The Role of Post-Menopausal Osteoporosis in Bone Metastatic Breast Cancer and the Effect of Monocyte Chemoattractant Protein-1

Background: Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer compared to other breast cancer subtypes. The frequency of TNBC expression is highest in young African-American women, leading to significant cancer health disparity in this population. Furthermore, TNBC is difficult to treat due to lack of known receptor targets at protein or gene level. Hence, it is imperative to identify novel therapeutic strategies for treatment of TNBC. Here we aim to show that the Monocyte Chemoattractant Protein -1 (MCP-1) is a reliable biochemical marker to assess TNBC progression. Experimental Design: We employed ELISA method to measure secreted MCP-1 in cell conditioned media, and real-time PCR to determine the mRNA status of MCP-1 in different TNBC cell lines. Boyden chamber assay was used to determine the effect of recombinant MCP-1 on cellular invasiveness. Immunohistochemistry staining was utilized for detecting protein of interest in tissue samples from breast cancer patients. MCP-1 knockdown was performed using lentiviral vector with shRNA targeting MCP-1 coding regions. RNAseq was performed with recombinant human MCP-1. Results: Our data show that MCP-1 is upregulated in TNBC cell lines, both transcriptionally and in secreted protein levels compared to ER-positive cell line, MCF-7. Breast cancer patients also showed high expression of MCP-1. MCP-1 treatment induced MDA-MB-231 and MCF-7 cell invasion, without affecting cell proliferation. Small-molecule antagonists against chemokine receptor 2 (CCR2), cognate receptor for MCP-1, and the MAP kinase pathway inhibitor U0106 negatively affected MDA-MB-231 cell invasion as measured by the Boyden chamber assay. This suggests that MCP-1-CCR2 axis may regulate invasiveness via the MAP kinase pathway. Knocking down MCP-1 by shRNA decreased cell invasion in TNBC cell line, BT-549, along with downregulation of key epithelial-to-mesenchymal transition markers, N-cadherin and Vimentin. Recombinant MCP-1 treatment in TNBC MDA-MB-231 cells upregulated genes associated with cytokine signaling. Conclusion: Our study suggests that a high MCP-1 level in TNBC cells may be responsible for increase in cell invasion via the MAP kinase pathway. Thus, MCP-1-mediated pathways could be potential therapeutic targets for the treatment of TNBC and reduce cancer health disparities. Citation Format: Pranabananda Dutta, Kimberly Paico, Yanyuan Wu, Marianna Sarkissyan, Jaydutt Vadgama. Tumor-derived MCP-1 regulates invasiveness in triple-negative breast cancer via the MAP kinase pathway [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B55.

Objective To investigate the effect of curcumin on tumor necrosis factor-alpha (TNF-α)-in-duced expression and release of monocyte chemoattractant protein-1 (MCP-1) in rat astrocytes.Methods The primary astrocytes were prepared from the cerebral cortex of 5 neonatal Sprague-Dawley rats and cultured.The cultured cells were identified by immunofluorescence staining with glial fibrillary acid protein.The cells were then divided into 6 groups (n =15 each):control group (group C),TNF-α group (group T),TNF-α+ different concentrations of curcumin groups (Cur5,Cur10 and Cur20 groups),and TNF-α+ solvent control group (D group).TNF-α with the final concentration of 20 ng/ml was added and the cells were incubated for 2h in T group.In Cur5,Cur10 and Cur20 groups,curcumin with the final concentrations of 5,10 and 20μmol/L was added,respectively,the cells were incubated for 24h and then the culture medium was abandoned,TNF-α with the final concentration of 20 ng/ml was added and the cells were then incubated for another 2h.In group D,dimethyl sulfoxide with the final concentration of 1 μl/ml was added,the cells were then incubated for 24h,then TNF-α with the final concentration of 20 ng/ml was added and the cells were incubated for another 2h.After treatment in each group,the expression of MCP-1 was determined by immunohistochemistry and the release of MCP-1 was determined by ELISA.Results Compared with group C,the expression and release of MCP-1 was significantly increased in the other five groups (P ＜ 0.05).Compared with group T,the expression and release of MCP-1 was significantly decreased in group Cur20 (P ＜ 0.05),and no significant changes in the expression and release of MCP-1 were found in Cur5,Cur10 and D groups (P ＞ 0.05).Compared with group Cur5,the expression and release of MCP1 was significantly decreased in group Cur20 (P ＜ 0.05),and no significant change in the expression and release of MCP-1 was found in group Cur10 (P ＞ 0.05).Conclusion Curcumin can inhibit TNF-α-induced expression and release of MCP-1 in rat astrocytes and the effect is dose-related and may be one of the mechanisms of curcumin-induced reduction of neurophathic pain. Key words: Curcumin; Astrocytes; Chemokine CCL2 ; Tumor necrosis factor-alpha

The proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in inflammatory and atherosclerotic lesions. GM-CSF is known to enhance monocytic expression of monocyte chemoattractant protein-1 (MCP-1). However, the molecular mechanism(s) by which GM-CSF up-regulates the MCP-1 expression remains to be clarified. Thus, in this study, we examined our hypothesis that GM-CSF up-regulates the MCP-1 expression via Jak2-Stat5 signaling pathway. In human monocytic cell line U937, GM-CSF increased MCP-1 expression in protein and mRNA levels. Furthermore, analysis of the GM-CSF promoter element revealed that the STAT5 (signal transducer and activator of transcription-5) transcription factor binding site, located between -152 and -144 upstream of the transcription start site, as well as Janus kinase-2-mediated Stat5 activation were necessary for the GM-CSF-induced transcriptional up-regulation of the MCP-1 gene. This GM-CSF-induced MCP-1 expression, measured as both protein and mRNA levels, was down-regulated by atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor. However, this decrease in MCP-1 expression was not at the transcriptional level of MCP-1 gene but rather at the level of the stability of MCP-1 mRNA. These results indicate that GM-CSF regulates MCP-1 expression via Janus kinase-2-Stat5 pathway and by a novel regulatory mechanism of statins to reduce inflammatory reactions by down-regulating the expression of monocytic MCP-1, which promotes atherogenesis.

Monocyte chemoattractant protein-1 (MCP-1) has been demonstrated to play a role in tumor progression. The present study examined the MCP-1 expression of colorectal liver metastases and determined whether MCP-1 is related to tumor progression and is a predictive marker for survival after hepatic resection of colorectal liver metastases. Eighty-seven patients with colorectal liver metastases were evaluated by immunohistochemistry of MCP-1, Angiopoietin-2, CD68, and CD34 for determination of microvessel density. Clinicopatholgical data were also examined. In a separate experiment, immunohistochemistry of MCP-1 was performed to investigate the expression of primary colorectal tumor according to the clinical stage. MCP-1 mRNA expression was determined in colorectal cancer cell lines. Forty-nine patients (56%) showed high expression of MCP-1 of colorectal liver metastases. High MCP-1 expression was related to multiple colorectal liver metastases. When the degree of MCP-1 expression increased, microvessel density count significantly increased compared with low MCP-1 expression. The MCP-1 expression correlated with Angiopoietin-2 expression. MCP-1 expression of the primary colorectal cancer increased as the clinical stage advanced. The increased MCP-1 mRNA expression was observed in cancer cell lines which have high metastasis potency. Univariate analysis demonstrated that the timing of metastases, tumor size, number of metastases, and MCP-1 expression were significant prognostic factors. Multivariate analysis demonstrated that MCP-1 expression was a significant prognostic factor in hepatic disease-free survival. The MCP-1 expression in colorectal liver metastases, at least in part, may be associated with angiogenesis and be a predictive marker for hepatic recurrence after hepatic resection for colorectal liver metastases.

