Clinical Breast Cancer Samples Research Articles

Repurposing hemostatic drug desmopressin in AVPR2-expressing triple-negative breast cancer: Target expression analysis and preclinical antineoplastic effects on tumor growth and metastatic progression.

e15087 Background: Desmopressin (dDAVP) is a hemostatic drug that acts as an agonist of the arginine vasopressin receptor 2 (AVPR2) that is present in vascular and malignant cells. Preclinical data show that dDAVP triggers cytostatic mechanisms, blocking angiogenesis and reducing metastases by limiting the survival of circulating tumor cells. Triple-negative breast cancer (TNBC) is associated with poor prognosis due to limited response to therapy and frequent early metastatic recurrence. Considering the unsatisfied clinical need for novel therapeutic options in TNBC, we evaluated the antitumoral activity of dDAVP on experimental models of TNBC, alone or in addition to chemotherapy. We also assessed the expression and clinical impact of AVPR2 in breast cancer samples. Methods: Human and murine TNBC cell lines were used for in vitro experiments. Target expression was assessed by qPCR and immunohistochemistry (IHC), in experimental or clinical tumors (NCT01606072). Effects of dDAVP were evaluated by proliferation, clonogenic, 3D growth and chemotaxis assays, qPCR arrays, and confocal microscopy. Local and distant tumor progression were assessed in vivo in nude or BALB/c mice. TIMER2.0 and UCSC Xena platforms (BRCA TCGA /n = 1260) were used for bioinformatic studies. Results: dDAVP significantly reduced growth and chemotaxis of AVPR2-expressing TNBC cells. Cytostatic effects of dDAVP were associated with altered actin cytoskeleton dynamics and differential expression of angiogenesis and metastasis-related genes. Cooperative effects were observed in vitro and in vivo after combining dDAVP with taxane or alkylating therapy. Tumor-bearing mice were treated with intravenous injections of dDAVP (0.3 µg/kg, thrice weekly) in combination with weekly cycles of paclitaxel (10 mg/kg i.p.) or carmustine (25 mg/kg i.p.). Combination regimens resulted in greater survival and tumor growth and metastasis inhibition. Bioinformatic analysis of the TCGA database showed that AVPR2 had a positive prognostic impact on overall survival in breast cancer patients, especially in the basal-like molecular subtype. AVPR2 also correlated with different immune cell infiltrates such as NK and dendritic cells, and CD4+ and CD8+ T cells. Moreover, AVPR2 expression was detected by IHC in 6/18 breast cancer clinical samples using a small cohort of surgical specimens. Conclusions: AVPR2 expression is associated with improved outcomes in patients with TNBC. Agonist activation of AVPR2 using dDAVP leads to cancer cell growth inhibition in TNBC cell lines and mouse models. We propose dDAVP as a potential novel therapeutic option for treating TNBC either as monotherapy or in combination with chemotherapy. The therapeutic impact of its addition to immune checkpoint inhibitors remains to be explored.

Molecular profiling using next generation sequencing with high-purity enrichment of circulating tumor cells.

e15054 Background: In the field of liquid biopsy, coupled with next generation sequencing (NGS), circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can serve as an alternative substrate to tumor tissue for mutation detection and companion diagnostic purpose. Emerging evidence suggests that complementary sequential CTCs and ctDNA profiling offers promising benefits for prognosis and treatment. In contrast to well-established ctDNA extraction methods, the process of isolation of intact CTCs for NGS analysis has not yet been fully elucidated. Here we show that isolation and harvesting of viable CTCs using CytoGen's Smart Biopsy™ System achieves high CTC purity for NGS analysis in breast cancer and non-small cell lung cancer (NSCLC). Methods: Targeted panel sequencing for CTC was developed to detect representative mutated genes using the Oncomine™ Comprehensive Assay Plus (501 genes) and the Oncomine™ Pan-Cancer Cell-Free Assay (52 genes). Analytical validation was performed with breast cancer cells MDA-MB-231 (50 to 5 cells) and NSCLC cells H358 (10 and 5 cells) which were spiked into 5 mL of healthy blood to mimic CTCs. CytoGen's Smart Biopsy™ System was applied to isolate the CTCs. Subsequently, CD45 depletion and whole genome amplification were performed. Sequencing was performed on Ion S5™ XL Systems using an Ion 550™ chip kit. The sequencing data were then analyzed using the Ion Torrent software with the default configurations. Results: As a result of targeted panel sequencing with Oncomine™ Comprehensive Assay Plus, representative breast cancer mutation genes (BRAF, KRAS, TP53, NF2 and NOTCH3) were detected in at least 20 MDA-MB-231 cells. In the results using Oncomine™ Pan-Cancer Cell-Free Assay, mutated genes (BRAF, KRAS and TP53) were successfully detected in as few as 5 MDA-MB-231 cells and, in addition, KRAS mutation was detected even with only 5 H358 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to detect representative mutated genes in clinical breast cancer and NSCLC patient samples with detection technology that require high sensitivity and specificity. Conclusions: Molecular analysis of CTCs is a challenge since CTCs are very rare in the blood and there are technical limitations in isolating CTCs with sufficient purity for NGS analysis. In this study, we developed the process for NGS analysis on very small number of high-purity CTCs using CytoGen's Smart Biopsy™ System. Taken together with the results from our ongoing clinical trials, this process could provide information for the diagnosis and appropriate treatment of breast cancer and NSCLC patients.

