymph Node Biopsy in Early Breast Cancer: a Real-world Multicenter Cross-sectional Study (CABS001 Study)

Sentinel Lymph Node Biopsy in Early Breast Cancer: a Real-world Multicenter Cross-sectional Study (CABS001 Study)

GREM1 is associated with metastasis and predicts poor prognosis in ER-negative breast cancer patients

BackgroundIn breast cancer, activation of bone morphogenetic protein (BMP) signaling and elevated levels of BMP-antagonists have been linked to tumor progression and metastasis. However, the simultaneous upregulation of BMPs and their antagonist, and the fact that both promote tumor aggressiveness seems contradictory and is not fully understood.MethodsWe analyzed the transcriptomes of the metastatic 66cl4 and the non-metastatic 67NR cell lines of the 4T1 mouse mammary tumor model to search for factors that promote metastasis. CRISPR/Cas9 gene editing was used for mechanistic studies in the same cell lines. Furthermore, we analyzed gene expression patterns in human breast cancer biopsies obtained from public datasets to evaluate co-expression and possible relations to clinical outcome.ResultsWe found that mRNA levels of the BMP-antagonist Grem1, encoding gremlin1, and the ligand Bmp4 were both significantly upregulated in cells and primary tumors of 66cl4 compared to 67NR. Depletion of gremlin1 in 66cl4 could impair metastasis to the lungs in this model. Furthermore, we found that expression of Grem1 correlated with upregulation of several stem cell markers in 66cl4 cells compared to 67NR cells. Both in the mouse model and in patients, expression of GREM1 associated with extracellular matrix organization, and formation, biosynthesis and modification of collagen. Importantly, high expression of GREM1 predicted poor prognosis in estrogen receptor negative breast cancer patients. Analyses of large patient cohorts revealed that amplification of genes encoding BMP-antagonists and elevation of the corresponding transcripts is evident in biopsies from more than half of the patients and much more frequent for the secreted BMP-antagonists than the intracellular inhibitors of SMAD signaling.ConclusionIn conclusion, our results show that GREM1 is associated with metastasis and predicts poor prognosis in ER-negative breast cancer patients. Gremlin1 could represent a novel target for therapy.

Preliminary study of contrast-enhanced ultrasound in combination with blue dye vs. indocyanine green fluorescence, in combination with blue dye for sentinel lymph node biopsy in breast cancer

BackgroundThis preliminary study aimed to examine the feasibility of sentinel lymph node biopsy (SLNB) using contrast-enhanced ultrasound (CEUS) vs. indocyanine green fluorescence (ICG), combined with blue dye in patients with breast cancer.MethodsThis was a retrospective study of consecutive female patients with invasive stage I-III (based on pre-operative physical examination and imaging) primary breast cancer at the Peking Union Medical College Hospital between 01/2013 and 01/2015 who underwent preoperative SLNB by ICG + blue dye or CEUS + blue dye. The numbers of detected SLNs, detection rates, and recurrence-free survival (RFS) rates were compared between the two groups.ResultsA total of 443 patients were included. The detection rates of SLNs in the CEUS + blue dye and ICG + blue dye groups were 98.4 and 98.1%, respectively (P = 0.814). The average numbers of SLNs detected per patient showed no significant difference between the two groups (3.06 ± 1.33 and 3.12 ± 1.31 in the CEUS + blue dye and ICG + blue dye groups, respectively; P = 0.659). After a median follow-up of 46 months, five patients in the CEUS + blue dye group and 15 in the ICG + blue dye group had recurrence. RFS rates showed no significant difference (P = 0.55).ConclusionThis preliminary study suggests that CEUS + blue dye and ICG + blue dye are both feasible for SLN detection in breast cancer.

1895P - Breast cancer organoids model treatment response of HER2 targeted therapy in HER2-mutant breast cancer