In addition to its chemotactic properties, recent evidence suggests that monocyte chemoattractant protein 1 (MCP-1) might participate in the fibrotic process by inducing the secretion of extracellular matrix (ECM) components. Since the factors that initiate the accumulation of inflammatory infiltrates and ECM deposits in systemic sclerosis (SSc) skin lesions are still unknown, this study was undertaken to examine the role of MCP-1 in SSc. In situ hybridization and immunohistochemistry studies for MCP-1 were performed on skin biopsy specimens from patients with SSc and healthy controls. To identify possible stimulators of MCP-1 overexpression in SSc lesions, cultured dermal fibroblasts were incubated with recombinant platelet-derived growth factor (PDGF) and analyzed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. The chemotactic effects of SSc fibroblasts were examined using a modified Boyden chamber assay. To analyze the fibrotic potential of MCP-1, cultured dermal fibroblasts were incubated with recombinant MCP-1, and type I procollagen was measured by radioimmunoassay and real-time PCR. MCP-1 was expressed by fibroblasts, keratinocytes, and perivascular infiltrates throughout the skin, in involved as well as uninvolved skin areas, from 10 of 11 SSc patients, whereas no expression of MCP-1 was found in healthy controls. Stimulation with PDGF resulted in a significant increase in MCP-1 messenger RNA and protein, with differences between healthy control fibroblasts and fibroblasts from SSc patients. The chemotactic activity for peripheral blood mononuclear cells of SSc fibroblast supernatants increased in a time-dependent manner. Antibodies blocking MCP-1 decreased the chemotactic activity of SSc fibroblasts by a mean +/- SD of 37 +/- 12%. Despite an increase in type I collagen levels over time, no effect of recombinant MCP-1 on the synthesis of type I collagen was observed. These data indicate that MCP-1 might contribute to the initiation of inflammatory infiltrates in SSc. Possible stimuli of MCP-1 in dermal SSc lesions include PDGF, which is known to be expressed in SSc. In contrast to previous findings in fibrotic lung diseases, no effect of MCP-1 on collagen synthesis was observed in SSc dermal fibroblasts in vitro.

Expression of monocyte chemoattractant protein-1 (MCP-1) and CC chemokine receptor 2 (CCR2) and its significance has been demonstrated in some cancer cells in recent clinical studies. However, the role of tumor MCP-1 and CCR2 expression in non-small cell lung cancer (NSCLC) remains unknown. The aim of the present study was to investigate the prognostic significance of MCP-1 and CCR2 expression in NSCLC cells. The relationship between MCP-1 and CCR2 expression in NSCLC cancer cells was examined by immunohistochemical staining of surgical specimens from 134 patients. Sixty-five of these patients had follow-up records. Kaplan-Meier analysis and Cox regression model were used to assess overall survival according to the presence or absence of MCP-1 and CCR2 expression in tumor cells. MCP-1 was detected in cancer cells of 107 NSCLC (79.9 %) and CCR2 was detected in cancer cells of 39 NSCLC (29.1 %). MCP-1 expression was correlated with sex, smoking habits, histology, and tumor size. Presence of MCP-1 in tumor cells was associated with better overall survival (P = 0.018). By multivariate analysis, MCP-1 expression in cancer cells showed an independent prognostic factor for overall survival (P = 0.002, hazard ratio [HR] = 0.256, 95 % confidence interval [CI] = 0.106-0.616). There was no significant relationship between CCR2 expression in tumor cells and clinical and pathological characteristics. Also, no significant positive correlation between MCP-1 and CCR2 expression was revealed by Spearman correlation analysis. Our data indicate that MCP-1 is overexpressed in NSCLC cells. Its expression in cancer cells is associated with better survival in NSCLC patients.

BMP7: A New Bone Metastases Prevention?

