Biomarker For Triple-negative Breast Cancer Research Articles

Immunotherapy in triple-negative breast cancer: Insights into tumor immune landscape and therapeutic opportunities.

Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer that represents 15–20% of breast tumors and is more prevalent in young pre-menopausal women. It is the subtype of breast cancers with the highest metastatic potential and recurrence at the first 5 years after diagnosis. In addition, mortality increases when a complete pathological response is not achieved. As TNBC cells lack estrogen, progesterone, and HER2 receptors, patients do not respond well to hormone and anti-HER2 therapies, and conventional chemotherapy remains the standard treatment. Despite efforts to develop targeted therapies, this disease continues to have a high unmet medical need, and there is an urgent demand for customized diagnosis and therapeutics. As immunotherapy is changing the paradigm of anticancer treatment, it arises as an alternative treatment for TNBC patients. TNBC is classified as an immunogenic subtype of breast cancer due to its high levels of tumor mutational burden and presence of immune cell infiltrates. This review addresses the implications of these characteristics for the diagnosis, treatment, and prognosis of the disease. Herein, the role of immune gene signatures and tumor-infiltrating lymphocytes as biomarkers in TNBC is reviewed, identifying their application in patient diagnosis and stratification, as well as predictors of efficacy. The expression of PD-L1 expression is already considered to be predictive of response to checkpoint inhibitor therapy, but the challenges regarding its value as biomarker are described. Moreover, the rationales for different formats of immunotherapy against TNBC currently under clinical research are discussed, and major clinical trials are highlighted. Immune checkpoint inhibitors have demonstrated clinical benefit, particularly in early-stage tumors and when administered in combination with chemotherapy, with several regimens approved by the regulatory authorities. The success of antibody–drug conjugates and research on other emerging approaches, such as vaccines and cell therapies, will also be addressed. These advances give hope on the development of personalized, more effective, and safe treatments, which will improve the survival and quality of life of patients with TNBC.

Personalised Therapies for Metastatic Triple-Negative Breast Cancer: When Target Is Not Everything.

Simple SummaryThe purpose of the present review is to shed light on new molecular biomarkers in triple-negative breast cancer (TNBC), showing emerging therapeutic approaches related to specific molecular signatures and their mechanisms of action. A general overview of ongoing clinical trials, future perspectives and differences in approval by American and European regulatory authorities is provided.Triple-negative breast cancer—defined by the absence of oestrogen/progesterone receptors and human epidermal growth factor receptor 2 expression—is a complex and heterogeneous type of tumour characterised by poor prognosis, aggressive behaviour and lack of effective therapeutic strategies. The identification of new biomarkers and molecular signatures is leading to development of new therapeutic strategies including immunotherapy, targeted therapy and antibody-drug conjugates (ADCs). Against a background where chemotherapy has always been considered the standard of care, evolution towards a precision medicine approach could improve TNBC clinical practice in a complex scenario, with many therapeutic options and new drugs. The aim of this review was to focus on emerging therapeutic targets and their related specific therapy, discussing available and emerging drugs, underlining differences in approval by American and European regulatory authorities and showing the future perspective in the large number of ongoing clinical trials.

LncRNA ACTA2-AS1 predicts malignancy and poor prognosis of triple-negative breast cancer and regulates tumor progression via modulating miR-532-5p

BackgroundDysregulation of ACTA2-AS1 and miR-532-5p and their functions in various cancers have been widely reported. Their potential of serving as biomarkers in triple-negative breast cancer (TNBC) remains unknown. This study aimed to evaluate the function of ACTA2-AS1 and miR-532-5p and their potential of serving as biomarkers in TNBC.ResultsThe TNBC tissues were collected from 119 patients, where the reduced level of ACTA2-AS1 and increased level of miR-532-5p were observed by PCR and showed a significantly negative correlation (P < 0.001). Both ACTA2-AS1 and miR-532-5p were closely associated with the malignant development and poor prognosis of TNBC patients. Moreover, in TNBC cell, overexpressing ACTA2-AS1 was found to suppress cell proliferation and metastasis, which was reversed by the upregulation of miR-532-5p.ConclusionsACTA2-AS1 and miR-532-5p could act as biomarkers of TNBC predicting the progression and prognosis of patients. ACTA2-AS1 served as a tumor suppressor of TNBC which was mediated by miR-532-5p.

Triple-Negative Breast Cancer circRNAome Reveals Hsa_circ_0072309 as a Potential Risk Biomarker.