BackgroundTo facilitate cancer precision medicine, breast cancer organoids have recently emerged as a useful pre-clinical model for retaining sufficient fidelity regarding histology, the transcriptome and genome. However, their potential to predict clinical treatment responses remains unclear. MethodsWe generated breast cancer organoids from breast cancer tissues and performed drug sensitivity test on these organoids based on genomic analysis data. ResultsA total of 25 fresh breast cancer tissues and biopsies from 22 patients were processed between November 2017 and February 2019. Breast cancer organoids were grown successfully from 10 out of 25 patients (40%). We performed histopathological analysis of H&E stained tissues and organoid sections and confirmed that the phenotypes of organoids matched the original histological breast cancer types. We also performed whole genome DNA sequencing (WGS) and RNA sequencing (RNA-seq) on breast cancer organoids and paired breast cancer tissues. One of these breast cancer organoids had HER2 mutations (previously shown to be an activating mutation) and retained expression of estrogen receptor (ER) and progesterone receptor (PR) (Luminal A subtype). In our previous work, we identified activating HER2 mutations (S310F D769Y V777L 778insGSP) in ER positive/HER2-amplification negative breast cancer, who had developed resistance to multi-line endocrine therapy. Two patients achieved a durable partial response (approximately 1 years) to trastuzumab combined with everolimus. We also showed that HER2 mutations were constitutively active, and T47D and MCF7 overexpressing HER2 mutations were sensitive to HER2 targeted therapies combined with everolimus. Hence, we tested the effects of trastuzumab and everolimus on this HER2-mutant breast cancer organoid in vitro and in vivo. HER2-targeted therapies combined with everolimus produced regression of the HER2-mutated organoid. ConclusionsThe breast cancer organoids may serve as a high-fidelity platform and recapitulate clinical treatment responses in personalized medicine. These data provide a strong preclinical rationale for combining HER2-targeted therapies with everolimus in HER2-mutant/ HER2-amplification negative breast cancer. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

177PD - Prospective, multicenter registry trial to evaluate the clinical feasibility of targeted axillary dissection (TAD) in patients (pts) with breast cancer (BC) and core biopsy proven axillary involvement (cN+)

BackgroundAs part of de-escalating strategies of the axillary treatment in lymph node (LN) positive BC pts, TAD was introduced (Caudle et al., JCO 2016). Despite this very interesting approach, some important aspects regarding the clinical feasibility after neoadjuvant chemotherapy (NACT) were missing if a safe procedure was to be guaranteed. The correct evaluation of pathologic complete response (pCR) has a prognostic impact in high risk BC pts (Katherine, CreATE X). This study aimed to investigate the clinical feasibility of clip placement in positive axillary LNs followed by NACT and TAD in BC patients. MethodsThe Senta trial is a prospective, multicentric registry study (N=598). Prior to NACT, pts with invasive BC received an ultrasound guided core biopsy with clip insertion into the suspicious axillary LN. After NACT, pts underwent axillary surgery according to the investigators discretion, with or without sentinel LN (SLN) biopsy. TAD included removal of the clipped LN and SLN biopsy (SLNB). The pathology of the clipped LN before and after NACT, other LNs, and the SLN were compared. The primary endpoint (EP) was the detection rate (DR) of the clipped LN after NACT. Secondary EP was the false negative rate (FNR) of TAD. ResultsBetween January 2017 and October 2018, 598 cN+ BC pts from 50 different German breast units had biopsy proven axillary LN involvement and received clip insertion into the affected LN. The complete data set from 378 pts was available. After NACT, the clipped N was detected in 78.6% of the patients (target lymph node biopsy = TLNB), and 58.9% of the whole cohort received SLNB as well. The SLN and TLN were identical in 48.0%. An axillary dissection was performed overall in 68%. The FNR of TLNB was 8.0% (CI 95%, 3.0–12.0) for the whole cohort. TAD followed by axillary LN dissection had a FNR of 4.4% (2 of 45, CI 95%, 0.02–9.0). ConclusionsPreliminary results demonstrated a high DR supporting the feasibility of this approach, however, the FNR of TAD (4.4%) was higher than reported earlier (FNR = 2.0%, Caudle et al.). This is of great importance for postneoadjuvant treatment decisions in case of non-pCR and should be discussed. Clinical trial identificationNCT03102307; release date: April 5, 2017. Legal entity responsible for the studyKliniken Essen-Mitte. FundingHas not received any funding. DisclosureM. Reinisch: Honoraria (self): Pfizer, Novartis, Eli Lilly, Roche; Advisory / Consultancy: Roche, Daiichi Sankyo, Hexal, Eli Lilly, Novartis; Travel / Accommodation / Expenses: Pfizer, Celgene, Novartis. J. Heil: Advisory / Consultancy: Roche, Siemens Healthcare Diagnostics; Research grant / Funding (self): BARD; Honoraria (self): Roche, Siemens Healthcare Diagnostics, BARD; Travel / Accommodation / Expenses: Celgene. C. Seiberling: Travel / Accommodation / Expenses: Teva. C. Ankel: Advisory / Consultancy, Travel / Accommodation / Expenses: PFM medicals. V. Hanf: Honoraria (self): Novartis. J. Holtschmidt: Travel / Accommodation / Expenses: Roche. S. Kuemmel: Advisory / Consultancy: Genentech, Genomic Health, Novartis, AstraZeneca, Amgen, Celgene, Somatex, Daiichi Sankyo, Puma Biotechnology, PFM Medical, Pfizer, MSD Oncology; Travel / Accommodation / Expenses: Roche, Daiichi Sankyo. All other authors have declared no conflicts of interest.