Background: Triple negative breast cancer (TNBC) poses a critical problem for targeted therapy due to lack of significant expression of estrogen, progesterone receptor or Her2/neu oncogene. Hence, it is imperative to identify novel therapeutic strategies to target TNBC. Our study is aimed to examine whether Monocyte Chemoattractant Protein -1 (MCP-1) is a specific marker for TNBC metastasis. Experimental Design: We employed ELISA to determine secreted MCP-1 in cell conditioned media, as well as Real-time PCR to determine the status of MCP-1 in TNBC cell lines. Boyden chamber assay was used to determine the effect of recombinant MCP-1 on cellular metastasis. Cellular proliferation was measured with MTT assay. Immunofluorescence staining was utilized for protein of interest in breast cancer cells. MCP-1 knockdown was performed using lentiviral vector with shRNA targeting MCP-1 coding regions. Results: Our data show that the key inflammatory chemokine MCP-1 is upregulated in TNBC cell lines both transcriptionally as well as in terms of secretion compared to ER-positive cell line, MCF-7. MCP-1 stimulation in MDA-MB231 and MCF-7cells does not affect cellular proliferation. However, MCP-1 enhances metastatic properties of MDA-MB-231 cells along with BT-549 cells. Inhibiting Chemokine receptor 2/4 (CCR2/4), cognate receptor for MCP-1, with small molecule antagonists negatively affects invasiveness in MDA-MB-231 cells as evidenced by Boyden chamber assay. Knocking down MCP-1 by shRNA decreases cell invasion in TNBC cell line, BT-549 along with downregulation of key epithelial to mesenchymal transition markers, N-cadherin and Vimentin. MCP-1 induced cell invasion in TNBC may involve activation of p44/p42 MAPK Thr202/Tyr204. Conclusion: Our study suggests that high MCP-1 levels in TNBC is driving up metastasis potential in cells. Thus MCP-1 and its mediated pathways could be potential therapeutic targets for the treatment of TNBC. Citation Format: Pranabananda Dutta, Kimberly Paico, Inez Yuwanita, Yanyuan Wu, Marianna Sarkissyan, Jaydutt Vadgama. Upregulation of MCP-1 regulates invasiveness in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1960. doi:10.1158/1538-7445.AM2017-1960

Background: In patients with early breast cancer, neoadjuvant therapy is widely performed as standard of care. While the molecular targeting strategy makes progress in HER2 positive breast cancer, the optimal regimens for ER+/HER2- breast cancer (BC) and triple negative breast cancer (TNBC) remain undecided. Furthermore, there are few strategies for patients who do not achieve complete pathological response (pCR) who have worse prognosis. To address these unmet clinical needs, there is urgent need to examine the genomic alterations and immune microenvironment of residual tumors after neoadjuvant therapy. Here, we conducted a comprehensive analysis integrating genomic, transcriptomic, and clinical data to investigate the difference between primary breast cancer and residual disease. Materials and Methods: Using the large prospectively ascertained ethnically diverse Chicago Multi-Ethnic (ChiMEC) cohort of 562 participants with integrated genomic data, we identified 176 patients with breast cancer who underwent sequencing with Tempus xT next-generation sequencing panel including DNA- and whole-transcriptome RNA-sequencing. These included 131 primary breast tumors and 45 residual tumors after neoadjuvant therapy. We compared mutation rates between primary tumor and residual tumor in ER+/HER2- BC and TNBC. We also investigated homologous recombination deficiency (HRD) scores, tumor mutational burden (TMB), degree of immune infiltration, and microsatellite instability (MSI), and their association with survival. Results: Out of the 176 patients, there were 72 ER+/HER2- BC, 42 TNBC, and 44 HER2 positive cases. Among patients with HR+/HER2- BC, residual tumors had higher mutation rates in PIK3CA (61% vs 33%), CDH1 (33% vs 17%), CCND1 (39% vs 7%), FGF3 (28% vs 0%), FGF4 (33% vs 2%), FGF19 (33% vs 2%), and GATA3 (44% vs 7%) than primary tumors, but lower rates in TP53 (28% vs 48%), MAP3K1 (6% vs 40%), KMT2D (0% vs 33%), MCL1 (0% vs 30%), SPEN (6% vs 20%), ZFHX3 (0% vs 20%), ARID1A (11% vs 16%), KMT2C (0% vs 19%), LZTR1 (0% vs 19%), BCORL1 (6% vs 17%), NOTCH3 (6% vs 17%), FAT1 (0% vs 17%) and LPR1B (0% vs 17%). Relative to primary TNBC tumors, residual TNBC tumors exhibited higher mutation rates in PTEN (10% vs 3%), CCNE1 (10% vs 3%), CIC (10% vs 0%), and KMT2D (10% vs 3%). Conversely, residual TNBC tumors had relatively lower rates of MCL1 (0% vs 21%), RB1 (0% vs 12%), CDH1 (0% vs 12%), KMT2C (11% vs 18%), and PIK3CA (0% vs 15%), CDKN1B (0% vs 12%) and ETV6 (0% vs 12%). There was a significant trend of higher TMB (>5.0 mutations/megabase, m/MB) associated with improved disease-free survival among the 176 patients (P=0.031). TMB was not significantly different between primary tumors and residual tumors, or across subtypes. Microsatellite instability (MSI) status was high in 3 patients (1.6%) and equivocal in 5 patients (2.7%). TMB in MSI-high or MSI-equivocal tumors was significantly higher than TMB in tumors with microsatellite stability (37.7m/MB vs 5.3m/MB, P<0.001). HRD scores were highest in TNBC, and lowest in ER+/HER2- breast cancers (P=0.022). There was no significant association between the HRD scores and survival. To date, tumors from 106 patients have undergone immune profiling, with estimation of immune cell infiltration, macrophages, B cells, CD4, CD8, and NK cells. Initial analysis showed no association between immune profiling and survival. Conclusion: These comprehensive analyses demonstrated that mutation status in HR+/HER2- breast cancers and TNBC differs between primary and residual tumors after neoadjuvant therapy. We identified genomic alterations and pathways in residual tumors that could be further explored as potential targets in the adjuvant setting to improve long term outcomes for patients who do not achieve pCR. Citation Format: Minoru Miyashita, Masaya Hattori, Yonglan Zheng, Toshio Yoshimatsu, Joshua SK Bell, Padma Sheila Rajagopal, Anna Woodard, Jean Baptiste Reynier, Elisabeth Sveen, Galina Khramtsova, Fang Liu, Abiola Ibraheem, Gini Fleming, Nora Jaskowiak, Rita Nanda, Benjamin Leibowitz, Nike Beaubier, Kevin White, Dezheng Huo, Olufunmilayo I Olopade. Unstable mutational profile and heterogeneity of residual breast tumor following neoadjuvant therapy from comprehensive genomic and transcriptomic sequencing [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-06.