Simple SummaryTriple Negative Breast Cancer (TNBC) is a highly aggressive type of cancer that lacks biomarkers for its early discovery, leading to overall poor prognosis after its diagnosis. Circular RNAs (circRNAs) are a new class of regulatory RNAs and are promising biomarkers for several human diseases, including TNBC. In this study, we profiled the expression of all circRNAs present in TNBC in order to identify new biomarkers for this disease and it was possible to observe that 16 were deregulated, among them hsa_circ_0072309. In two distinct sets of samples, hsa_circ_0072309 was able to distinguish TNBC from healthy controls, making it a promising risk biomarker for this disease. Additionaly, since circRNAs are known to interact with RNA-Binding Proteins (RBPs), we investigated its probable function in this cancer and found that by interacting with such RBPs, this circRNA is acting in several cancer-related biological pathways. Recognizing these differentially expressed circRNAs and identifying their role can lead to a better understanding of dysregulated pathways in TNBC and ultimately allow the development of personalized therapies in this molecular subtype of breast cancer. Circular RNAs (circRNAs) are a class of long non-coding RNAs that have the ability to sponge RNA-Binding Proteins (RBPs). Triple-negative breast cancer (TNBC) has very aggressive behavior and poor prognosis for the patient. Here, we aimed to characterize the global expression profile of circRNAs in TNBC, in order to identify potential risk biomarkers. For that, we obtained RNA-Seq data from TNBC and control samples and performed validation experiments using FFPE and frozen tissues of TNBC patients and controls, followed by in silico analyses to explore circRNA-RBP interactions. We found 16 differentially expressed circRNAs between TNBC patients and controls. Next, we mapped the RBPs that interact with the top five downregulated circRNAs (hsa_circ_0072309, circ_0004365, circ_0006677, circ_0008599, and circ_0009043) and hsa_circ_0000479, resulting in a total of 16 RBPs, most of them being enriched to pathways related to cancer and gene regulation (e.g., AGO1/2, EIF4A3, ELAVL1, and PTBP1). Among the six circRNAs, hsa_circ_0072309 was the one that presented the most confidence results, being able to distinguish TNBC patients from controls with an AUC of 0.78 and 0.81, respectively. This circRNA may be interacting with some RBPs involved in important cancer-related pathways and is a novel potential risk biomarker of TNBC.

Biomimetic nanobubbles for triple-negative breast cancer targeted ultrasound molecular imaging

Triple-negative breast cancer (TNBC) is a highly heterogeneous breast cancer subtype with poor prognosis. Although anatomical imaging figures prominently for breast lesion screening, TNBC is often misdiagnosed, thus hindering early medical care. Ultrasound (US) molecular imaging using nanobubbles (NBs) capable of targeting tumor cells holds great promise for improved diagnosis and therapy. However, the lack of conventional biomarkers in TNBC impairs the development of current targeted agents. Here, we exploited the homotypic recognition of cancer cells to synthesize the first NBs based on TNBC cancer cell membrane (i.e., NBCCM) as a targeted diagnostic agent. We developed a microfluidic technology to synthesize NBCCM based on the self-assembly property of cell membranes in aqueous solutions. In vitro, optimal NBCCM had a hydrodynamic diameter of 683 ± 162 nm, showed long-lasting US contrast enhancements and homotypic affinity. In vivo, we demonstrated that NBCCM showed increased extravasation and retention in a TNBC mouse model compared to non-targeted NBs by US molecular imaging. Peak intensities and areas under the curves from time-intensity plots showed a significantly enhanced signal from NBCCM compared to non-targeted NBs (2.1-fold, P = 0.004, and, 3.6-fold, P = 0.0009, respectively). Immunofluorescence analysis further validated the presence of NBCCM in the tumor microenvironment. Circumventing the challenge for universal cancer biomarker identification, our approach could enable TNBC targeting regardless of tumor tissue heterogeneity, thus improving diagnosis and potentially gene/drug targeted delivery. Ultimately, our approach could be used to image many cancer types using biomimetic NBs prepared from their respective cancer cell membranes.

Circular RNAs as novel biomarkers in triple-negative breast cancer: a systematic review.