A 3'-pre-tRNA-derived small RNA tRF-1-Ser regulated by 25(OH)D promotes proliferation and stemness by inhibiting the function of MBNL1 in breast cancer.

We explored the potential novel anticancer mechanisms of 25-hydroxyvitamin D (25(OH)D), a vitamin D metabolite with antitumour effects in breast cancer. It is stable in serum and is used to assess vitamin D levels in clinical practice. Transfer RNA-derived small RNAs are small noncoding RNAs that generate various distinct biological functions, but more research is needed on their role in breast cancer. Small RNA microarrays were used to explore the novel regulatory mechanism of 25(OH)D. High-throughput RNA-sequencing technology was used to detect transcriptome changes after 25(OH)D treatment and tRF-1-Ser knockdown. RNA pull-down and high-performance liquid chromatography-mass spectrometry/mass spectrometry were used to explore the proteins bound to tRF-1-Ser. In vitro and in vivo functional experiments were conducted to assess the influence of 25(OH)D and tRF-1-Ser on breast cancer. Semi-quantitative PCR was performed to detect alternative splicing events. Western blot assay and qPCR were used to assess protein and mRNA expression. The expression of tRF-1-Ser is negatively regulated by 25(OH)D. In our breast cancer (BRCA) clinical samples, we found that the expression of tRF-1-Ser was higher in cancer tissues than in paired normal tissues, and was significantly associated with tumour invasion. Moreover, tRF-1-Ser inhibits the function of MBNL1 by hindering its nuclear translocation. Functional experiments and transcriptome data revealed that the downregulation of tRF-1-Ser plays a vital role in the anticancer effect of 25(OH)D. In brief, our research revealed a novel anticancer mechanism of 25(OH)D, unveiled the vital function of tRF-1-Ser in BRCA progression, and suggested that tRF-1-Ser could emerge as a new therapeutic target for BRCA.

Enhancing Specific Fluorescence In Situ Hybridization with Quantum Dots for Single-Molecule RNA Imaging in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

Abstract 1030: Concordance between the DESTINY-Breast04 clinical trial assay (4B5[CDx]) and other HER2 IHC assays for HER2-low breast cancer in real-world practice: First phase of a large-scale, multicenter global ring study