The power of integrated technology – is there a place for it in midwifery practice?

Data on the role of the microbiota in cancer have accumulated in recent years, with particular interest in intratumoral bacteria. Previous results have shown that the composition of intratumoral microbiome is different depending on the type of primary tumour and that bacteria from the primary tumour could migrate to metastatic sites.Seventy-nine patients with breast, lung, or colorectal cancer and available biopsy samples from lymph node, lung, or liver site, treated in the SHIVA01 trial were analysed. We performed bacterial 16S rRNA gene sequencing on these samples to characterise the intratumoral microbiome. We assessed the association between microbiome composition, clinicopathological characteristics, and outcomes.Microbial richness (Chao1 index), evenness (Shannon index) and beta-diversity (Bray Curtis distance) were associated with biopsy site (p = 0.0001, p = 0.03 and p < 0.0001, respectively) but not with primary tumour type (p = 0.52, p = 0.54 and p = 0.82, respectively). Furthermore, microbial richness was inversely associated with tumour-infiltrating lymphocytes (TILs, p = 0.02), and PD-L1 expression on immune cells (p = 0.03), or assessed by Tumor Proportion Score (TPS, p = 0.02) or Combined Positive Score (CPS, p = 0.04). Beta-diversity was also associated with these parameters (p < 0.05). Patients with lower intratumoral microbiome richness had shorter overall survival (p = 0.03) and progression-free survival (p = 0.02) in multivariate analysis.Biopsy site, rather than primary tumour type, was strongly associated with microbiome diversity. Immune histopathological parameters such as PD-L1 expression and TILs were significantly associated with alpha and beta-diversity supporting the cancer-microbiome-immune axis hypothesis.

Micro-costing vaginal birth at home, in a birth centre and in a hospital in New South Wales for women at low risk of complications

Data on the role of the microbiota in cancer have accumulated in recent years, with particular interest in intratumoral bacteria. Previous results have shown that the composition of intratumoral microbiome is different depending on the type of primary tumour and that bacteria from the primary tumour could migrate to metastatic sites.Seventy-nine patients with breast, lung, or colorectal cancer and available biopsy samples from lymph node, lung, or liver site, treated in the SHIVA01 trial were analysed. We performed bacterial 16S rRNA gene sequencing on these samples to characterise the intratumoral microbiome. We assessed the association between microbiome composition, clinicopathological characteristics, and outcomes.Microbial richness (Chao1 index), evenness (Shannon index) and beta-diversity (Bray Curtis distance) were associated with biopsy site (p = 0.0001, p = 0.03 and p < 0.0001, respectively) but not with primary tumour type (p = 0.52, p = 0.54 and p = 0.82, respectively). Furthermore, microbial richness was inversely associated with tumour-infiltrating lymphocytes (TILs, p = 0.02), and PD-L1 expression on immune cells (p = 0.03), or assessed by Tumor Proportion Score (TPS, p = 0.02) or Combined Positive Score (CPS, p = 0.04). Beta-diversity was also associated with these parameters (p < 0.05). Patients with lower intratumoral microbiome richness had shorter overall survival (p = 0.03) and progression-free survival (p = 0.02) in multivariate analysis.Biopsy site, rather than primary tumour type, was strongly associated with microbiome diversity. Immune histopathological parameters such as PD-L1 expression and TILs were significantly associated with alpha and beta-diversity supporting the cancer-microbiome-immune axis hypothesis.