More effective prognostic and diagnostic tools are urgently required for early detecting and treating triple-negative breast cancer, which is the most acute type of breast cancer because of its lower survival rate, aggressiveness, and non-response to various common treatments. So, it remains the most harmful malignancy for women worldwide. Recently, circular RNAs, as a group of non-coding RNAs, with covalently closed loop and high stability have been discovered, which can modulate gene expression through competing with endogenous microRNA sponges. This finding provided further insight into novel approaches for controlling genes affected in many disorders and malignancies. This review concentrates on the dysregulated expression of circRNAs like their diagnostic and prognostic values in TNBC. This review aims to focus on the abnormal expression of circRNAs and their diagnostic and prognostic values in TNBC. We used PubMed, Embase, and Web of Science databases and ClinicalTrials.gov to systematically search for all relevant clinical studies. This review is based on articles published in databases up to April 2022 with the following keywords: "Circular RNA", "CircRNA", "Triple-Negative Breast Cancer" and "TNBC". We conducted a review of published CircRNA profiled-research articles to identify candidate CircRNA biomarkers for TNBC. The review is registered on JBI at https://jbi.global/systematic-review-register . Accumulating evidence has shown that several circRNAs are downregulated and some are upregulated in TNBC. The results of these studies confirm that circRNAs might be potential biomarkers with the diagnostic, prognostic, and therapeutic target value for TNBC. We also consider the connection between circRNAs and TNBC cell proliferation, apoptosis, metastasis, and chemotherapy resistance and sensitivity.

Effector memory cytotoxic CD3+/CD8+/CD45RO+ T cells are predictive of good survival and a lower risk of recurrence in triple-negative breast cancer

Triple-negative breast cancer (TNBC) with high tumour-infiltrating lymphocytes (TILs) has been associated with a promising prognosis. To better understand the prognostic value of immune cell subtypes in TNBC, we characterised TILs and the interaction between tumour cells and immune cell subtypes. A total of 145 breast cancer tissues were stained by multiplex immunofluorescence (mIF), including panel 1 (PD-L1, PD-1, CD3, CD8, CD68 and CK) and panel 2 (Foxp3, Granzyme B, CD45RO, CD3, CD8 and CK). Phenotypes were analysed and quantified by pathologists using InForm software. We found that in the ER-negative (ER <1% and HER2-negative) group and the ER/PR-low positive (ER 1–9% and HER2-negative) group, 11.2% and 7.1% of patients were PD-L1+ by the tumour cell score, 29.0% and 28.6% were PD-L1+ by the modified immune cell score and 30.8% and 32.1% were PD-L1+ by the combined positive score. We combined ER-negative and ER/PR-low positive cases for the survival analysis since a 10% cut-off is often used in clinical practice for therapeutic purposes. The densities of PD-L1+ tumour cells (HR: 0.366, 95% CI: 0.138–0.970; p = 0.043) within the tumour compartment and CD3+ immune cells in the total area (tumour and stromal compartments combined) (HR: 0.213, 95% CI: 0.070–0.642; p = 0.006) were favourable prognostic biomarkers for overall survival (OS) in TNBC. The density of effector/memory cytotoxic T cells (CD3+CD8+CD45RO+) in the tumour compartment was an independent prognostic biomarker for OS (HR: 0.232, 95% CI: 0.086–0.628; p = 0.004) and DFS (HR: 0.183, 95% CI: 0.1301–0.744; p = 0.009) in TNBC. Interestingly, spatial data suggested that patients with a higher density of PD-L1+ tumour cells had shorter cell-cell distances from tumour cells to cytotoxic T cells (p < 0.01). In conclusion, we found that phenotyping tumour immune cells by mIF is highly informative in understanding the immune microenvironment in TNBC. PD-L1+ tumour cells, total T cells and effector/memory cytotoxic T cells are promising prognostic biomarkers in TNBC.

Reduced miR-371b-5p expression drives tumor progression via CSDE1/RAC1 regulation in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer; however, specific prognostic biomarkers have not yet been developed. In this study, we identified dysregulated microRNAs (miRNAs) in TNBC by profiling miRNA and mRNA expression. In patients with TNBC, miR-371b-5p expression was reduced, and miR-371b-5p overexpression significantly mitigated TNBC cell growth, migration, and invasion. In addition, we found that expression of cold shock domain-containing protein E1 (CSDE1), a direct target gene of miR-371b-5p, was upregulated in TNBC cells, and inhibition of CSDE1 expression alleviated TNBC cell growth by regulating RAC1 transcription. Mechanistically, CSDE1, phosphorylated C-terminal domain (p-CTD) of RNA polymerase II (RNAPII), and CDK7 form a complex, and downregulation of CSDE1 leads to weak interaction between RNAPII p-CTD and CDK7, resulting in a decrease in RNAPII p-CTD expression to reduce RAC1 transcript levels in CSDE1-deficient TNBC cells. Our data demonstrate that miR-371b-5p is a tumor-suppressive miRNA that regulates the CSDE1/Rac1 axis and could be a potential prognostic biomarker for TNBC.

