Tissues Of Breast Cancer Patients Research Articles

Investigation of promoter methylation patterns association with genes expression profile of ISL1, MGMT and DMNT3b in tissue of breast cancer patients.

Cancer initiation and progression could influenced by both genetic and epigenetic events revealing of the overlap between epigenetic and genetic alteration can give important insights into cancer biology. In this experiment ISL1, MGMT, DMNT3b genes were candidate to investigate both methylation status and expression profile by using methylation-specific PCR and real time PCR in 40 breast cancer patients, respectively, also we have assessed relation of the promoter methylation status and expression variation of the target genes. The mean level of methylation of ISL1 and MGMT in tumor tissues were significantly greater than normal tissues. In Contrast, DMNT3b gene was showed lower mean level of methylation in tumor tissue compared to normal tissues, however, this was not statistically significant. Relative expression analysis was displayed a significant reduction in expression level of ISL1 and MGMT in tumor tissues. Furthermore, there was a meaningful association between down expression of ISL1 with histological grade, Her2 and ER status. Moreover, MGMT down expression was significantly associated with tumor sizes. Any remarkable relation was not observed between DMNT3b expression level and clinic pathological features. At the end, significant relation between methylation status and expression level has been revealed. In this study all observed results were exactly in line with the results were obtained from articles which were based on the methylation research and illustrate that the real-time PCR and methylation methods are in correlated with each other, furthermore, selected genes are capable to use as a potential biomarkers, however, more research on extended cases are needed.

Creation of a collection of frozen tumor tissue and deemed healthy tissues of breast cancer patients

<h3>ЦЕЛЬ ИССЛЕДОВАНИЯ</h3> Создать коллекцию образцов опухолевых и условно-здоровых тканей больных раком молочной железы для изучения молекулярных механизмов, которые потенциально могут вызывать резистентность к выбранной терапии, а также для более глубокого изучения молекулярной гетерогенности метастазирующего рака молочной железы. <h3>МАТЕРИАЛ И МЕТОДЫ</h3> Создание коллекции произведено в соответствии с разработанными стандартными операционными процедурами, включавшими сбор клинической информации и информированного добровольного согласия на передачу биологического материала, аналитический этап, контроль качества биообразцов, а также хранение в парах жидкого азота в криохранилище биобанка ФГБУ «НМИЦ онкологии» Минздрава России при температуре –196 °C. <h3>РЕЗУЛЬТАТЫ</h3> Результатом работы стало создание аннотированной коллекции замороженных образцов опухолевых и условно-здоровых тканей, включающей 1 400 образцов. Биологические образцы получены от 350 пациенток старше 18 лет, находившихся на лечении в ФГБУ «НМИЦ онкологии» Минздрава России по поводу рака молочной железы. Все пациентки подписали информированное добровольное согласие на передачу материала в биобанк с возможностью последующего использования в научно-исследовательских целях. Биологические образцы, а также сопутствующая клиническая информация деперсонализированы и депонированы в биобанке ФГБУ «НМИЦ онкологии» Минздрава России. <h3>ЗАКЛЮЧЕНИЕ</h3> Созданная коллекция является ценным ресурсом для фундаментальных исследований, поскольку обеспечит бесперебойный доступ исследователей к большому количеству биологических образцов, дополненных клинической информацией.

Identification of Circular RNA hsa-circ-0006969 as a Novel Biomarker for Breast Cancer

Background: To investigate the characteristics of circular RNA hsa-circ-0006969 in breast cancer and identify it as a novel biomarker for breast cancer. Methods: Three breast cancer (BC) patient tissues were selected to perform human circRNA microarray analysis. GeneSpring 13.0 (Agilent) software was applied for analyzing the data. Another 116 BC patients were recruited for verification. Hsa-circ-0006969 was found as a potential circRNA for BC diagnostic biomarker. The structure of hsa-circ-0006969 was predicted by circPrimer1.2 software. MiRanda v3.3, RNA hybrid 2.1, and Cytoscape 3.6.0 were used for predicting the networks of circRNA-miRNA. T-test, Curve regression, and ROC analysis were applied to certify the diagnostic values of hsa-circ-0006969. Using SPSS25.0 software to perform the Statistical analysis. Results: 546 higher expression and 1475 lower expression circRNAs were identified. Four circRNAs were filtrated for further verification. Hsa-circ-0006969 was significantly low expressed in BC tissues and peripheral blood. Hsa-circ-0006969 was confirmed as higher diagnostic correlation with BC tissues (AUC = 0.965) and peripheral blood (AUC = 0.842), with Grade 1 (AUC = 0.639), ER-positive (AUC = 0.612), TNM I (AUC = 0.693), TNM II (AUC = 0.712), TNM III (AUC = 0.757), TNM early stage(I, II) (AUC = 0.709). Hsa-circ-0006969 presented more effective diagnostic values for tumor metastasis (AUC = 0.784) compared with CA153 (AUC = 0.752). Conclusion: Hsa-circ-0006969 could be a novel biomarker for the diagnosis and treatment of BC.