The ecology of birth: Aligning with evolution in honour of the whole woman

Data on the role of the microbiota in cancer have accumulated in recent years, with particular interest in intratumoral bacteria. Previous results have shown that the composition of intratumoral microbiome is different depending on the type of primary tumour and that bacteria from the primary tumour could migrate to metastatic sites.Seventy-nine patients with breast, lung, or colorectal cancer and available biopsy samples from lymph node, lung, or liver site, treated in the SHIVA01 trial were analysed. We performed bacterial 16S rRNA gene sequencing on these samples to characterise the intratumoral microbiome. We assessed the association between microbiome composition, clinicopathological characteristics, and outcomes.Microbial richness (Chao1 index), evenness (Shannon index) and beta-diversity (Bray Curtis distance) were associated with biopsy site (p = 0.0001, p = 0.03 and p < 0.0001, respectively) but not with primary tumour type (p = 0.52, p = 0.54 and p = 0.82, respectively). Furthermore, microbial richness was inversely associated with tumour-infiltrating lymphocytes (TILs, p = 0.02), and PD-L1 expression on immune cells (p = 0.03), or assessed by Tumor Proportion Score (TPS, p = 0.02) or Combined Positive Score (CPS, p = 0.04). Beta-diversity was also associated with these parameters (p < 0.05). Patients with lower intratumoral microbiome richness had shorter overall survival (p = 0.03) and progression-free survival (p = 0.02) in multivariate analysis.Biopsy site, rather than primary tumour type, was strongly associated with microbiome diversity. Immune histopathological parameters such as PD-L1 expression and TILs were significantly associated with alpha and beta-diversity supporting the cancer-microbiome-immune axis hypothesis.

Axillary dissection in sentinel lymph node positive breast cancer: Is the staging information worthwhile for patients?

The Z0011 randomized trial demonstrated no significant difference in axillary recurrence rate or survival with or without axillary dissection in patients with a positive sentinel node biopsy. However, there is continuing controversy regarding the generalizability of its results, and axillary dissection provides additional pathologic staging information that may guide adjuvant therapy. Thus, axillary dissection after positive sentinel node biopsy is being further investigated in an actively recruiting randomized trial. We elicited patients' preferences for axillary dissection versus no axillary dissection after positive sentinel node biopsy for early breast cancer. Patients who had undergone axillary dissection after positive sentinel node biopsy as part of breast conserving therapy were provided with a validated, self-rated questionnaire. The questionnaire comprised two trade-off questions to determine the maximum chance of developing arm side-effects from axillary dissection to justify the benefit of additional axillary staging information. Social, demographic, and clinical details were collected. Ninety-nine of the 126 eligible patients returned the questionnaire and 76 completed the trade-off assessment. The median age of participants was 62 years. The median numbers of sentinel and axillary nodes removed were 2 and 12, respectively. Forty-seven percent of participants had arm swelling or tenderness of any severity. Seventy-five percent of participants would have axillary dissection even if the chance of arm side-effects like they had experienced was 100%. Most patients with early breast cancer preferred axillary dissection after positive sentinel node biopsy for the additional staging information even though there was no survival benefit from axillary dissection.

The time of sentinel lymph node biopsy and neoadjuvant chemotherapy in clinical negative axilla breast cancer: before or after?

The time of sentinel lymph node biopsy and neoadjuvant chemotherapy in clinical negative axilla breast cancer: before or after?

Molecular detection of Epstein-Barr virus in breast cancer among Sudanese female population: a case-control study

Background: Breast cancer is the most common cancer in women worldwide and in Sudan. Breast cancer occurs due to a multifactorial process and infection with an oncogenic virus has been recently investigated as a possible risk factor for breast cancer. For nearly two decades, studies have incriminated Epstein-Barr virus (EBV) in the etiology of breast cancer. However, the results are unconvincing, and their interpretation has remained a matter of debate. The aim of this study was to detect EBV in breast cancer biopsies obtained from Sudanese female patients. Methods: A descriptive, hospital-based, case-control study, conducted at Faculty of Medical Laboratory Science, University of Khartoum, Khartoum, Sudan. Archival blocks were obtained from 115 patients with breast cancer and 115 controls during the period between November 2016 till March 2017. Results: Among 115 breast cancer tissue specimens, EBV DNA was identified in 42/115 (36.5 %) samples and was not identified in 73/115 (63.5 %) tissue samples. The highest frequency of EBV detection was among 41–60 year-olds (23/42, 54.7 %), followed by 21–40 year-olds (12/42, 28.5 %) and 61–80 year-olds (5/42, 11.9 %). In the control group, the majority were diagnosed with fibroadenoma (70.4%), followed by fibrocystic changes (10.4%) and lactating changes (0.9%). Conclusion: The data obtained in this study demonstrated that EBV was present in a high percentage of our study population; however, the exact role of EBV in Sudanese breast cancer needs to be studied more in depth.