Development of an immune-related prognostic biomarker for triple-negative breast cancer

Purpose: Oncology studies employing digital dissection methodologies have provided some insight on the biological features of tumor microenvironment of Triple-negative breast cancer (TNBC), but molecular diagnostics rarely have therapeutic impact. We aimed to identify a novel prognostic biomarker to investigate immune characteristics of TNBC using transcriptomic features. Patients and Methods: We extracted whole transcriptome from breast cancer tissue of 30 TNBC patients and then used bioinformatics approaches to characterize the different immune cell contents in tumor tissue and para-cancerous tissue. We extract 2 indicators to describe the major differences in immune infiltration in the microenvironment between tumor tissue and para-cancerous tissue. We then combined the 2 indicators that represent the levels of increased and decreased infiltration in each sample to obtain the Immune Infiltration Score (IIS). Then we compared the tumor-infiltrating immune cell contents and immune infiltrating status in TNBC samples with CIBERSORT and ESTIMATE score to validate the IIS. Finally, 132 TNBC patients from the Cancer Genome Atlas program (TCGA) dataset was used to validate the predictive power of IIS. Results: 4 types of upregulated and 4 types of downregulated immune cells were identified in the tumor tissue samples of the TNBC patients. Then we developed a novel biomarker, IIS. Results showed that IIS score can clearly separate cancer and para-cancerous tissue. Using the same cutoff value of 0 in the TNBC-TCGA cohort, we show that those patients with a higher IIS had significantly higher PD-L1 expression and shorter progression-free survival time than those with a lower IIS value, indicating IIS score can be generalized to other TNBC datasets. Conclusion: we explored the immune infiltration landscape in 30 TNBC patients and provided IIS as a novel and reliable biomarker to evaluate the progression-free survival and prognosis of the TNBC patients.

CmP Signaling Network Leads to Identification of Prognostic Biomarkers for Triple-Negative Breast Cancer in Caucasian Women.

Objective: Triple-negative breast cancer (TNBC) constitutes ∼15% of all diagnosed invasive breast cancer cases with limited options for treatment since immunotherapies that target ER, PR, and HER2 receptors are ineffective. Progesterone (PRG) can induce its effects through either classic, nonclassic, or combined responses by binding to classic nuclear PRG receptors (nPRs) or nonclassic membrane PRG receptors (mPRs). Under PRG-induced actions, we previously demonstrated that the CCM signaling complex (CSC) can couple both nPRs and mPRs into a CmPn signaling network, which plays an important role during nPR(+) breast cancer tumorigenesis. We recently defined the novel CmP signaling network in African American women (AAW)-derived TNBC cells, which overlapped with our previously defined CmPn network in nPR(+) breast cancer cells. Methods: Under mPR-specific steroid actions, we measured alterations to key tumorigenic pathways in Caucasian American women (CAW)- derived TNBC cells, with RNAseq/proteomic and systems biology approaches. Exemption from ethics approval from IRB: This study only utilized cultured NBC cell lines with publicly available TNBC clinical data sets. Results: Our results demonstrated that TNBCs in CAW share similar altered signaling pathways, as TNBCs in AAW, under mPR-specific steroid actions, demonstrating the overall aggressive nature of TNBCs, regardless of racial differences. Furthermore, in this report, we have deconvoluted the CmP signalosome, using systems biology approaches and CAW-TNBC clinical data, to identify 21 new CAW-TNBC-specific prognostic biomarkers that reinforce the definitive role of CSC and mPR signaling during CAW-TNBC tumorigenesis. Conclusion: This new set of potential prognostic biomarkers may revolutionize molecular mechanisms and currently known concepts of tumorigenesis in CAW-TNBCs, leading to hopeful new therapeutic strategies.