Abstract Background: DESTINY-Breast04 (DB-04) showed that trastuzumab deruxtecan improves survival vs chemotherapy in patients with human epidermal growth factor receptor 2 (HER2)-low (immunohistochemistry [IHC] 1+ or IHC 2+ with in situ hybridization negative) metastatic breast cancer (BC). The VENTANA 4B5(CDx) IHC assay, now approved in the United States (US) as a companion diagnostic, was used as the clinical trial assay (CTA) in DB-04. Real-world differentiation of HER2-low vs HER2 IHC 0 BC with non-CTAs is difficult. This is the first phase of a study assessing agreement between CTA and non-CTA scores. Methods: 50 clinical BC samples with HER2 IHC scores of 0, 1+, 2+, and 3+ were sent to laboratories in Europe, Canada, and the US for HER2 IHC testing per ASCO/CAP 2018 guidelines. After routine protocol scoring, pathologists were trained and rescored the samples 2 weeks later. Post training scores were compared with reference CTA scores determined by a central laboratory and a panel of experts. The primary endpoint was positive percentage agreement (PPA) and negative percentage agreement (NPA) between CTA and non-CTA scores, assessed after training, considering HER2-low as positive and HER2 IHC 0 as negative. Results: A total of 3449 post training non-CTA scores were available for analysis. PPA for HER2-low vs HER2 IHC 0 was 87.5% and NPA was 61.9%, with Cohen κ of 0.51 (Table). HER2-low vs HER2 IHC 0 agreement between CTA and non-CTA varied across subgroups. PPA >95% was shown with 4B5 Laboratory Developed Tests (LDTs) and HercepTest (Omnis), NPA of 80% with Leica, and overall agreement >80% with HercepTest (Link48), 4B5 LDTs, and German, French, and Italian laboratories. Conclusions: The degree of concordance between the CTA and non-CTAs varied among assay types and laboratory locations. A low NPA for some non-CTAs suggests the need for further assay optimization for HER2 IHC 0 evaluation. Post training non-CTA results HER2-low vs HER2 IHC 0 BC Post training scores PPA (95% CI), % NPA (95% CI), % Overall percentage agreement (95% CI), % Cohen κ (95% CI) Overall non-CTA test results (N = 3449) 87.5 (86.0-89.0) 61.9 (58.9-64.9) 78.7 (77.1-80.1) 0.51 (0.48-0.54) Non-CTA subgroups HercepTest (Omnis) (n = 467) 95.5 (92.3-97.7) 36.9 (28.9-45.4) 75.2 (70.8-79.4) 0.37 (0.28-0.46) HercepTest (Link48) (n = 790) 88.5 (85.2-91.2) 64.3 (57.8-70.4) 80.3 (77.2-83.2) 0.55 (0.48-0.61) Leica (n = 96) 59.3 (45.0-72.4) 80.0 (61.4-92.3) 66.7 (55.5-76.6) 0.35 (0.17-0.53) Non-4B5 LDTs (n = 1609) 83.8 (81.2-86.1) 67.8 (63.5-72.0) 78.2 (75.9-80.3) 0.52 (0.47-0.57) 4B5 LDTs (n = 487) 96.0 (93.0-98.0) 58.8 (50.4-66.8) 83.0 (79.1-86.4) 0.59 (0.51-0.68) Country subgroups Germany (n = 340) 93.1 (88.5-96.3) 64.4 (54.2-73.6) 83.1 (78.3-87.2) 0.61 (0.51-0.70) France (n = 342) 95.4 (91.5-97.9) 64.4 (54.4-73.6) 84.7 (80.1-88.6) 0.64 (0.55-0.73) Italy (n = 395) 89.9 (85.2-93.5) 72.5 (63.6-80.3) 83.9 (79.6-87.6) 0.64 (0.55-0.72) Spain (n = 567) 83.0 (78.4-86.9) 64.2 (56.6-71.3) 76.3 (72.3-80.0) 0.48 (0.40-0.56) US/Canada (n = 1029) 86.6 (83.6-89.2) 61.8 (56.1-67.3) 78.2 (75.4-80.8) 0.50 (0.44-0.56) Europe, other (n = 776) 85.0 (81.3-88.2) 52.8 (46.2-59.3) 73.8 (70.3-77.1) 0.40 (0.32-0.47) Citation Format: Hans-Ulrich Schildhaus, Sunil Badve, Corrado D’Arrigo, Gelareh Farshid, Annette Lebeau, Vicente Peg, Fréderique Penault-Llorca, Josef Rueschoff, Wentao Yang, Neil Atkey, Jessica Baumann, Elisabeth Beyerlein, Amy Hanlon Newell, Alexander Penner, Akira Moh, Guiseppe Viale. Concordance between the DESTINY-Breast04 clinical trial assay (4B5[CDx]) and other HER2 IHC assays for HER2-low breast cancer in real-world practice: First phase of a large-scale, multicenter global ring study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1030.

MicroRNA-561-3p indirectly regulates the PD-L1 expression by targeting ZEB1, HIF1A, and MYC genes in breast cancer

Globally, breast cancer is the second most common cause of cancer-related deaths among women. In breast cancer, microRNAs (miRNAs) are essential for both the initiation and development of tumors. It has been suggested that the tumor suppressor microRNA-561-3p (miR-561-3p) is crucial in arresting the growth of cancer cells. Further research is necessary to fully understand the role and molecular mechanism of miR-561 in human BC. The aim of this study was to investigate the inhibitory effect of miR-561-3p on ZEB1, HIF1A, and MYC expression as oncogenes that have the most impact on PD-L1 overexpression and cellular processes such as proliferation, apoptosis, and cell cycle in breast cancer (BC) cell lines. The expression of ZEB1, HIF1A, and MYC genes and miR-561-3p were measured in BC clinical samples and cell lines via qRT-PCR. The luciferase assay, MTT, Annexin-PI staining, and cell cycle experiments were used to assess the effect of miR-561-3p on candidate gene expression, proliferation, apoptosis, and cell cycle progression. Flow cytometry was used to investigate the effects of miR-561 on PD-L1 suppression in the BC cell line. The luciferase assay showed that miRNA-561-3p targets the 3′-UTRs of ZEB1, HIF1A and MYC genes significantly. In BC tissues, the qRT-PCR results demonstrated that miR-561-3p expression was downregulated and the expression of ZEB1, HIF1A and MYC genes was up-regulated. It was shown that overexpression of miR-561-3p decreased PD-L1 expression and BC cell proliferation, and induced apoptosis and cell cycle arrest through downregulation of candidate oncogenes. Furthermore, inhibition of candidate genes by miR-561-3p reduced PD-L1 at both mRNA and protein levels. Our research investigated the impact of miR-561-3p on the expression of ZEB1, HIF1A and MYC in breast cancer cells for the first time. Our findings may help clarify the role of miR-561-3p in PD-L1 regulation and point to this miR as a potential biomarker and novel therapeutic target for cancer immunotherapy.