Evaluation of the association between centrosome amplification in tumor tissue of breast cancer patients and changes in the expression of CETN1 and CNTROB genes

One of the contributing factors of cancer is the chromosomal aberration caused by centrosome amplification (CA). Changes in the expression of centrosomal genes can cause centrosomal abnormalities. Among the genes involved are CETN1 and CNTROB. Considering the importance and prevalence of breast cancer the aim of this study was to investigate the expression of these genes and its association with CA in breast cancer in women. In this case-control study, 40 breast cancer patients were referred to Mehr hospital selected and invasive breast cancer was confirmed by pathological tests. IHC method was done on paraffin-embedded breast cancer tissue to evaluate centrosome amplification by targeting Pericentrin antibody. Real-time PCR performed to gene expression of CETN1 and CNTROB. The data were analyzed by t-test and ANOVA test. The results showed that CA in breast tumor cells significantly was more than adjacent normal cells (p ≤ 0.05). Results of CETN1 and CNTROB gene expression showed increase expression in tumor tissue compared to control, respectively (p = 0.01, P = 0.02). Our finding showed that alterations of CETN1 and CNTROB gene expression in cancer tissues of patients were excessive in comparison with normal tissues, which could cause cell proliferation, tumor growth and metastasis. It is suggested to find basic strategies for diagnosis and treatment of breast cancer needed more samples of breast cancer, and other genes involved in the function of centrosomes.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) has been frequently overexpressed in many types of malignancy, suggesting its oncogenic function. It recognizes phosphorylated serine or threonine (pSer/Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. The oncogenic transcription factor, Nrf2harbors the pSer/Thr-Pro motif. This prompted us to investigate whether Pin1 could bind to Nrf2 and influence its stability and function in the context of implications for breast cancer development and progression. The correlation between Pin1 and Nrf2 in the triple-negative breast cancer cells was validated by RNASeq analysis as well as immunofluorescence staining. Interaction between Pin1 and Nrf2 was assessed by co-immunoprecipitation and an in situ proximity ligation assay. We found that mRNA and protein levels of Pin1 were highly increased in the tumor tissues of triple-negative breast cancer patients and the human breast cancer cell line. Genetic or pharmacologic inhibition of Pin1 enhanced the ubiquitination and degradation of Nrf2. In contrast, the overexpression of Pin1 resulted in the accumulation of Nrf2 in the nucleus, without affecting its transcription. Notably, the phosphorylation of Nrf2 at serine 215, 408, and 577 is essential for its interaction with Pin1. We also identified phosphorylated Ser104 and Thr277 residues in Keap1, a negative regulator of Nrf2, for Pin1 binding. Pin1 plays a role in breast cancer progression through stabilization and constitutive activation of Nrf2 by competing with Keap1 for Nrf2 binding.

Genome-wide CRISPR/Cas9 knockout screening uncovers ZNF319 as a novel tumor suppressor critical for breast cancer metastasis

Breast cancer is prone to relapse and metastasize to many vital organs, contributing to most of the breast cancer-related death and accentuating the importance of systematic identification of key factors regulating the metastasis of breast cancer. In this study, we performed a genome-wide CRISPR/Cas9 knock out screen in an orthotopic murine model of breast cancer for essential genes monitoring the progression and metastasis of breast cancer. We found one member of the zinc finger protein (ZNF) family, i.e., ZNF319, was among the top candidate genes. We further confirmed the lower expression of ZNF319 in the tumor tissue of breast cancer patients by analyzing tissue sections with IHC staining and TCGA database. Consistently, higher expression of ZNF319 correlates with better clinical outcome in almost all subtypes of breast cancer. Moreover, knocking down or overexpressing ZNF319 in breast cancer cells dramatically affects the breast cancer growth and metastasis capacity both in vitro and in vivo, suggesting ZNF319 functions as a strong suppressor of breast cancer progression. Lastly, the transcriptome analysis on ZNF319-silenced breast cancer cells shows that ZNF319 is involved in multiple crucial signaling pathways and biological processes, especially in cell cycle and proliferation. GO and KEGG analyses of our RNA-seq results reveal the up-regulation of E2F and G2/M related genes in ZNF319-silenced cells, suggesting that ZNF319 monitors the cell cycle during the breast cancer progression through the regulation of the E2F target genes and G2/M checkpoint. In summary, our study identifies ZNF319 as a novel metastasis suppressor gene arresting tumor cell cycle in breast cancer and thus presents a novel potential therapeutic target for breast cancer treatment.