Circular RNA circPTK2 modulates migration and invasion via miR-136/NFIB signaling on triple-negative breast cancer cells in vitro

Triple-negative breast cancer (TNBC) is an aggressive breast type of cancer with poor prognosis and high mortality rates. CircRNAs have been widely investigated, due to their crucial role in cancer progression. We aimed to elucidate the function of circPTK2 (has_circ_0003221) in TNBC and explore the mechanism in progression of TNBC. qPCR was performed to validate the expression of circPTK2 and related mRNA in TNBC tissue and cell lines. CCK-8, EdU, Transwell assay were conducted to detect the proliferation, migration and invasion of circPTK2 and miR-136 on TNBC cells. RIP and dual-luciferase reporter assay were used to confirm the interaction among circPTK2, miR-136 and NFIB. Si-circPTK2, mimic and inhibitor of miR-136 were transfected in TNBC cells to confirm the mechanism of circPTK2 and miR-136 in TNBC cells. CircPTK2 were downregulated in TNBC tissues and cell lines. CircPTK2 significantly promoted the proliferation, migration, and invasion of TNBC cells. CircPTK2 was confirmed to be a sponge of miR-136, and directly regulated NFBI and AKT/PI3K pathway. A rescue assay validated circPTK2/miR-136/NFIB axis in TNBC cells. CircPTK2 promoted TNBC progression and development. circPTK2/miR-136/NFIB might be an effective biomarker for TNBC.

Histamine H4 Receptor Expression in Triple-negative Breast Cancer: An Exploratory Study.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. There are neither universally accepted prognostic markers nor molecular targets related to TNBC. The histamine H4 receptor (H4R) has been characterized in TNBC experimental models, demonstrating its critical role in tumor development and progression. In this study, H4R expression was compared in breast cancer subtypes and correlated with clinical features using The Cancer Genome Atlas data (Pan-Cancer Atlas). The H4R status was further evaluated by immunohistochemistry in 30 TNBC human samples in relation to clinicopathological parameters. Results indicate that H4R was downregulated in basal-like/TNBC compared with luminal A and normal breast-like tumors. The higher expression of H4R was associated with improved progression-free and overall survival outcomes in basal-like/TNBC. H4R immunoreactivity was detected in about 70% of tumors, and its expression was positively correlated with the levels in the histologically normal peritumoral tissue. High H4R expression in peritumoral tissue correlated with reduced number of lymph node involvement and unifocal TNBC, while it was associated with increased patient survival. In conclusion, the H4R might represent a potential prognostic biomarker in TNBC. Further studies in large cohorts are needed to better understand the significance of H4R in breast cancer biology.

Abstract P1-08-24: Platelet-to-lymphocyte ratio is worth using with tumor-infiltrating lymphocytes to predict good response to neoadjuvant chemotherapy in triple negative breast cancer: A study on 120 patients

Abstract Introduction: Triple negative breast cancer (TNBC) is highly heterogeneous, but still most of the patients are treated by the anthracycline/taxane-based neoadjuvant therapy (NACT). Tumor-infiltrating lymphocytes (TILs) are a strong predictive and prognostic biomarker in TNBC, however not always available due to organizational and analytical issues. Peripheral blood counts, which reflect the systemic inflammatory/immune status, are easier to obtain than TILs. We investigated whether baseline white cell or platelet counts, as well as Neutrophil-to-Lymphocyte Ratio (NLR) or Platelet-to-Lymphocyte Ratio (PLR) could replace baseline TILs as predictive or prognostic biomarkers in a series of TNBC treated by standard NACT. Patients and methods: One hundred twenty patients uniformly treated by FEC/taxane NACT in a tertiary cancer care center were retrospectively analyzed. The presence of pathological complete response (pCR: ypT0/Tis, ypN0) or the presence of pCR and small residual disease (ypT0/Tis/T1ab, ypN0) were considered as good responses in data analysis. Baseline/pre-NACT blood count, NLR, PLR and TILs were evaluated as predictors of response, distant recurrence rate and distant recurrence-free survival (DRFS). TILs were assessed on breast tumor biopsies according to the recommendations of the International Immuno-Oncology Biomarker Working Group (www.tilsinbreastcancer.org). Results: TILs ≥30% and ≥1.5% were best predictors of pCR and distant recurrence risk, respectively (p=0.007, p=0.012). However, in this cohort, pCR status was not significantly associated with recurrence. Only the ensemble of patients with pCR and small residual disease had lower recurrence risk and longer survival DRFS (p=0.042, p=0.024, respectively) than the rest of the cohort (larger residual disease). The only parameter which could predict the pCR/small residual disease status was PLR: patients with values lower than 133.25 had significantly higher chance of reaching that status after NACT (p=0.045). However, no direct correlation could be established between baseline PLR and metastatic recurrence. No correlation either was found between TIL and individual blood counts, or between TILs and NLR or PLR. Conclusion: In this cohort, TILs retained their pCR predictive value, however PLR was better predictor of the ensemble of responses which had good outcome in terms of less distant recurrences or longer DRFS (pCR or small residual disease). Thus, baseline PLR is worth further, prospective investigation together with baseline TILs, as it might indicate a good TNBC response to NACT when TILs are unavailable. Citation Format: Nina Radosevic-Robin, Sejdi Lusho, Xavier Durando, Marie-Ange Mouret-Reynier, Myriam Kossai, Nathalie Lacrampe, Ioana Molnar, Frederique Penault-Llorca, Catherine Abrial. Platelet-to-lymphocyte ratio is worth using with tumor-infiltrating lymphocytes to predict good response to neoadjuvant chemotherapy in triple negative breast cancer: A study on 120 patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-24.