Tumor Cell-Derived Exosomal miR-191-5p Activates M2-Subtype Macrophages Through SOCS3 to Facilitate Breast Cancer.

Differential activation of macrophages is associated with poor progression of breast cancer (BC). Many reports have elucidated the important involvement of exosomes produced by cancer cells in remodeling the macrophage activation phenotype to promote tumor expansion and invasion. However, the underlying mechanisms by which exosomes secreted by BC cells facilitate macrophage M2 polarization remain enigmatic and worth exploring. In this study, quantitative real-time PCR (RT-qPCR) was used to investigate miR-191-5p expression in BC tumor tissues and cells. Cell counting kit 8 (CCK-8), transwell, and flow cytometry were applied to assess the functional role of miR-191-5p in BC. Isolated nano-vesicles were identified using transmission electron microscopy and western blotting. We also observed that miR-191-5p was significantly elevated in BC clinical samples and that inhibition of miR-191-5p hindered the growth and metastasis of BC cells. Importantly, BC cells successfully accelerated macrophage M2-like polarization by directly transferring exosomes to macrophages, resulting in increased miR-191-5p levels in macrophages. Mechanistically, exosomal miR-191-5p directly inhibited the suppressors of cytokine signaling 3 (SOCS3) expression in macrophages and aggravated macrophage M2 polarization. Similarly, si-SOCS3 transfected macrophages boosted BC cell migration and invasion in a positive feedback manner. Overall, our results manifested a pro-growth and pro-metastatic role between the two cells by elucidating the crucial role of exosomal miR-191-5p in stimulating M2 macrophage polarization and mediating communication between BC cells and macrophages. These findings opened up new horizons for the development of BC therapeutic strategies.

MYSM1 acts as a novel co-activator of ERα to confer antiestrogen resistance in breast cancer

Endocrine resistance is a crucial challenge in estrogen receptor alpha (ERα)-positive breast cancer (BCa). Aberrant alteration in modulation of E2/ERα signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Herein, we demonstrate that MYSM1 as a deubiquitinase participates in modulating ERα action via histone and non-histone deubiquitination. MYSM1 is involved in maintenance of ERα stability via ERα deubiquitination. MYSM1 regulates relevant histone modifications on cis regulatory elements of ERα-regulated genes, facilitating chromatin decondensation. MYSM1 is highly expressed in clinical BCa samples. MYSM1 depletion attenuates BCa-derived cell growth in xenograft models and increases the sensitivity of antiestrogen agents in BCa cells. A virtual screen shows that the small molecule Imatinib could potentially interact with catalytic MPN domain of MYSM1 to inhibit BCa cell growth via MYSM1-ERα axis. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ERα action and provide a potential therapeutic target for endocrine resistance in BCa.

Abstract B085: Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer

Abstract Purpose: All patients with metastatic breast cancer (BC) expressing estrogen receptor α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of cases, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Clinical evidence suggests adverse outcomes for these patients. Our hypothesis was that ESR1 mutations may confer resistance to subsequent treatment lines, specifically chemotherapy. Experimental Design: MCF-7 cells harboring ESR1 mutations (Y537S and D538G) were used to characterize proliferation, apoptosis, and tumor response to chemotherapy in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter, ChIP assays and pharmacological inhibition. Results: Our results revealed that the mutated-ER (mut-ER) cells displayed relative chemoresistance, demonstrated by higher viability and reduced apoptosis. In vivo mouse model confirmed these findings. To elucidate the underlying mechanism, we examined MDR1 expression and JNK pathway activity. We observed elevated MDR1 expression in mut-ER cells, which was also reflected in clinical BC samples. Additionally, increased occupancy of the MDR1 promoter by c-Jun was observed in mut-ER cells. Importantly, inhibition of the JNK pathway resulted in decreased MDR1 expression, particularly in D538G cells. Moreover, JNK inhibition increased apoptosis and reduced viability in all cells, with a more pronounced effect in D538G cells. Conclusions: Taken together, our data indicate that ESR1 mutations confer chemoresistance in BC through the activation of the JNK/MDR1 axis. These findings suggest that targeting this pathway could be a potential strategy for novel treatments aimed at restoring chemotherapy sensitivity and improving outcomes for patients with metastatic BC. Citation Format: Marwa Taya, Keren Merenbakh-Lamin, Asia Zubkov, Zohar Honig, Ori Mayer, Noam Shomron, Ido Wolf, Tami Rubinek. Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B085.