Purpose: Metaplastic breast cancer (BC) is an uncommon yet aggressive histologic subtype of BC. We sought to identify factors associated with its diagnosis and compare the management and outcomes of metaplastic BC with those of other BCs and triple negative invasive ductal carcinoma in particular given how often it has a triple negative phenotype.Patients and Methods: We identified women diagnosed with invasive BC in 2010-2014 in the National Cancer Data Base, and used univariate analysis to compare baseline patient and tumor characteristics by BC subtype. Overall survival (OS) was estimated with the Kaplan-Meier method, and multivariate Cox proportional hazards models were used to identify independent predictors of OS.Results: Of 247,355 cases, 2,084 (0.8%) were metaplastic BC, 55,998 (23%) triple negative BC, and 77% other BC. Relative to non-metaplastic BC, women with metaplastic BC were more likely to be older at diagnosis (median age, 62 vs. 59 years), have ≥1 comorbid conditions (22% vs. 18%), and be on Medicare (41% vs. 33%; P<0.001). Metaplastic BCs tended to be basal-like (77%), and relative to triple-negative or other BC, metaplastic BC was associated with higher clinical T status (cT3-4, 18% vs. 11%, 8%), no clinical nodal involvement (cN0, 86%, 77%, 80%), no lymphovascular invasion (72%, 65%, 62%), and high-grade tumors (71%, 77%, 35%) (P<0.001). Most metaplastic BCs were treated with mastectomy (58%), sentinel lymph node dissection (65%), chest wall or breast irradiation (74%), and chemotherapy (75%) as adjuvant therapy (60%). At a median follow-up time of 44.5 months, OS rates were lower for metaplastic BC than for triple-negative or other BC across all clinical stages at 5 years (stage I, 85%, 87%, 91%; II, 73%, 77%, 87%; III, 43%, 53%, 75%) and at 3 years (Stage IV, 15%, 22%, 64%; P<0.001). On multivariate analysis, increasing age, advanced clinical stage, lymphovascular invasion, axillary (vs. sentinel) node dissection, and no radiation or chemotherapy were associated with worse outcomes in metaplastic BC. Extent of surgery affected survival for triple-negative and other BC but not for metaplastic BC.Conclusion: Outcomes for metaplastic BC continue to be worse than those for other BC subtypes despite modern treatments. Optimizing systemic therapy options, which was a significant predictor of survival, should be a priority in managing metaplastic BC.