Abstract P4-05-12: Kinetics of endocan in the peripheral blood before and after the resection of primary breast tumors

Abstract Objectives: This study aims to investigate whether endocan levels in the peripheral blood change after the removal of primary breast tumors and allow predicting postoperative prognosis for breast cancer. Significance: An early prediction of postoperative recurrence and metastases with liquid biopsy will lead to better management and quality of life of breast cancer patients. Background: Cancer recurrence comes down to a poor prognosis in breast cancer patients. Although removal of primary breast cancer can achieve a successful outcome, metastasis and local recurrence remain a critical clinical problem in later years. Although the overall survival of breast cancer patients varies among molecular subtypes, the individual patients have different prognostic outcomes; some patients relapse within a few months, and others have not seen any recurrence for several years. Our previous study indicated that endocan had the potential to be a prognostic biomarker for triple-negative breast cancer. It is critical to show changes in endocan levels in the peripheral blood after the surgical excision of the primary tumor, which, in turn, will corroborate our hypothesis that plasma endocan is a beneficial biomarker for a postoperative prognosis for breast cancer patients. We included all the breast cancer subtypes in this study as the information on endocan levels in the peripheral blood from breast cancer patients has been limited thus far. Methods: To detect metastatic recurrence of breast cancer cells noninvasively, MDA-MB-231BR was transfected with the mVenus-Akaluc gene. MDA-MB-231BR spontaneously overexpresses the ESM1 gene. MDA-MB-231BR/mVenus-Akaluc was inoculated into the mammary fat pad of female athymic nu/nu mice and NSG mice. Once tumors top 200 mm3, they were surgically removed. Metastatic recurrence was visualized by the IVIS imager. Blood was withdrawn from the tail of each mouse before and after the resection of the primary tumors in a timely manner, and plasma samples were stored at -80°C until usage. Endocan in plasma samples was quantitated with a commercial ELISA kit. Tumor burden was estimated from luminescence signals of images obtained by IVIS. We obtained IRB approval from both institutions for the following research. Blood samples were collected from breast cancer patients who had given informed consent at the Breast Cancer, Showa University Hospital, and the plasma samples have been stored at -80°C until usage. Endocan in plasma sample was determined with a commercial ELISA kit. Results and discussion: Before removing primary breast tumors, the median endocan level in plasma was 0.73 ng/mL (range, 0.38-1.54 ng/mL) in nude mice. After the removal, endocan remained below the detection limits even though small metastases were detected by IVIS imager at later time points. These data indicate that loss of primary tumors results in an out-of-detection range in endocan levels in the blood. In contrast, NSG mice displayed inconsistent results: out of eight mice, three mice showed a decrease in endocan, three had an increase in endocan, and two had no change three days postoperatively. IVIS images of NSG mice indicated that distant metastases were already detected even when some mice underwent tumor resection. In NSG mice, the occurrence of tumor metastases was much earlier, and metastatic tumor burdens were much more significant than in nude mice, which might lead to a different outcome. Endocan levels in plasma from breast cancer patients at the postoperative period were not always dropped, which was similar to the results obtained from NSG mice. The rationale behind the inconsistent kinetics of endocan remains to be clarified. Conclusion: Our results from NSG mice and clinical data in patients’ plasma may have more relevance to prognostic outcomes in breast cancer patients. Citation Format: Kentaro Daiki, Yoko Kanada, Aya Nagata, Seigo Nakamura, Yoshinori Kato. Kinetics of endocan in the peripheral blood before and after the resection of primary breast tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-05-12.