Patterns in the Use of Axillary Operations for Patients with Node-Positive Breast Cancer After Neoadjuvant Chemotherapy: A National Cancer Database (NCDB) Analysis.

The American College of Surgeons Oncology Group (ACOSOG) Z1071 and Sentinel Neoadjuvant (SENTINA) trials of sentinel node biopsy for node-positive breast cancer treated with neoadjuvant chemotherapy (NAC) demonstrated false-negative rates that varied on the basis of surgical technique. This study evaluated trends in axillary operations before and after publication of these trials. This study analyzed patients from National Cancer Database (NCDB) with clinical T0 through T4, N1 and N2, M0 breast cancer who received NAC from 1 January 2012 to 31 December 2015 and sentinel lymph node biopsy (SNB) or axillary lymph node dissection (ALND). The patients were divided into the following groups: SNB, ALND, and (SNB + ALND). Of the 32,036 evaluable patients identified in this study. 5565 had SNB, 19,930 had ALND, and 6541 had SNB + ALND. Compared with the ALND group, the SNB group was younger, had more invasive ductal cancers, and had lower clinical T- and N-stage disease (p < 0.001). The patients in the SNB group had a higher rate of estrogen receptor-positive and triple-negative breast cancers, but a lower rate of human epidermal growth factor receptor 2 (HER2)-positive cancer (p < 0.001). The nodal pathologic complete response (PCR) rate, defined as no residual invasive cancer, was 66.5% in the SNB group and 33.1% in the ALND group. Since 2013, the rate of ALND has decreased from 88.7 to 77.1% in both community and academic institutions (p < 0.001). Since publication of the ACOSOG Z1071 and SENTINA trials, the national rates of ALND in node positive breast cancer treated with NAC have decreased despite reported false-negative SNB rates and lack of prospective outcome data regarding the oncologic safety of ALND omission.

Increased Soluble CrkL in Serum of Breast Cancer Patients Is Associated with Advanced Disease

Over-expression of Crk-like protein (CrkL), an intracellular adaptor protein, in breast cancer biopsies has been linked to poor prognosis. CrkL can be secreted from cancer cells binding to β1 integrin on the cell membrane. In this study, we evaluated, for the first time, the levels of soluble CrkL in serum of breast cancer patients. Expression of CrkL and secreted fractions from human breast cancer cell lines and clinical patient samples were assessed by immunohistochemistry and Enzyme Linked Immuno-Sorbent Assay (ELISA). CrkL levels in tissues and sera of patients with different disease stages were compared and statistically analyzed by Chi-square test and Student’s t-test. Culture media from human breast cancer cell lines SUM159, MDA-MB231, and MCF7 showed over a 21-, 15-, and 11-fold higher concentration of soluble CrkL as compared to normal breast epithelium cell line MCF10A. Expression of CrkL was elevated in 85% of breast tumor tissue sections. Serum levels of CrkL were significantly higher in breast cancer patients than in healthy donors. All patients with metastatic disease had significantly elevated concentration of soluble CrkL in the serum with on average three-fold increase from the baseline. The data suggest that soluble fraction of CrkL can be further evaluated as a serum biomarker for advanced disease in breast cancer patients.

Abstract LB-224: Tyrosine phosphorylation of p27Kip1 associates with Palbociclib responsiveness in breast cancer