Adipocyte-derived exosomes may promote breast cancer progression in type 2 diabetes.

Obesity and metabolic diseases, such as insulin resistance and type 2 diabetes (T2D), are associated with metastatic breast cancer in postmenopausal women. Here, we investigated the critical cellular and molecular factors behind this link. We found that primary human adipocytes shed extracellular vesicles, specifically exosomes, that induced the expression of genes associated with epithelial-to-mesenchymal transition (EMT) and cancer stem–like cell (CSC) traits in cocultured breast cancer cell lines. Transcription of these genes was further increased in cells exposed to exosomes shed from T2D patient–derived adipocytes or insulin-resistant adipocytes and required the epigenetic reader proteins BRD2 and BRD4 in recipient cells. The thrombospondin family protein TSP5, which is associated with cancer, was more abundant in exosomes from T2D or insulin-resistant adipocytes and partially contributed to EMT in recipient cells. Bioinformatic analysis of breast cancer patient tissue showed that greater coexpression of COMP (which encodes TSP5) and BRD2 or BRD3 correlated with poorer prognosis, specifically decreased distant metastasis–free survival. Our findings reveal a mechanism of exosome-mediated cross-talk between metabolically abnormal adipocytes and breast cancer cells that may promote tumor aggressiveness in patients with T2D.

Circular RNA circ-ERBB2 promotes HER2-positive breast cancer progression and metastasis via sponging miR-136-5p and miR-198

BackgroundCircular RNAs (circRNAs) are pivotal regulators of various human cancers and circ-ERBB2 is abnormally expressed in breast cancer cells. However, the role and mechanism of circ-ERBB2 in HER2-positive breast cancer are still unknown.MethodsThe circ-ERBB2 expressions in the tumor tissues of HER2-positive breast cancer patients were tested using quantitative real-time PCR. The circ-ERBB2 function was investigated by cell counting kit 8 assay, Transwell, flow cytometry and Western blot. Mechanistically, fluorescence in situ hybridization, RNA immunoprecipitation, RNA pull-down and dual-luciferase reporter gene assays were conducted to confirm the interaction between circ-ERBB2 and miR-136-5p or miR-198 in HER2-positive breast cancer cells.ResultsCirc-ERBB2 was elevated in the tumor tissues of HER2-positive breast cancer patients. Functionally, the interference with circ-ERBB2 repressed HER2-positive breast cancer cell proliferation, migration, invasion and accelerated cell apoptosis. Furthermore, the mechanistic analysis corroborated that circ-ERBB2 acted as a competing endogenous RNA for miR-136-5p or miR-198 to relieve the repressive influence of miR-136-5p or miR-198 on its target transcription factor activator protein 2C (TFAP2C). Meanwhile, in vivo assays further corroborated the oncogenic function of circ-ERBB2 in HER2-positive breast cancer.ConclusionsCirc-ERBB2 accelerated HER2-positive breast cancer progression through the circ-ERBB2/miR-136-5p/TFAP2C axis or the circ-ERBB2/miR-198/TFAP2C axis.

Evaluation of Angiogenesis by Using CD105 and CD34 in Sudanese Breast Cancer Patients.