Background: Triple negative breast cancer (TNBC) is a molecularly complex and heterogeneous subtype of breast cancer with distinct biological features and clinical behavior. TNBC is associated with increased risk of metastasis and recurrence. To identify the underlying transcriptional changes and differentially expressed genes in metastatic progression of TNBC, we compared the transcriptomic profile of primary and matched metastatic tumors using massively parallel RNA sequencing. Methods: We performed gene expression profiling using paraffin embedded formalin fixed tissues of 38 TNBC patients obtained from University Hospital Zurich, Switzerland and Stavanger University Hospital, Norway. Of these, 18 were primary TNBC tumors that did not metastasize; 20 patients metastasized to lymph nodes or to distant organs (16 paired primary and metastatic tumors). Absolute fold change of ≥ 2 and a p-value &amp;lt;0.05 was used as the cutoff to identify significant differentially expressed genes (DEGs). Functional analysis was performed on DEGs. We also compared gene-expression profiles for hypoxia related- and EMT-related genes between our comparison groups. CIBERSORT analysis was used to estimate relative fractions of 22 immune cell types in each RNA-seq sample. Results: We identified 2442 DEGs between primary TNBC tumors and corresponding metastatic lesions and 1561 DEGs between metastasis-free and metastatic primary tumors. Of the 2442 DEGs between primary and metastatic tumors, we found 579 genes to be upregulated and 1863 genes to be downregulated in primary tumors compared to matched metastatic tumors. A total of 530 genes were found to be upregulated and 1031 genes were downregulated in metastasis-free compared to metastatic primary TNBC tumors. Of the 27 gene hypoxia signature, only 1 gene (MPRS17) was significantly different between primary and corresponding metastatic tumors and 3 hypoxia-related genes (KCTD11, FOSL1 and SDC1) showed significant differences between primary tumors with and without metastasis. We found expression for 1 EMT related gene (TWIST1) to be different between primary and matched metastatic tumors whereas 4 EMT related genes (CDH2, VIM, SNAI1 and FOXC2) were found to be significantly different between primary tumors that metastasized compared to primary tumors that remained metastasis-free. The relative fractions of 3 immune cell types, T cells CD4 memory resting, M2 macrophages and M0 macrophage were similar between the primary tumor and their matched metastatic lesions. Conclusions: Our results show that distinct patterns of gene expression exist between primary TNBCs and matched metastatic tumors. Further studies are warranted to explore whether these discrete expression profiles underlie or derive from disease status. Citation Format: Jaspreet Kaur. Differential gene expression profiling of matched primary and metastatic triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 758.