The oncogene Musashi1 encodes novel miRNAs in breast cancer

RNA-binding protein Musashi1 (MSI1) shows an increased expression level in several cancers and has been introduced as a prognostic marker in some malignancies. It is expected that if any miRNA is encoded by this gene, it might have a role in cancer development or could be considered as a prognostic biomarker. Accordingly, in this study, we aimed to find novel miRNA(s) inside the intronic regions of the MSI1 gene. Here, we report two novel miRNAs within intron 4 of MSI1 gene, named MSM2 and MSM3, which were selected among several miRNA precursors predicted by bioinformatic studies. For experimental analysis, corresponding precursor miRNAs were transfected into HEK293T cells and exogenous expression of the mature miRNAs were detected. Two mature miRNAs, MSM3-3p and MSM3-5p were generated by MSM3 precursor and one, MSM2-5p was derived from MSM2. Besides, endogenous expression of MSM2-5p and MSM3-3p was detected in MCF-7 and SH-SY5Y cell lines. Expression of both mature miRNAs was also detected in clinical samples of breast cancer. Additionally, the interaction between the MSM3-3p and 3′UTR region of PDE11A was confirmed by dual luciferase assay. Overall, our data demonstrated that MSI1 gene encodes two novel miRNAs in breast cancer cells.

A filter-electrochemical microfluidic chip for multiple surface protein analysis of exosomes to detect and classify breast cancer

Breast cancer (BC) is a complex disease with high variability and no specific tumor markers available for diagnosis. Exosomes contain rich maternal tumor information and are a novel non-invasive biomarker with the potential for cancer diagnosis and prognosis. However, analysis of exosomal protein markers in blood samples is challenging due to lengthy sample workups and insufficient sensitivity. To address this difficulty, we developed a novel filter-electrochemical microfluidic chip (FEMC) to detect and classify BC directly in whole blood without requiring heavy purification methods. In our system, exosome enrichment was performed using a dual filtration system. The target was directed through a curved channel onto four screen-printed electrodes (SPEs), where it was captured by the previously modified antibodies. Simultaneously, Zr-MOFs encapsulated with a large number of methylene blue molecules (MB@UiO-66) were absorbed on the surface of exosomes due to the high affinity for phosphate groups. This process leads to the amplification of electrical signals. The approach demonstrated that the utilization of BC exosome-associated tumor biomarkers (i.e., PMSA, EGFR, CD81, and CEA), enabled the classification of various BC mouse models samples and clinical BC samples. The entire FEMC assay was completed in 1 h with a limit of detection of 1 × 104 particles/mL. Thus, the FEMC assay can provide real-time detection information, allowing timely and better-informed opportunities for clinical BC diagnosis and typing.

Detection of homologous recombination deficiency (HRD) using a novel genomic and epigenomic liquid biopsy assay in patients with breast cancer.

556 Background: Homologous recombination and repair (HRR) deficiency (HRD) is characterized by genomic instability associated with dysfunction in BRCA1/2 or other HRR genes. Patients with breast cancer harboring an HRD phenotype with or without HRR mutations have derived clinical benefit from PARPi therapy. We have previously shown that GuardantINFINITY, a novel genomic and epigenomic liquid biopsy assay, can identify HRR SNVs, indels, rearrangements, copy number loss, reversions, and BRCA1 promoter methylation for patient selection and resistance monitoring, a major challenge for PARPi treatment. Here, we present a method of predicting HRD status by cfDNA using GuardantINFINITY in patients with advanced breast cancer. Methods: We developed a probabilistic genomic model to predict HRD status, inferred from genome-wide somatic SNV, indel, and CNV signatures indicative of BRCA1/2 deficiency, including large-scale state transitions (LST), whole-genome tumor loss of heterozygosity (LOH), telomeric allelic imbalance (TAI). A second probabilistic model, based on targeted measurements of genomic and epigenetic changes was learned from a subset of clinical samples to enhance the genomic model. The model was trained and tested on a cohort of over 12,000 GuardantOMNI and GuardantINFINITY clinical breast cancer samples to assess the sensitivity of accurately detecting deficiency in select HRR-genes ( BRCA1/2, PALB2, RAD51D). The aggregated predictive model was validated on an independent cohort of breast cancer samples. Results: The model based on genomic and epigenetic signals identified HRR-gene deficiency in a breast cancer cohort with a estimated 95% LoD of 22.5% tumor fraction and an AUC of 0.75 across all tumor fractions, an improvement in sensitivity over when only the genomic model is used (AUC of 0.7, 95% LoD of 28%). Specificity remained high for both models (100%, n = 83) in cancer free samples. Application of this model in a cohort of 1,101 patients with unselected breast cancer identified 390 patients with an HRD phenotype, of whom 192 patients had a known HRR-gene mutation. Of the 201 patients with a known pathogenic mutation in an HRR gene, 49 also had co-occurring LoH in the same gene, indicating biallelic loss. Conclusions: In this analysis, we demonstrate that a probabilistic model of genomic and methylation predictors can detect HRD status in patients with breast cancer from cfDNA using GuardantINFINITY. Additional analytical and clinical studies to further evaluate this model are ongoing. With HRD prediction, GuardantINFINITY provides a comprehensive minimally-invasive solution for PARPi and DNA damage treatment selection, longitudinal monitoring, and an exploratory platform for investigating epigenetic signals that may underpin resistance.