Secreted Non-Coding RNAs: Functional Impact on the Tumor Microenvironment and Clinical Relevance in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast carcinoma characterized by poor prognosis and high rate of metastasis. Current treatment is based on chemo- and/or radiotherapy and surgery. TNBC is devoid of estrogen, progesterone and HER2 receptors. Although precision medicine has come a long way to ameliorate breast cancer disease management, targeted therapies for the treatment of TNBC patients are still limited. Mounting evidence has shown that non-coding RNAs (ncRNAs) drive many oncogenic processes at the basis of increased proliferation, invasion and angiogenesis in TNBC, strongly contributing to tumor progression and resistance to treatments. Many of these ncRNAs are secreted in the tumor microenvironment (TME) and impinge on the activity of the diverse immune and stromal cell types infiltrating the TME. Importantly, secreted ncRNAs may be detected as circulating molecules in serum/plasma from cancer patients and are emerging a promising diagnostic/therapeutic tools in TNBC. This review aims to discuss novel insights about the role of secreted circulating ncRNAs in the intercellular communication in the tumor microenvironment and their potential clinical use as diagnostic and prognostic non-invasive biomarkers in TNBC.

PSAT1 gene as a biomarker for targeting triple negative breast cancer in presence of Rapamycin

The Triple-Negative Breast Cancers (TNBCs) poor survival outcomes are related to not responding to hormonal therapy or targeting HER2 protein. The aim of this study was to introduce a drug among the conventional chemotherapy drugs based on the transcripts of the TNBC patients. The transcriptome of MDA-MB-468 cell line as a TNBC model was analyzed in the presence of 17 common drugs in fifteen GEO datasets. Then, decreased gene lists in the presence of each drug were compared in different subtypes of breast cancer based on TCGA data. The effect of selected drug on survival and apoptosis rates of MDA-MB-468 and MCF-7 cells (as ER+/PR + model) were compared. The outcomes showed that the expression of the decreased gene by Rapamycin among other drugs increased in TNBC subtype compared to the other two subtypes including triple-positive and ER+/PR+. The expression of PSAT1 in TNBC group was significant higher than normal and other subtypes (p ≤ 0.0001), and was introduced as a biomarker in TNBC that its expression was down-regulated by Rapamycin based on in silico studies. In the presence of 3000 µg/mL Rapamycin, there was a sharp decrease in MDA-MB-468 cell viability (p ≤ 0.0001) and a significant increase in apoptosis compared to MCF-7 cells treated with the same concentration of the drug. Moreover, RT-qPCR revealed PSAT1 expression more decreased under Rapamycin-treatment in TNBC cells compared to ER + PR + cells. The results of this study show that Rapamycin is a suitable drug for targeting TNBC based on its transcriptome and biomarker. Supplemental data for this article is available online at

Machine learning assisted analysis of breast cancer gene expression profiles reveals novel potential prognostic biomarkers for triple-negative breast cancer

Tumor heterogeneity and the unclear metastasis mechanisms are the leading cause for the unavailability of effective targeted therapy for Triple-negative breast cancer (TNBC), a breast cancer (BrCa) subtype characterized by high mortality and high frequency of distant metastasis cases. The identification of prognostic biomarker can improve prognosis and personalized treatment regimes. Herein, we collected gene expression datasets representing TNBC and Non-TNBC BrCa. From the complete dataset, a subset reflecting solely known cancer driver genes was also constructed. Recursive Feature Elimination (RFE) was employed to identify top 20, 25, 30, 35, 40, 45, and 50 gene signatures that differentiate TNBC from the other BrCa subtypes. Five machine learning algorithms were employed on these selected features and on the basis of model performance evaluation, it was found that for the complete and driver dataset, XGBoost performs the best for a subset of 25 and 20 genes, respectively. Out of these 45 genes from the two datasets, 34 genes were found to be differentially regulated. The Kaplan-Meier (KM) analysis for Distant Metastasis Free Survival (DMFS) of these 34 differentially regulated genes revealed four genes, out of which two are novel that could be potential prognostic genes (POU2AF1 and S100B). Finally, interactome and pathway enrichment analyses were carried out to investigate the functional role of the identified potential prognostic genes in TNBC. These genes are associated with MAPK, PI3-AkT, Wnt, TGF-β, and other signal transduction pathways, pivotal in metastasis cascade. These gene signatures can provide novel molecular-level insights into metastasis.

Tumor suppressor PLK2 may serve as a biomarker in triple-negative breast cancer for improved response to PLK1 therapeutics.