&lt;b&gt;Background and Objective:&lt;/b&gt; Angiogenesis is a mechanism by which new blood vessels are developed in healing and tumour tissues, where it is necessary for regeneration growth, tumour cells survival and metastasis. This study aimed to assess the angiogenesis mechanism among Sudanese females with breast cancer using anti-CD34 and anti-CD105 markers. &lt;b&gt;Materials and Methods:&lt;/b&gt; Three hundred female representative Formalin-Fixed Paraffin-Embedded (FFPE) breast tissue blocks were included in this study. Of the 300 representative tissue blocks, 200 were breast cancer patient's tissues (confirmed cases) and 100 were normal breast tissues (controls). Their ages mean±SD, 47.3±12.9 years. &lt;b&gt;Results:&lt;/b&gt; The results showed the MVD of CD34 significantly increased in malignant lesions as compared to normal breast tissues. The mean of MVD CD34 and MVD CD105 showed statistical differences among different histologic types of breast cancer. Also, a strong positive correlation was detected between the manual and automated MVD counting methods. Also, the current study revealed no significant differences were observed in mean MVD counting for both markers and menopausal status or the age groups of the study population. &lt;b&gt;Conclusion:&lt;/b&gt; The MVD is a good tool for assessing prognostic markers. The CD105 marker has a high specificity to the new evolving tumour vessels and is a useful predictor for angiogenesis and breast cancer metastasis.

The Utility of SOX2 and AGR2 Biomarkers as Early Predictors of Tamoxifen Resistance in ER-Positive Breast Cancer Patients.

Background Despite the undeniable benefit of tamoxifen therapy for ER-positive breast cancer patients, approximately one-third of those patients either do not respond to tamoxifen or develop resistance. Thus, it is a crucial step to identify novel, reliable, and easily detectable biomarkers indicating resistance to this drug. Objective The aim of this work is to explore SOX2 and AGR2 biomarker expression in the tumor tissue of ER-positive breast cancer patients in combination with the evaluation of serum AGR2 level of these patients in order to validate these biomarkers as early predictors of tamoxifen resistance. Methods This study was conducted on 224 ER-positive breast cancer patients. All patients were primarily subjected to serum AGR2 levelling by ELISA and their breast cancer tissue immunostained for SOX2 and AGR2. After 5 years of follow-up, the patients were divided into 3 groups: group 1 was tamoxifen sensitive and groups 2 and 3 were tamoxifen resistant. Time to failure of tamoxifen treatment was considered the time from the beginning of tamoxifen therapy to the time of discovery of breast cancer recurrence or metastases (in months). Results SOX2 and AGR2 biomarkers expression and serum AGR2 level were significantly higher in groups 2 and 3 in comparison to group 1, while the relationship between Her2 neu expression and Ki67 index in the 3 different groups was statistically nonsignificant. Lower SOX2 and AGR2 expression and low AGR2 serum levels in the studied patients of groups 2 and 3 were significantly associated with longer time-to-failure of tamoxifen treatment. According to the ROC curve, the combined use of studied markers validity was with a sensitivity of 100%, specificity of 96%, PPV 96%, and NPV 100% (p < 0.001; AUC: 0.984). Conclusions Integrated use of SOX2 and AGR2 biomarkers with serum AGR2 assay holds a promising hope for their future use as predictive markers for early detection of tamoxifen resistance in ER-positive breast cancer patients.

MiR-370-3p as a Novel Biomarker Promotes Breast Cancer Progression by Targeting FBLN5.

miRNAs play a crucial part in multiple biological processes of cell proliferation, migration, apoptosis, and chemoresistance. In cancer, miRNAs can be divided into oncogenes or tumor suppressors on the basis of their functions in the carcinogenic process. The purpose of this study was to explore the roles and clinical diagnostic value of miR-370-3p in breast cancer. Our results demonstrated that miR-370-3p significantly promoted proliferation, metastasis, and stemness of breast cancer in vitro and in vivo. In particular, clinical data revealed that high expression of serum miR-370-3p and exosomal miR-370-3p from breast cancer patients was remarkably correlated with lymphatic metastasis and tumor node metastasis (TNM) stages. Mechanistically, miR-370-3p inhibited FBLN5 expression and activated the NF-κB signaling pathway to promote breast cancer cell proliferation, migration, and stemness. FBLN5 expression was significantly decreased in breast cancer cells and tumor tissues of breast cancer patients. Our research identified that miR-370-3p promoted breast cancer progression by inhibiting FBLN5 expression and activating the NF-κB signaling pathway. Serum exosomal miR-370-3p would provide a potential biomarker for the diagnosis of breast cancer.