Targeting long non-coding RNA MALAT1 reverses cancerous phenotypes of breast cancer cells through microRNA-561-3p/TOP2A axis

Non-coding RNAs, including Inc-RNA and miRNA, have been reported to regulate gene expression and are associated with cancer progression. MicroRNA-561-3p (miR-561-3p), as a tumor suppressor, has been reported to play a role in preventing cancer cell progression, and MALAT1 (Lnc-RNA) have also been demonstrated to promote malignancy in various cancers, such as breast cancer (BC). In this study, we aimed to determine the correlation between miR-561-3p and MALAT1 and their roles in breast cancer progression. The expression of MALAT1, mir-561-3p, and topoisomerase alpha 2 (TOP2A) as a target of miR-561-3p was determined in BC clinical samples and cell lines via qRT-PCR. The binding site between MALAT1, miR-561-3p, and TOP2A was investigated by performing the dual luciferase reporter assay. MALAT1 was knocked down by siRNA, and cell proliferation, apoptotic assays, and cell cycle arrest were evaluated. MALAT1 and TOP2A were significantly upregulated, while mir-561-3p expression was downregulated in BC samples and cell lines. MALAT1 knockdown significantly increased miR-561-3p expression, which was meaningfully inverted by co-transfection with the miR 561-3p inhibitor. Furthermore, the knockdown of MALAT1 by siRNA inhibited proliferation, induced apoptosis, and arrested the cell cycle at the G1 phase in BC cells. Notably, the mechanistic investigation revealed that MALAT1 predominantly acted as a competing endogenous RNA in BC by regulating the miR-561-3p/TOP2A axis. Based on our results, MALAT1 upregulation in BC may function as a tumor promoter in BC via directly sponging miRNA 561-3p, and MALAT1 knockdown serves a vital antitumor role in BC cell progression through the miR-561-3p/TOP2A axis.

Genetically predicted vitamin C levels significantly affect patient survival and immunotypes in multiple cancer types.

Recent observational studies and meta-analyses have shown that vitamin C reduces cancer incidence and mortality, but the underlying mechanisms remain unclear. We conducted a comprehensive pan-cancer analysis and biological validation in clinical samples and animal tumor xenografts to understand its prognostic value and association with immune characteristics in various cancers. We used the Cancer Genome Atlas gene expression data involving 5769 patients and 20 cancer types. Vitamin C index (VCI) was calculated using the expression of 11 genes known to genetically predict vitamin C levels, which were classified into high and low subgroups. The correlation between VCI and patient overall survival (OS), tumor mutational burden (TMB), microsatellite instability (MSI), and immune microenvironment was evaluated, using Kaplan-Meier analysis method and ESTIMATE (https://bioinformatics.mdanderson.org/estimate/). Clinical samples of breast cancer and normal tissues were used to validate the expression of VCI-related genes, and animal experiments were conducted to test the impact of vitamin C on colon cancer growth and immune cell infiltration. Significant changes in expression of VCI-predicted genes were observed in multiple cancer types, especially in breast cancer. There was a correlation of VCI with prognosis in all samples (adjusted hazard ratio [AHR] = 0.87; 95% confidence interval [CI] = 0.78-0.98; P = 0.02). The specific cancer types that exhibited significant correlation between VCI and OS included breast cancer (AHR = 0.14; 95% CI = 0.05-0.40; P < 0.01), head and neck squamous cell carcinoma (AHR = 0.20; 95% CI = 0.07-0.59; P < 0.01), kidney clear cell carcinoma (AHR = 0.66; 95% CI = 0.48-0.92; P = 0.01), and rectum adenocarcinoma (AHR = 0.01; 95% CI = 0.001-0.38; P = 0.02). Interestingly, VCI was correlated with altered immunotypes and associated with TMB and MSI negatively in colon and rectal adenocarcinoma (P < 0.001) but positively in lung squamous cell carcinoma (P < 0.05). In vivo study using mice bearing colon cancer xenografts demonstrated that vitamin C could inhibit tumor growth with significant impact on immune cell infiltration. VCI is significantly correlated with OS and immunotypes in multiple cancers, and vitamin C might have therapeutic potential in colon cancer.