Abstract PURPOSE: Cdk4 inhibitors (CDK4i), such as palbociclib, are approved in combination with hormonal therapy as a front line treatment for metastatic HR+, Her2- breast cancer. However, despite these advances, many patients demonstrate either de novo or acquired resistance. This highlights the need to identify biomarkers to pinpoint patients who would respond to this therapy. In Rb+ tumors, which comprise the majority of HR+ breast cancers, Ki67, Cyclin D, cdk4, and p16 levels do not identify responsive subgroups. Cyclin D or cdk4 levels themselves are not reliable because tyrosine phosphorylated p27Kip1 (pY88) is required for activation of the cyclin D-cdk4 (DK4) complex. We have previously shown that the level of pY88 correlates with cdk4 activity: the more pY88 detected, the greater the specific activity of cdk4 (Patel, et. al. MCR, 2018). We developed dual immunohistochemical staining for p27 and pY88 and demonstrated that pY88 status, which is negative in benign breast epithelium, stratified tumors across all hormone receptor groups (Gottesman, et.al, MCR, 2018). Lack of pY88 suggested that DK4 was inactive, and that these samples would not have the active cdk4 target. We have grown tumor resection material in explant culture, treated with palbociclib, and stained with Ki67 as a marker of response. Explants from the no pY88 group were non-responsive, while explants from the low or high pY88 group responded to drug. Thus, we hypothesized the pY88-p27 status may serve as a biomarker for patients that can respond to cdk4i therapy. RESULTS: We analyzed paraffin-embedded archival breast cancer biopsies from a 13 patient cohort of HR+, Her2- patients who had received Palbociclib/Letrozole in the front line metastatic setting. 62% of these patients progressed within 8 months of treatment, while 38% were on drug for 12-38 months. Using our staining method, we stratified patients into three groups based on pY88 status: 23% had no staining (Group 0), 38% had a low percentage of pY88+ cells (Group 1), and 38% had a high percentage of pY88+ cells (Group 2, &amp;gt;30% of cells pY88+). We then compared the time until disease progression, when they were removed from drug, for each group. Patients with a pY88 score of 0 had disease progression within 6-8 months, suggesting that they exhibited primary resistance. Patients with a pY88 score of 2 also showed disease progression within 6 months. This suggested that the Y88 high group may have too much active cdk4 target to respond to pharmacological drug dose, a hypothesis supported by our breast cancer cell line data. However, patients with a low pY88 score were all still on drug after 12+ months with either reduced tumor or stable disease. Additional patients continue to be recruited. CONCLUSION: Our data suggest that pY88-p27 status, as a surrogate marker for cdk4 activity, associates with responsiveness to CDK4i treatment. Clinical use of the pY88 biomarker may identify patients responsive or resistant to Cdk4 targeting drugs. Citation Format: Stacy W. Blain, Susan R.S. Gottesman, Jonathan Somma, Vladislav Tsiperson, Julia McGuinness, Matthew Ingham, Kevin Kalinsky, Gary Schwartz, Elina Tress, Hanina Hibshoosh, Mohamed Mh Kahila, Bachar Samra Samra, Evelyn Taiwo, Steve Xie. Tyrosine phosphorylation of p27Kip1 associates with Palbociclib responsiveness in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-224.

Human cytomegalovirus infection is correlated with enhanced cyclooxygenase-2 and 5-lipoxygenase protein expression in breast cancer

PurposeWhile enhanced expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) and their derived metabolites is associated with breast cancer (BC) risk, the precise link between BC carcinogenesis and enhanced inflammatory activity remains to be clarified. Human Cytomegalovirus (HCMV) may induce expression of COX-2 and 5-LO and is frequently found in breast cancer biopsies. Thus, we investigated whether there is an association between HCMV proteins and expression of COX-2 and 5-LO in human BC tissue and BC cell lines.Materials and methodsParaffin embedded biopsies obtained from 49 patients with breast cancer and 26 tissue samples from adjacent, benign breast tissues were retrospectively examined for HCMV-immediate early (IE), HCMV-Late (LA), COX-2, and 5-LO proteins by immunohistochemistry. In vitro, uninfected and HCMV-infected BC cell lines were examined for COX-2 and 5-LO transcripts and proteins by PCR and flow cytometry.ResultsExtensive expression of COX-2, 5-LO and HCMV-IE proteins were preferentially detected in BC samples. We found a statistically significant concordant correlation between extensive HCMV-IE and COX-2 (P < 0.0001) as well as with HCMV-IE and 5-LO (P = 0.0003) in infiltrating BC. In vitro, HCMV infection induced COX-2 and 5-LO transcripts and COX-2 proteins in MCF-7 cells (P =0.008, P =0.018, respectively). In MDA-MB-231 cells that already had high base line levels of COX-2 expression, HCMV induced both COX-2 and 5-LO proteins but not transcripts.ConclusionOur findings demonstrate a significant correlation between extensive HCMV-IE protein expression and overexpression of COX-2 and 5-LO in human breast cancer.

Methylene blue 1% as a sensitive and safe alternative for sentinel lymph node biopsy in early stage breast cancer: Results of a large pilot study.

Methylene blue 1% as a sensitive and safe alternative for sentinel lymph node biopsy in early stage breast cancer: Results of a large pilot study.