Crown-like structures in breast adipose tissue of breast cancer patients: associations with CD68 expression, obesity, metabolic factors and prognosis

Crown-like structures of the breast (CLS-B), defined by the clustering of macrophages (identified using CD68 immunohistochemical staining) to surround a dying adipocyte, are a sign of adipose-tissue inflammation. In human cohorts, CLS-B positively correlates with older age, obesity, dyslipidemia and higher levels of glucose, insulin, C-reactive protein and IL-6. In an existing cohort of early-stage breast cancer patients, CLS-B were identified using H&E stained histologic sections (hCLS-B), and by CD68 immunohistochemistry (CD68 + CLS-B). We examined associations of H&E and CD68-detected CLS-B with clinicopathologic features using χ2 tests, with metabolic factors using Wilcoxon rank sum tests and with disease free and overall survival using Cox regression models. hCLS-B were detected in 59 of 163 patients with slides (36.2%) and CD68 + CLS-B in 37 of 119 patients with paraffin blocks (31.1%). hCLS-B were positively correlated with higher weight (p = 0.003), BMI (p = 0.0008) and C-reactive protein (p = 0.045). CD68 + CLS-B were positively correlated with higher weight (p = 0.006), BMI p = 0.001), leptin (p = 0.034), insulin (p = 0.008) and Homeostasis Model Assessment (p = 0.027). CD68 + CLS-B were associated with poor distant disease-free with a hazard ratio (HR) of 2.81, 95% confidence interval (CI) 1.20–6.57, and overall survival with HR 3.97 (1.66–9.48), while hCLS-B were not associated with either: HR for distant recurrence 0.59 (0.26–1.30); HR for death 1.04 (0.50–2.16). The presence of hCLS-B and of CD68 + CLS-B were associated with obesity; CD68 + CLS-B were associated with insulin resistance and adverse prognosis. Similar patterns were not seen for hCLS-B. Research is needed to understand the biologic basis for these differences.

A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akthigh/p-gH2AXlow, MCL1high/p-gH2AXlow and GRP78high/VIMhigh breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis.

Breast Cancer, Diabetes Mellitus and Glucagon-Like Peptide-1 Receptor Toward Exploring Their Possible Associations.

Diabetes Mellitus (DM) has been one of the well known risk factors of breast cancer (BC) development and also associated with adverse clinical outcomes of BC patients. Glucagon-like peptide-1 (GLP-1) receptor agonists have been used as antidiabetic therapeutic agents and recent epidemiological studies have reported their use to be correlated with increased BC risks. However, biological or pathological details have remained unknown. Therefore, in this study, we examined the status of GLP-1 receptor (GLP-1R) in BC with and without DM and correlated the findings with the clinicopathological factors of the patients to explore the possible involvement of GLP-1 in BC pathology. We immunolocalized GLP-1R in cancer and adjacent non-pathological breast tissues in BC patients with DM (125 cases) and without DM (58 cases). We then compared the status of GLP-1R with that of fibroblast growth factor 7 (FGF7) and fibroblast growth factor receptor 2 (FGFR2), Ki-67 labeling index (Ki-67 LI) and disease free survival (DFS) of the patients and also between cancerous and non-pathological breast tissues. GLP-1R immunoreactivity was significantly higher (p = 0.044) in the patients with DM than without in carcinoma tissues. However, this was detected only in invasive carcinoma (p < 0.01) and not in non-invasive carcinoma nor non-pathological mammary glands. FGF7 was significantly correlated with the status of GLP-1R in BC (p = 0.045). In addition, in ER positive BC cases, those with GLP-1R positive status tended to have higher Ki-67 LI of more than 14% (p = 0.070). These findings all demonstrated the possible association between GLP-1R status and biological features of BC, especially of invasive BC in DM patients.

Abstract LB122: The complete estrogen receptor antagonist (CERAN) OP-1250 shrinks ER+ brain metastases in an intracranial xenograft tumor model expressing mutant ESR1