Polo-like kinase (PLK) family members play important roles in cell cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Paradoxically, frequent loss of chromosome 5q11-35 which includes PLK2 is observed in basal-like breast cancer. In this study, we found that PLK2 was tumor suppressive in breast cancer, preferentially in basal-like and triple-negative breast cancer (TNBC) subtypes. Knockdown of PLK1 rescued phenotypes induced by PLK2-loss both in vitro and in vivo. We also demonstrated that PLK2 directly interacted with PLK1 at prometaphase through the kinase but not the polo-box domains of PLK2, suggesting PLK2 functioned at least partially through the interaction with PLK1. Furthermore, an improved treatment response was seen in both Plk2-deleted/low mouse preclinical and PDX TNBC models using the PLK1 inhibitor volasertib alone or in combination with carboplatin. Re-expression of PLK2 in an inducible PLK2-null mouse model reduced the therapeutic efficacy of volasertib. In summary, this study delineates the effects of chromosome 5q loss in TNBC that includes PLK2, the relationship between PLK2 and PLK1, and how this may render PLK2-deleted/low tumors more sensitive to PLK1 inhibition in combination with chemotherapy.

Differential expression of microRNA between triple negative breast cancer patients of African American and European American descent

ABSTRACT There are racial disparities in the outcome of triple negative breast cancer (TNBC) patients between women of African ancestry and women of European ancestry, even after accounting for lifestyle, socioeconomic and clinical factors. MicroRNA (miRNA) are non-coding molecules whose level of expression is associated with cancer suppression, proliferation and drug resistance; therefore, these have potential for biomarker applications in cancers including TNBC. Historically, miRNAs up-regulated in African American (AA) patients have received less attention than for patients of European ancestry. Using laser capture microdissection (LCM) to acquire ultrapure tumor cell samples, miRNA expression was evaluated in 15 AA and 15 European American (EA) TNBC patients. Tumor sections were evaluated using RNA extraction followed by miRNA analysis and profiling. Results were compared based on ethnicity and method of tissue fixation. miRNAs that showed high differential expression in AA TNBC patients compared to EA included: miR-19a, miR-192, miR-302a, miR-302b, miR-302c, miR-335, miR-520b, miR-520f and miR-645. LCM is a useful technique for isolation of tumor cells. We found a greater abundance of RNA in frozen samples compared to formalin fixed, paraffin embedded samples. miRNA appears to be a useful biomarker for TNBC to improve diagnosis and treatment.

Radiomics features based on automatic segmented MRI images: Prognostic biomarkers for triple-negative breast cancer treated with neoadjuvant chemotherapy

PurposeTo establish radiomics prediction models based on automatic segmented magnetic resonance imaging (MRI) for predicting the systemic recurrence of triple-negative breast cancer (TNBC) in patients after neoadjuvant chemotherapy (NAC). Materials and methodsA total of 147 patients with TNBC who underwent NAC between January 2009 and December 2018 were enrolled in this study. Clinicopathologic data were collected, and the differences between the recurrent and nonrecurrent patients were analyzed by univariate and multivariate analyses. Patients were randomly divided into training and testing sets. The training set consisted of 104 patients (recurrence: 22, nonrecurrence: 82), and the testing set consisted of 43 patients (recurrence: 9, nonrecurrence: 34). To establish the radiomics prediction model, we used a deep learning segmentation model to automatically segment tumor areas on dynamiccontrast-enhanced-MRI images of pre- and post-NAC magnetic resonance examinations. Radiomics features were then extracted from the tumor areas. Three MRI radiomics models were developed in the training set: a radiomics model based on pre-NAC MRI features (model 1), a radiomics model based on post-NAC MRI features (model 2), and a radiomics model based on both pre- and post-NAC MRI features (model 3). A clinical model for predicting systemic recurrence was built in the training set using independent clinical prediction factors. Receiver operating characteristic curve analysis was used to evaluate the performance of the radiomics and clinical models. ResultsThe clinical model yielded areas under the curve (AUCs) of 0.747 in the training set and 0.737 in the testing set in terms of predicting systemic recurrence. Models 1, 2, and 3 yielded AUCs of 0.879, 0.91, and 0.963 in the training set and 0.814, 0.802, and 0.933 in the testing set, respectively, in terms of predicting systemic recurrence. All of the radiomics models had achieved higher AUCs than the clinical model in the testing set. DeLong test was used to compare the AUCs between the models and indicated that the predictive performance of model 3 was better than the clinical model, and the difference was statistically significant (p < 0.05). ConclusionThe radiomics models built based on the combination of pre- and post-NAC MRI features showed good performance in predicting whether patients with TNBC will have systemic recurrence within 3 years post-NAC. This can help us non-invasively identify which patients are at high risk of recurrence post-NAC, so that we can strengthen follow-up and treatment of these patients. Then the prognosis of these patients might be improved.