Detection of ERBB2 and CEN17 signals in fluorescent in situ hybridization and dual in situ hybridization for guiding breast cancer HER2 target therapy

The overexpression of the human epidermal growth factor receptor 2 (HER2) is a predictive biomarker in therapeutic effects for metastatic breast cancer. Accurate HER2 testing is critical for determining the most suitable treatment for patients. Fluorescent in situ hybridization (FISH) and dual in situ hybridization (DISH) have been recognized as FDA-approved methods to determine HER2 overexpression. However, analysis of HER2 overexpression is challenging. Firstly, the boundaries of cells are often unclear and blurry, with large variations in cell shapes and signals, making it challenging to identify the precise areas of HER2-related cells. Secondly, the use of sparsely labeled data, where some unlabeled HER2-related cells are classified as background, can significantly confuse fully supervised AI learning and result in unsatisfactory model outcomes. In this study, we present a weakly supervised Cascade R-CNN (W-CRCNN) model to automatically detect HER2 overexpression in HER2 DISH and FISH images acquired from clinical breast cancer samples. The experimental results demonstrate that the proposed W-CRCNN achieves excellent results in identification of HER2 amplification in three datasets, including two DISH datasets and a FISH dataset. For the FISH dataset, the proposed W-CRCNN achieves an accuracy of 0.970±0.022, precision of 0.974±0.028, recall of 0.917±0.065, F1-score of 0.943±0.042 and Jaccard Index of 0.899±0.073. For DISH datasets, the proposed W-CRCNN achieves an accuracy of 0.971±0.024, precision of 0.969±0.015, recall of 0.925±0.020, F1-score of 0.947±0.036 and Jaccard Index of 0.884±0.103 for dataset 1, and an accuracy of 0.978±0.011, precision of 0.975±0.011, recall of 0.918±0.038, F1-score of 0.946±0.030 and Jaccard Index of 0.884±0.052 for dataset 2, respectively. In comparison with the benchmark methods, the proposed W-CRCNN significantly outperforms all the benchmark approaches in identification of HER2 overexpression in FISH and DISH datasets (p<0.05). With the high degree of accuracy, precision and recall , the results show that the proposed method in DISH analysis for assessment of HER2 overexpression in breast cancer patients has significant potential to assist precision medicine.

O → S Acyl Transfer Directing Human Sera Glycoprotein Immobilization for Breast Cancer Diagnosis.

Breast cancer (BC) is one of the most common malignancies in the world with aberrantly expressed glycans. The different types and stages still limit a comprehensive method in the prediagnosis of BC patients. In this research, a synthetic boronic acid-disulfide (BASS) probe has been developed for the two steps of O → S → N acyl transfer in glycoprotein recognition and labeling. The specificity and sensitivity of this method have been carefully studied in the case of immunoglobulin G, and the labeling efficiency was determined up to 60%. The BASS-functionalized slide is a powerful platform for monitoring the alteration of glycan patterns in human sera. Compared to the samples from healthy individuals, sera of BC patients gave specific patterns to eight lectins binding. The BASS-directed glycoprotein strategy promises a rapid sensing platform for a high-throughput screening of clinical BC samples and could be easily applied to other cancer prediagnoses.

Anterior gradient-2 regulates cell communication by coordinating cytokine-chemokine signaling and immune infiltration in breast cancer.

Anterior gradient-2 (AGR2) is crucial to breast cancer progression. However, its role in the tumor immune microenvironment remains unclear. RNA sequencing expression profiles and associated clinical information were downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases, respectively. The AGR2 expression patterns were verified using clinical samples of breast cancer. Based on single-cell transcriptomic data, AGR2 expression patterns were identified and cell communication analysis was carried out. Furthermore, the roles of AGR2 in breast tumor progression were explored by a series of functional experiments. We found that DNA methylation was an important mechanism for regulating the expression patterns of AGR2. Patients with AGR2 low expression displayed an immune "hot" and immunosuppressive phenotype characterized by high abundance of tumor immune cell infiltration and increased enrichment scores for transforming growth factor-β (TGF-β) and epithelial-mesenchymal transition pathways, whereas patients with AGR2 high expression showed an opposite immunologic feature with a lack of immune cell infiltration, suggestive of an immune "cold" and desert phenotype. Moreover, single-cell analysis further revealed that AGR2 in malignant cells alters cell-cell interactions by coordinating cytokine-chemokine signaling and immune infiltration. Notably, two immunotherapy cohorts revealed that AGR2-coexpressed genes could serve as prognostic indicators of patient survival. In conclusion, AGR2 could promote breast cancer progression by affecting the tumor immune microenvironment. Patients with AGR2 low expression could be suitable for combination treatment with immune checkpoint inhibitor agents and TGF-β blockers. Therefore, this study provides a theoretical foundation for developing a strategy for personalized immunotherapy to patients with breast cancer.