Abstract Estrogen receptor-positive (ER+) breast cancer can undergo metastatic spread to the brain, which occurs in up to 15% of HER2-/ER+ metastatic breast cancer patients. Although endocrine therapy is not explicitly approved for brain metastasis and clinical trials are lacking, anecdotal reports of clinical responses by endocrine agents motivate further investigation. One such agent is the ER antagonist tamoxifen, which has previously been shown to reach high levels in brain tissue and ER+ brain metastases in patients with breast cancer*. We previously reported that OP-1250, a novel, oral complete ER antagonist (CERAN), penetrates the brain and is efficacious at shrinking tumors in a xenograft brain metastasis model in which patient derived human ER+ tumors were intracranially transplanted into immunocompromised mice**. Here we compare the ability of OP-1250 to shrink brain tumors in this model with tamoxifen and other agents. Whereas tamoxifen indeed had therapeutic effects in this brain xenograft model, the effect was not as profound as that of OP-1250, which was able to completely clear the tumors in many animals, as determined by MRI. * Lien et al. Distribution of tamoxifen and metabolites into brain tissue and brain metastases in breast cancer patients. Br. J. Cancer, 1991, 63, 641-645.**Hodges-Gallagher, et al. OP-1250 a complete estrogen receptor antagonist that penetrates the brain and prevents lethality from an intracranial xenograft tumors expressing mutant ESR1, abstract, AACR 2020 Citation Format: Leslie Hodges-Gallagher, Alison D. Parisian, Richard Sun, David C. Myles, Pamela M. Klein, Cyrus L. Harmon, Peter J. Kushner. The complete estrogen receptor antagonist (CERAN) OP-1250 shrinks ER+ brain metastases in an intracranial xenograft tumor model expressing mutant ESR1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB122.

Abstract 2264: Molecular detection and genotyping of human papillomavirus in presence of some oncogenic viruses among Egyptian breast cancer women

Abstract Introduction High risk human papillomaviruses (HR-HPVs) has a possible role in development and pathogenesis of breast cancer (BC) disease. Other oncogenic viruses showed to be linked to BC such as Epstein Barr virus (EBV), and Human mammary tumor virus (HMTV). Presence of high risk HPV genotypes, EBV or HMTV could affect the severity course of BC. Therefore, the aim of the current study is to explore distribution of HPV with regards to its genotypes and the presence of EBV and HMTV among Egyptian BC women with its correlation to age and hormonal status. Patients &amp; Methods This study was conducted on 40 fresh tissues of BC patients and blood samples from 30 apparently healthy controls. All samples were investigated for detection of HPV, EBV and HMTV DNAs using qualitative PCR. Detection of HPV viral load was performed using quantitative real time PCR. HPV genotypes were determined on 10 purified positive PCR product using Sanger sequencing of HPV L1 region. The obtained sequences were compared to the known HPV DNA sequences at National center for biotechnology information (NCBI) and/or Papillomavirus Episteme (Pave) data bases using their online BLAST and taxonomy tools. Data were correlated with age and hormonal status. Results All the tested 3 oncogenic viruses were not detected into 30 controls. HPV, EBV and HMTV were detected in 20 (50%), 23 (57.5%), 28 (70%) respectively of the 40 BC patients by using PCR assay. HPV median load of the 20 positive samples was 4800 copies/μL. Seven different HPV genotypes were detected included high risk genotypes ((HPV 18 in 3 samples), 70 and 52), low risk genotype (11) detected into only one sample along with 3 other genotypes (102, 114 and 151). One sample showed no significant similarity with any sequence registered in the used databases. Presence of EBV and HMTV DNAs and high risk HPV genotypes was more associated with young age (lower than 50 years), P value = 0.015, 0.001 and 0.05, respectively but were not associated with hormonal status of BC patients. Conclusion HMTV was the most detected viral DNAs among BC patients, High risk HPVs were identified into 10% of studied Egyptian BC patients and were associated with young age. Further larger sample size study is needed to evaluate role of HPV genotypes on pathogenesis and outcome of BC disease. STDF Acknowledgment This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No.22944. Citation Format: Nasra F Abdel Fattah, Shimaa A Metwally, Samah A Loutfy, Maha A Abo-Shadi, Ahmed B Barakat, Omar A Rabee, Ahmed M Osman, Amany M Helal, Tarek M Hashem, Manar M Moneer. Molecular detection and genotyping of human papillomavirus in presence of some oncogenic viruses among Egyptian breast cancer women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2264.