Increased expression of the HDAC9 gene is associated with antiestrogen resistance of breast cancers.

Estrogens play a pivotal role in breast cancer etiology, and endocrine therapy remains the main first line treatment for estrogen receptor‐alpha (ERα)‐positive breast cancer. ER are transcription factors whose activity is finely regulated by various regulatory complexes, including histone deacetylases (HDACs). Here, we investigated the role of HDAC9 in ERα signaling and response to antiestrogens in breast cancer cells. Various Michigan Cancer Foundation‐7 (MCF7) breast cancer cell lines that overexpress class IIa HDAC9 or that are resistant to the partial antiestrogen 4‐hydroxy‐tamoxifen (OHTam) were used to study phenotypic changes in response to ER ligands by using transcriptomic and gene set enrichment analyses. Kaplan–Meier survival analyses were performed using public transcriptomic datasets from human breast cancer biopsies. In MCF7 breast cancer cells, HDAC9 decreased ERα mRNA and protein expression and inhibited its transcriptional activity. Conversely, HDAC9 mRNA was strongly overexpressed in OHTam‐resistant MCF7 cells and in ERα‐negative breast tumor cell lines. Moreover, HDAC9‐overexpressing cells were less sensitive to OHTam antiproliferative effects compared with parental MCF7 cells. Several genes (including MUC1, SMC3 and S100P) were similarly deregulated in OHTam‐resistant and in HDAC9‐overexpressing MCF7 cells. Finally, HDAC9 expression was positively associated with genes upregulated in endocrine therapy‐resistant breast cancers and high HDAC9 levels were associated with worse prognosis in patients treated with OHTam. These results demonstrate the complex interactions of class IIa HDAC9 with ERα signaling in breast cancer cells and its effect on the response to hormone therapy.

Diagnostic accuracy of the Videssa protein-based liquid biopsy for breast cancer in suspicious mammography.

e13034 Background: The Videssa protein-based blood test (Provista Diagnostics) is a combined proteomic biomarker assay that aims to detect established breast cancer – rendering it useful in patients with abnormal or difficult-to-interpret mammograms. Since the incidence of malignancy for BI-RADS 4 biopsies is approximately 20%, a significant percentage of patients undergo needle biopsy unnecessarily. The Videssa assay provides an alternative diagnostic method by way of a non-invasive liquid biopsy. Methods: Our goal was to assess whether this liquid biopsy combined with 3D tomographraphic mammography could accurately predict disease status to reduce the amount of unnecessary tissue biopsies. An IRB-approved, prospective, single-arm study had patients with BI-RADS4 lesions with calcifications requiring tissue biopsy undergo the blood test prior to stereotactic core biopsy. Subsequent results and biopsy pathology were correlated. Results: 46 patients were initially entered with BI-RADS4 calcifications. 9 patients had DCIS (19.6%) and 37 patients had benign calcifications (80.4%). No patient had invasive cancer. Liquid biopsy results were elevated in 2 patients with DCIS (22% sensitivity) and in 5 patients with benign biopsies (10.8%). At interim analysis, the liquid biopsy demonstrated a 28.5% positive predictive value (PPV) and 82% negative predictive value (NPV). Tumor grade did not affect results. We subsequently discontinued using the blood test for BI-RADS4 calcifications and enrolled 13 patients with non-palpable solid lesions to determine the correlation. The liquid biopsy was elevated in 1 of 4 patients with invasive cancer (25% sensitivity) and was not elevated in all 9 patients with benign biopsies (100% NPV). At one-year follow-up, 3 of the 5 patients with an elevated blood assay and negative biopsy have had no incidence of cancer on subsequent imaging. The remaining 2 patients are scheduled for follow-up. Conclusions: In our study, the NPV of the Videssa liquid biopsy is not robust enough to defer tissue biopsy in any patient with indeterminate calcifications on imaging, nor is it specific enough to help predict results in high risk solid lesions. We do not see this liquid biopsy changing our current practice.