Prognostic and Predictive Value of LIV1 Expression in Early Breast Cancer and by Molecular Subtype.

LIV1 is a transmembrane protein that may become a new therapeutic target through the development of antibody-drug conjugates (ADCs). Few studies are available regarding the assessment of LIV1 expression in clinical breast cancer (BC) samples. We analyzed LIV1 mRNA expression in 8982 primary BC. We searched for correlations between LIV1 expression and clinicopathological data, including disease-free survival (DFS), overall survival (OS), pathological complete response to chemotherapy (pCR), and potential vulnerability and actionability to anti-cancer drugs used or under development in BC. Analyses were performed in the whole population and each molecular subtype separately. LIV1 expression was associated with good-prognosis features and with longer DFS and OS in multivariate analysis. However, patients with high LIV1 expression displayed a lower pCR rate than patients with low expression after anthracycline-based neoadjuvant chemotherapy, including in multivariate analysis adjusted on grade and molecular subtypes. LIV1-high tumors were associated with higher probabilities of sensitivity to hormone therapy and CDK4/6 inhibitors and lower probabilities of sensitivity to immune-checkpoint inhibitors and PARP inhibitors. These observations were different according to the molecular subtypes when analyzed separately. These results may provide novel insights into the clinical development and use of LIV1-targeted ADCs by identifying prognostic and predictive value of LIV1 expression in each molecular subtype and associated vulnerability to other systemic therapies.

Homologous recombination deficiency prediction using low-pass whole genome sequencing in breast cancer

Homologous recombination repair deficiency (HRD) results in a defect in DNA repair and is a frequent driver of tumorigenesis. Poly(ADP-ribose) polymerase inhibitors (PARPi) or platinum-based therapies have increased theraputic effectiveness when treating HRD positive cancers. For breast cancer and ovairan cancer HRD companion diagnostic tests are commonly used. However, the currently used HRD tests are based on high-depth genome sequencing or hybridization-based capture sequencing, which are technically complex and costly. In this study, we modified an existing method named shallowHRD, which uses low-pass whole genome sequencing (WGS) for HRD detection, and estimated the performance of the modified shallowHRD pipeline. Our shallowHRD pipeline achieved an AUC of 0.997 in simulated low-pass WGS data, with a sensitivity of 0.981 and a specificity of 0.964; and achieved a higher HRD risk score in clinical BRCA-deficient breast cancer samples (p = 5.5 × 10−5, compared with BRCA-intact breast cancer samples). We also estimated the limit of detection the shallowHRD pipeline could accurately predict HRD status with a minimum sequencing depth of 0.1 ×, a tumor purity of > 20%, and an input DNA amount of 1 ng. Our study demostrates using low-pass sequencing, HRD status can be determined with high accuracy using a simple approach with greatly reduced cost.

Label-free detection of MiRNA biomarkers using broadband multi-resonant infrared metasurfaces for early breast cancer diagnosis.

Breast cancer is the most prevalent cancer globally. Early detection is crucial and can be achieved by detecting cancer biomarkers in blood, such as circulating miRNAs (microRNAs). In this study, we present a label-free detection method based on broadband multi-resonant infrared metasurface for surface-enhanced infrared absorption (SEIRA) spectroscopy to detect miRNAs. The SEIRA resonances were optimized to match the miRNA biomarker fingerprint regions in the range of 800 to 2000 cm-1 and 2800 to 3500 cm-1, resulting in a simulated resonance enhancement of up to 103 times. Nine patient samples (six cancerous and three non-cancerous) were measured using SEIRA multi-well sensor chips. A novel analysis method, SEIRA-AR, was also developed to benchmark the results against industry standards, such as quantitative reverse transcription polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Our results showed an excellent linear correlation with a Pearson's r value of up to 0.99 and an R Squared value of up to 0.98. This study represents the first use of a SEIRA sensor for biomarker detection on clinical breast cancer samples and introduces an analysis method that produces results comparable to industry standards. Our findings pave the way for routine cancer diagnosis in the future. Additionally, the method discussed can be generalized to other biosensing activities involving two-step binding processes with complementary molecule-capturing agents.