Copper-64-Labeled Daratumumab As a PET Imaging Tracer for Multiple Myeloma: Potential Use for Minimal Residual Disease Detection

Introduction : Minimal residual disease (MRD) is known to be an independent predictor of progression free survival (PFS) in multiple myeloma (MM). Methods of MRD detection include flow cytometry, PCR based sequencing, and imaging. However, irrespective of the sensitivity of MRD detection, techniques based on bone marrow biopsies cannot account for a “patchy,” non-homogenous bone marrow infiltration. Positron emission tomography (PET) coupled with computed tomography (CT) using 18fluoro-deoxyglucose as the radiotracer (18FDG-PET) is a commonly used imaging method that is much more sensitive than traditional radiographs, but it has several limitations; for one, lesions with a low metabolic rate may result in false negatives. Also, diffuse tracer uptake by normal marrow can obscure focal MM lesions. Daratumumab (Dara), a human anti-CD38 IgG1 (ĸ subclass) antibody against the highly expressed plasma cell receptor CD38, has been recently approved by the FDA for the treatment of MM. Based on the idea that Dara can selectively target MM cells irrespective of their metabolism, our group labeled Dara with the positron-emitting isotope copper-64 (64Cu-DOTA-Dara) in order to generate a novel imaging agent that can detect MM cells with high specificity. Our group already safely used 64Cu-labeled trastuzumab to detect HER2-positive tissue in breast cancer patients (Mortimer JE et. al. 2014), and here we provide the first pre-clinical evidence that 64Cu-labeled antibodies can be considered powerful imaging tools for MM detection.Methods :Commercially availableDara was conjugated with the NHS active ester of DOTA (Macrocyclics). DOTA-conjugated antibody was incubated with 64Cu, chased with 1 mM diethylenetriamine pentaacetic acid (DTPA), and purified on a preparative size-exclusion column. The immunoreactivity of 64Cu-DOTA-Dara was also performed using purified CD38-Fc (Chimerigen Laboratories) in an HPLC size shift assay. To assess whether 64Cu-DOTA-Dara can localize to MM cells in vivo, 5 x 106 human MM1S GFP+/Luc+ cells were injected intravenously into 6 NOD-SCID mice. At 14 days, when the bioluminescence signal in the bones of the mice were detected, mice were injected with 64Cu-DOTA-Dara (50 µCi, 10 µg), and PET imaging was performed at 21 and 44 hr after the injection (Figure 1A). Cancer free mice were used as controls (Figure 1B). 18F-FDG PET/CT scans in the same group of mice were also performed. Biodistribution studies and histological analysis were also conducted.Results : 64Cu-DOTA labeling did not affect Dara immunoreactivity against the antigen in the HPLC size shift assay. The biodistribution studies show that 64Cu-DOTA-Dara was highly detectable in the femurs, tibias, sternum and skull, known sites of MM cell invasion, which were negative in cancer-free control mice. The radionuclide was also highly detected in the lung, liver, and spleen, relative to controls. In contrast, the majority of 64Cu-DOTA-Dara remained in the blood of the control mice. Moreover, PET images show that 64Cu-DOTA-Dara PET/CT displays a higher resolution and specificity in detecting MM cells in the bones not only compared to 18F-FDG PET/CT, but even compared to bioluminescence signals from the MM cell line. To assess the specificity of 64Cu-Dara signal compared to 18F-FDG, studies using step sections in histological analysis are in process.Conclusions : Our data support the use of 64Cu-DOTA-Dara as a novel sensitive imaging agent to detect MM. The relatively short half life of 64Cu (12.7 hours) limits potential marrow toxicity and is sufficiently long for PET/CT imaging up to 48 hours after administration of the radionuclide. A pilot study assessing the safety and feasibility of 64Cu-DOTA-Dara imaging in human subjects is under way. [Display omitted] DisclosuresKeats:Amgen: Research Funding. Krishnan:Takeda: Speakers Bureau; Onyx: Speakers Bureau; Sutro: Consultancy; Celgene: Consultancy, Equity Ownership, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Inhibition of Ras and STAT3 activity of 4-(tert-butyl)-N-carbamoylbenzamide as antiproliferative agent in HER2-expressing breast cancer cells.