Rationale and purpose: Triple negative breast cancers (TNBCs), characterized by lack of hormone receptors' expression in absence of HER2 amplification, partially overlap with the basal-like subtype, with frequent occurrence in BRCA1 mutation carriers (BRCA1+). TNBCs are often associated with earlier onset, interval cancer diagnosis, larger size and aggressive clinical course with peak risk of recurrence at 1-3 years and increased mortality rate in the first 5 years. Thus, when screening women at high risk of breast cancer, special attention should be paid to patient outcome for TNBCs in comparison with non-TNBCs. Our aim was to compare TNBCs and non-TNBCs diagnosed during a prospective, non-randomized, multimodality screening study – including clinical breast examination (CBE), mammography, ultrasound (US) and magnetic resonance imaging (MRI) – on women at familial/genetic high risk of breast cancer conducted in 18 centres from June 2000 to March 2008 (ISS-HIBCRIT-1; Sardanelli F et al, Invest Radiol 2011). Methods: Comparisons were performed using Mann-Whitney U, Fisher exact and χ2 tests. Results: Among the 44 patients diagnosed with invasive cancers, 14 (31%) were TNBCs and 30 (69%) non-TNBCs, the former being 13 invasive ductal (IDC) and 1 atypical medullary carcinoma, the latter also including 15/30 lobular subtypes and/or DCIS component (p=0.005). Of the 14 TNBCs, 10 (71%) were found in BRCA1+, 2 (14%) in BRCA2+ and 2 (14%) in BRCA-untested women with strong family history of breast/ovarian cancer; the same data for 30 non-TNBCs were 9 (30%), 6 (20%) and 15 (50%) respectively (p=0.028). We had only three interval cancers, all TNBCs. The median age at diagnosis was 49 years (range 36-62) for TNBCs and 53 years (range 35-72) for non-TNBCs (p=n.s). TNBCs presented a higher rate (11/14, 79%) of pathological grade 3 IDCs compared with non-TNBCs (8/30, 27%) (p=0.002). The mean tumor size was 1.6 cm for TNBCs and 1.2 cm for non-TNBCs (p=n.s). Nodal status was negative in 12/14 (86%) TNBCs and in only 16/30 (53%) non-TNBCs (p=0.038). MRI similarly outperformed CBE, mammography and US in both TNBCs and non-TNBCs. Clinical course and survival could be monitored for 40/44 patients (91%), 13 TNBCs and 27 non-TNBCs, with a follow-up of 5.8 and 6.3 years (p=n.s) respectively. The rate of disease-free patients for over 5 years was 8/13 (62%; mean disease-free interval 7.0 years, range 5.0-8.0) for TNBCs and 17/27 (63%; mean 7.2 years, range 5.2-9.9) for non-TNBCs. Death due to BC occurred for 2/13 TNBC (15%, at 3.5 and 4.2 years) and 3/27 non-TNBC patients (11%; at 2.0, 4.9 and 5.7 years). The rate of locoregional relapse was 1/13 (8%, at 4.4 years) and 5/27 (19%; mean time of 5.0 years, range 2.4-7.0) respectively. Distant recurrence was reported for only 2 non-TNBC patients. Conclusion: TNBCs showed stronger association with BRCA1+ status, lower rate of lobular subtypes or DCIS component, and less frequent nodal involvement. Despite a more frequent pathological grade 3 and the tendency to be diagnosed as interval cancers, under the current treatment protocols TNBCs showed relapse and BC-related death rates and over-5-year disease-free intervals similar to those of non-TNBCs. These data provide outcome evidence supporting the value of entering women at high risk of TNBC (in particular BRCA1+) in intensive screening programs including MRI. Citation Format: Franca Podo, Filippo Santoro, Siranoush Manoukian, Clelia de Giacomi, Laura Cortesi, Lorenzo Preda, Stefano Corcione, Francesco Sardanelli. High-risk patients found affected with breast cancer during a multimodality screening program: Triple negative versus non-triple negative breast cancers. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B13.