Predicting response to neoadjuvant chemotherapy in nonmetastatic hormone receptor-positive breast cancer using 21-gene Breast Recurrence Score test.

e12093 Background: The Recurrence Score (RS) result based on the 21-gene Oncotype DX Breast Recurrence Score assay is standard of care in deciding adjuvant chemo-hormonal therapy versus hormone therapy alone in hormone-receptor positive (HR+), HER 2 negative, node–negative breast cancer. This study explores the role of RS result in predicting the response to neoadjuvant chemotherapy (NACT). Methods: In this retrospective single institution cohort study, electronic medical records of 148 women with HR+, HER 2 negative, non-metastatic breast cancer who received NACT from 2006 onward were screened. 38 patients were excluded due to lack of tissue for testing. Pretreatment biopsy blocks were sent to Genomic Health, Inc. for Oncotype Dx testing. Low RS result was defined as ≤25. Pathologic complete response (pCR) was defined as no residual tumor. Partial response (PR) was residual tumor with &gt; 25% decrease in the largest dimension. No response (NR) was defined as &lt; 25% decrease in the tumor post NACT. Progression (PD) was defined as increase in size of original tumor or new site(s) of disease. Results: Of the 110 patients studied, 58% were postmenopausal women. Fifty percent were African American, 12% were Caucasian and 27% were Hispanic. Invasive ductal carcinoma was the predominant histology (86%). Most patients had &gt; T2 disease (97%) with 73% being clinically node positive. Adriamycin based NACT regimen was used in treating 86.3% of the women. Forty patients (36.4%) had tumor with RS≤25. NR/PD was significantly higher in tumors with RS≤25 (27/40) vs RS &gt; 25 (13/70) (OR: 9.1, 95% CI: 3.7-22.2, P&lt; 0.001). pCR was seen in 16% with RS &gt; 25 and 0% with RS ≤25. Response to NACT (pCR/PR) was 32.5% in RS≤25 vs 81.4% in RS &gt; 25. In tumors with response, RS &gt; 25 was associated with a greater percent decrease in the tumor size compared to RS≤25 (median decrease of 71% vs 52%, P= 0.033). Conclusions: HR+, HER 2 negative, RS≤25 breast cancer is associated with increased rates of NR/PD and is unlikely to respond to NACT. Recurrence Score result determination in pretreatment breast cancer biopsy samples can be an effective tool to select patients with non-metastatic breast cancer for NACT. Studies are needed to determine novel neoadjuvant therapeutic approaches in patients who are candidates for neoadjuvant therapy but are unlikely to benefit from NACT.

Heterogeneity in signaling pathway activity within primary breast cancer and between primary and metastases.

589 Background: Treatment with targeted drugs aims to block tumor driving signaling pathway(s). Drug choice is often based on a single preoperative primary breast cancer biopsy. It is important that biopsied cancer tissue is representative for the primary tumor (PT) or metastases to treat. Little is known about pathway heterogeneity within the PT, and between PT and metastatic tumors. A novel analysis method was developed to identify and quantify activity of signal transduction pathways in cancer tissue, based on Bayesian models that infer a pathway activity score from transcription factor target gene mRNA levels (Cancer Res 2014;74:2936-45). Methods: Pathway analysis, originally developed for AffymetrixU133Plus2.0, was adapted to RT-qPCR for use on formalin fixed paraffin embedded tissue. Estrogen (ER) and androgen (AR) receptor, PI3K, Hedgehog (HH), TGFβ and Wnt pathway activities were analyzed. Samples were from multiple locations (“quadrants”) in 15 luminal A, 9 luminal B, and 8 ER-negative primary breast cancers; from subdivided quadrant samples (4 “subquadrants”) of respectively 9, 4, and 4 PTs; and from 13 distant and 24 lymph node (LN) metastases of respectively 9 and 7 matched luminal PTs. Analysis of pathway activity score (PAS) variance was performed with linear mixed models with subgroup-dependent standard deviations. Results: In primary breast cancer intra-tumor PAS variance was not larger at macroscale (“quadrant”) than at microscale (“subquadrant”). For ER, AR, HH, and Wnt pathways, PAS variation was higher between distant metastases and PT than within the PT (p &lt; 0.0002). For HH and Wnt pathways, PAS variation was higher between LN metastases than within the PT (p &lt; 0.002). Correlation between primary and metastatic pathway activities ranged from -0.34 for ER to 0.47/0.50 for TGFβ/HH pathways. Conclusions: A single location tissue sample was representative for the whole primary tumor with respect to signaling pathway activity, suggesting one biopsy as generally sufficient for (neo)adjuvant therapy choice. Pathway activities varied between primary cancer and metastases, indicating the necessity of metastatic sample analysis (biopsies or liquid biopsy) to improve therapy choice.