Human epidermal growth factor receptor type 2 (HER2)-expressing breast cancer patients indicate poor prognosis in disease progression. HER2 overexpression can increase activities of Ras-mitogen activated protein kinase (Ras-MAPK) pathway and Janus Kinase (JAK)-STAT3, increasing breast cancer cell proliferation as demonstrated by marker Ki67. Therapeutic options for HER2-expressing breast cancer arelimited and have major side effects, so anticancer development as an antiproliferative is needed. From previous research, synthetic chemical 4-(tert-butyl)-N-carbamoylbenzamide (4TBCB) compound has cytotoxic activity in vitro on HER2-expressing breast cancer cells. This study wanted to determine the mechanism 4TBCB compound in inhibiting HER2 signaling through Rat Sarcoma (Ras) and signal transducer and activator of transcription 3 (STAT3) pathway in HER2-expressing breast cancer cells. Breast cancer cells were isolated from the biopsy tissue of breast cancer patients. The isolated cells were cultured and given 4TBCB test compound with three concentrations (0.305, 0.61, and 1.22mM) and lapatinib 0.05mM as a comparison compound. Cancer cell cultures were stained with monoclonal antibodies phosphorylated HER2 (pHER2), phosphorylated Ras (pRas), phosphorylated STAT3 (pSTAT3), and Ki67. The expression of pHER2, pRas, pSTAT3, and Ki67 proteins was observed using the immunofluorescence method and the results were compared with control cells, namely cancer cells that were not given 4TBCB and lapatinib but stained with monoclonal antibodies. 4TBCB compounds (0.61 and 1.22mM) and lapatinib can reduce pHER2, pRas, pSTAT3, and Ki67 expressions compared to control cells. 4TBCB compounds (0.61 and 1.22mM) can reduce pHER2, pRas, pSTAT3, Ki67 expressions and predicted to inhibit HER2 signaling through the Ras and STAT3 pathways in HER2-expressing breast cancer cells.

Higher PD-L1 Immunohistochemical Detection Signal in Frozen Compared to Matched Paraffin-Embedded Formalin-Fixed Tissues.

Purpose: Response to anti-PD-L1/PD-1 immunotherapy correlates with PD-L1 expression in breast cancer. However, the prevalence of PD-L1 positive breast cancer is variable, which could be due to differences in the population/cohort of patients tested or the preservation/detection technology used. To investigate this variability, we examined the effect of two tissue preservation methods on PD-L1 immunohistochemical detection in breast cancer. Methods: We compared PD-L1 expression in patient-matched frozen (FR) and formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. PD-L1 expression was assessed using tumor proportion score (TPS, simply PD-L1 score), and case positivity was determined with PD-L1 score ≥5. Results: In FFPE tissues, PD-L1 was positive in 7–10% of tested patients, depending on the antibody used. In patient-matched FR tissues, the same antibodies showed positive PD-L1 expression in 20–30% of cases. The impact of the antibody tested on the rate of PD-L1 positivity (% of PDL1 positive cases) was minor, as evident in the near perfect concordance between PD-L1 score obtained using the different antibodies whether tested in FR or FFPE tissues. However, there was a systematic drop by an average of 13–20% in the PD-L1 score obtained in FFPE tissues compared to their patient-matched FR tissues. Conclusions: In the tested patient-matched cohort, there was consistently a higher PD-L1 score in FR than FFPE tissues, regardless of the antibody used, demonstrating a significant effect on PD-L1 detection due to the preservation method. These findings should inspire further work to improve the sensitivity of PD-L1 detection and possibly search for more sensitive antibodies in FFPE tissues.

Urokinase type plasminogen activator receptor (uPAR) and human epidermal growth factor receptor 2 (HER2) expression in metastasis of breast cancer.

The plasminogen urokinase activation system consists of urokinase plasminogen activator (uPA), its receptor uPAR, and plasminogen activator inhibitor type 1 (PAI-1), which are considered to have a relationship with cancer aggressiveness. Several studies have found correlations between HER2 mRNA and uPAR in disseminated tumor cells (DTCs) in breast cancer patients. They are associated with a more aggressive primary tumor phenotype and recurrence/metastasis. This study aims to determine the relationship between the expression of urokinase-type plasminogen activator receptor (uPAR) and human epidermal growth factor receptor type 2 (HER2) with the incidence of distant metastases in breast cancer. This study was an observational study using a cross-sectional method and was conducted at Wahidin Sudirohusodo Hospital and the network. Immunohistochemical methods carry out examination of uPAR and HER2 expression from tissues of breast cancer patients. The relationship of uPAR, HER2 expression, and metastasis was tested with the Mann Whitney test. The study results found that the proportion of patients with metastasis was significantly higher in high uPAR expression compared to low uPAR (77.8% compared to 36.8%). The negative HER2 expression was significantly higher in the low uPAR expression than the high uPAR (78.9% compared to 33.3%). In comparison, the positive HER2 expression was significantly higher in the high uPAR expression than the low uPAR (66.7% compared to 21.1%). In positive HER2 expression, the mean percentage of uPAR expression was significantly higher in metastases than those without metastasis (72.7% compared to 42.1%). uPAR expression is associated with metastasis in HER2 positive breast cancer.