Background: Bone is the most common site of metastasis in breast cancer, and patients developing skeletal-related events show poor prognosis. Bone metastasis is a complex process involving interactions between many different molecules, which include receptors and proteins related to chemokine signaling, cell adhesion and migration, or bone metabolism regulation. We aimed to demonstrate the correlation between bone metastasis and expression of these molecules in breast cancer. Methods: Clinicopathologic parameters were collected from 1,440 stage I-III breast cancer patients (1,436 female and 44 male, median age 50 years, range 25-90) who underwent curative surgery at Seoul National University Hospital from October 2000 to December 2013. Immunohistochemistry (IHC) for CXCR4, CXCR6, MGP, osteocalcin, periostin, PTH1R, PTHLH and RANKL were performed on tissue microarray (TMA) constructed from the resected breast cancer tissue. The expression levels of each marker were assessed as H-scores, and we applied cut-off values of 0, 100, 200, and 300 to dichotomize into positive or negative. Results: There were 966 patients (67.1%) with hormone receptor (HR)-positive and HER2-negative breast cancer, 225 patients (15.6%) with HER2-positive breast cancer, and 249 patients (17.3%) with triple negative breast cancer (TNBC). As of February 2024, at median follow-up duration of 138 months, 196 patients (13.6%) had experienced recurrence. This included 65 patients with local recurrence and 131 patients with distant metastasis (49 patients had bone-only metastasis and 82 patients had visceral metastasis as well). There were 186 patients (12.9%) who died from any cause. CXCR6 H-scores were higher in HR-positive breast cancer (mean H-score 33.90) compared to HER2-positive breast cancer (mean H-score 15.58, p=0.0006) or TNBC (mean H-score 16.78, p=0.0056). CXCR6 positivity was significantly associated with longer overall survival (OS) (5-year survival rate (5YSR) 95.8% vs. 95.0%, p=0.049 for cutoff 0). In HR-positive breast cancer, CXCR6 positivity was also correlated with longer bone-specific relapse-free survival (RFS) (5YSR 98.0% vs. 96.8%, p=0.024 for cutoff 0). In contrast, CXCR6 positivity was correlated with shorter bone-specific RFS in HER2-positive breast cancer and TNBC across various cutoff values. RANKL positivity was correlated with shorter OS (5YSR 95.6% vs. 93.9%, p=0.049 for cutoff 200) and RFS (5YSR 92.8% vs. 88.5%, p=0.026 for cutoff 200). In contrast, bone-specific RFS prolonged as RANKL positivity increased (5YSR 97.4% vs. 96.8%, p=0.044 for cutoff 100). The positive correlation to bone-specific RFS was maintained in RANKL-positive, HR-positive breast cancer (5YSR 98.2% vs. 96.3%, p=0.018 for cutoff 100). However, in TNBC, the RANKL score was negatively correlated with RFS across various cutoff values. Periostin was significantly less expressed in TNBC (mean H-score 23.79) compared to other subtypes (mean H-score 48.37, p&amp;lt;0.001 for HR-positive breast cancer and mean H-score 49.14, p&amp;lt;0.001 for HER2-positive breast cancer). Periostin positivity was related to longer OS (5YSR 97.7% vs. 95.6%, p=0.03 for cutoff 0) and bone-specific RFS (5YSR 99.5% vs. 96.7%, p=0.014 for cutoff 100) in HR-positive breast cancer, but not in other subtypes. Conclusion: Expression levels of CXCR6, RANKL, and periostin were correlated with prognosis and bone-specific survival in breast cancer. Interestingly, the clinical impact of each marker differed with respect to each breast cancer subtypes, possibly suggesting a different underlying mechanism of bone metastasis in HR-positive breast cancer compared to the others. Further investigations into the differences between subtypes and establishing proper cutoff values are warranted. Citation Format: H. Yi, K. Lee, C. Park, D. Lee, S. Cho, J. Koh, H. Ryu, S. Im. Prognostic Role of CXCR6 and RANKL for Bone Metastasis in Early Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-01-18.

Epigenetic Regulation of Cancer Stem Cell Genes in Triple-Negative Breast Cancer

Aberrant monocyte chemoattractant protein-1 (MCP-1) and CC chemokine receptor 2 (CCR2) expression in malignant tissues have been reported; however, their role in hematological malignancies prognosis remains little known. The aim of this study was to investigate the prognostic value of MCP-1 and CCR2 expression in patients with diffuse large B cell lymphoma (DLBCL). The study included 221 patients with DLBCL. MCP-1 and CCR2 expression was analyzed by immunohistochemical staining and its correlations with clinicopathologic features and prognosis were evaluated. High expression of MCP-1 or CCR2 was correlated with clinicopathological characteristics, and an adverse prognostic factor for overall survival (OS) and progression-free survival (PFS) of DLBCL patients. Also, significant positive correlation between MCP-1 and CCR2 expression was revealed (r = 0.545, P < 0.001). Patients with high MCP-1 or high CCR2 expression had significantly poorer OS and PFS than those with low MCP-1 or low CCR2 expression (OS: P < 0.001, P < 0.001; PFS: P < 0.001, P < 0.001), respectively, even in the rituximab era, and MCP-1 or CCR2 expression could further identify high-risk patients otherwise classified as low/intermediate risk by the International Prognostic Index (IPI) alone. Furthermore, incorporation of MCP-1 or CCR2 expression into the IPI score could improve prognostic value for OS. This is the first report describing the clinicopathological features and survival outcome according to expression of MCP-1 and CCR2 in DLBCL.