Identification of miR-1256 Signature as a Promising Diagnostic Biomarker in FFPE Tissues of Breast Cancer Patients

Researchers have recently begun to investigate microRNAs (miRNAs) as a potential new class of biomarkers. In this study, we examine whether miR-3652 may be regarded as a powerful biomarker for predicting the prognosis of breast cancer (BC). In this study, the expression level of miR-3652 was detected in 30 FFPE tissue pairs of tumoral samples and their adjacent non-tumoral tissues using qRT-PCR. The clinicopathological characteristics of patients relative to miR-3652 expression level, along with fold change analysis employing the 2-ΔΔCT method, were also investigated. All statistical analyses were conducted using GraphPad Prism version 8.4.3 (686). We found that the miR-3652 level in BC FFPE tissues is notably increased compared to matched non-cancerous specimens. Also, we analyzed the association between the expression level of miR-3652 and the clinicopathological parameters of BC patients. The overexpression of miR-3652 exhibited no significant association with age, tumor stage, ER status, PR status, TNM stage, or calcification. All HER2-negative status patients suggested a potential trend (P = 0.017), indicating that low miR-3652 expression was more common in HER2-negative status subjects. Moreover, the p-value, AUC, Std. Error, sensitivity, and specificity are (p < 0.018, 0.6772, 0.06905, 0.53, and 0.80, respectively), which signifies a moderate capacity of the test to differentiate between patients with tumors and controls. The results of fold change analysis employing the 2-ΔΔCT method indicated a (6.89)-fold elevation in miR-3652 expression in tumors relative to controls. This suggests that miR-3652 may have an oncogenic role in carcinogenesis, potentially promoting BC development. In conclusion, our results demonstrated that elevated miR-3652 expression associates with the progression of BC, and this indicates that miR-3652 may possess an oncogenic function in tumorigenesis, possibly facilitating BC progression. Additional studies are required.

BackgroundAn absence of reliable molecular markers has hampered individualised breast cancer treatments, and a major limitation for translational research is the lack of fresh tissue. There are, however, abundant banks of formalin-fixed paraffin-embedded (FFPE) tissue. This study evaluated two platforms available for the analysis of DNA copy number and gene expression using FFPE samples.MethodsThe cDNA-mediated annealing, selection, extension, and ligation assay (DASL™) has been developed for gene expression analysis and the Molecular Inversion Probes assay (Oncoscan™), were used for copy number analysis using FFPE tissues. Gene expression and copy number were evaluated in core-biopsy samples from patients with breast cancer undergoing neoadjuvant chemotherapy (NAC).ResultsForty-three core-biopsies were evaluated and characteristic copy number changes in breast cancers, gains in 1q, 8q, 11q, 17q and 20q and losses in 6q, 8p, 13q and 16q, were confirmed. Regions that frequently exhibited gains in tumours showing a pathological complete response (pCR) to NAC were 1q (55%), 8q (40%) and 17q (40%), whereas 11q11 (37%) gain was the most frequent change in non-pCR tumours. Gains associated with poor survival were 11q13 (62%), 8q24 (54%) and 20q (47%). Gene expression assessed by DASL correlated with immunohistochemistry (IHC) analysis for oestrogen receptor (ER) [area under the curve (AUC) = 0.95], progesterone receptor (PR)(AUC = 0.90) and human epidermal growth factor type-2 receptor (HER-2) (AUC = 0.96). Differential expression analysis between ER+ and ER– cancers identified over-expression of TTF1, LAF-4 and C-MYB (p ≤ 0.05), and between pCR vs non-pCRs, over-expression of CXCL9, AREG, B-MYB and under-expression of ABCG2.ConclusionThis study was an integrative analysis of copy number and gene expression using FFPE core biopsies and showed that molecular marker data from FFPE tissues were consistent with those in previous studies using fresh-frozen samples. FFPE tissue can provide reliable information and will be a useful tool in molecular marker studies.Trial RegistrationTrial registration number ISRCTN09184069 and registered retrospectively on 02/06/2010.

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.

Background: All initial therapeutic decisions in early breast cancer are commonly based on the expression profiles of estrogen (ER), progesterone (PR) and the human epidermal growth factor 2 (HER2) receptors. However, breast cancer is a very heterogeneous disease, and receptor changes were manifold reported during progression. Only little data is known about receptor changes after upfront therapy. Here, we compared receptor expression profiles between core needle biopsy (CNB) tissue and primary tumor tissue after different treatment regimes. Methods: In a German single center study, we retrospectively analyzed 248 breast cancer patients during primary treatment regime between 2014 and 2020. Patients had either neoadjuvant chemotherapy, neoadjuvant endocrine therapy or no upfront therapy. Tumor material was obtained by core needle biopsy (CNB) at primary diagnosis and during primary oncological surgery of the axilla and breast. Analysis of histological subtype, grading, Ki67 index and expression profiling of ER, PR, HER2, was performed using formalin-fixed, paraffin-embedded (FFPE) specimens. Immunohistochemical examination was performed according to the ASCO/CAP guidelines using the Ventana-platform. Tumors were grouped into different intrinsic subtypes according to the international St. Gallen classification in either Luminal A, Luminal B, HER2-enriched or triple negative. 35 patients were excluded because the intrinsic subtype was not specified in the CNB and final postoperative specimen, therefore 213 patients were included in the primary analysis. Results: In the primary analysis (n=213), median age was 53 years (IQR 43 - 64). Based on the initial CNB receptor profile 105 (49%) patients had a Luminal A carcinoma, 46 (22%) patients had a Luminal B HER2 negative carcinoma, 22 (10%) patients had a Luminal B HER2 positive carcinoma, 6 (3%) patients had a HER2 enriched carcinoma and 33 (16%) patients had a triple negative carcinoma. In the primary analysis 84 (39%) patients had neoadjuvant chemotherapy, 48 (23%) patients had neoadjuvant endocrine therapy and 81 (38%) patients had no upfront therapy. Overall, 77 (36%) patients had an intrinsic subtype change between CNB and definitive surgical treatment, 139 (64 %) patients had no subtype change. There were 44 (52%) changes after neoadjuvant chemotherapy, 17 (35%) changes after neoadjuvant endocrine therapy and 16 (20%) subtype changes after no upfront therapy (p<0.0001 for the effect of neoadjuvant chemotherapy). ER receptor status changed in 5 (6%) patients after neoadjuvant chemotherapy, 6 (13%) changes were observed after neoadjuvant endocrine therapy and 2 (2%) ER changes occurred after no upfront therapy. Concerning the PR receptor there were 23 (27%) receptor changes after neoadjuvant chemotherapy, 17 (35%) changes after neoadjuvant endocrine therapy and 9 (11%) changes after no upfront therapy (p<0.0001 for the effect of neoadjuvant endocrine therapy). Regarding the HER2 receptor, there were 18 (21%) receptor status changes after neoadjuvant chemotherapy, 2 (4%) changes after neoadjuvant endocrine therapy and 2 (2%) subtype changes after no upfront therapy (p=0.0001 for the effect of neoadjuvant chemotherapy).Neoadjuvant chemotherapy led significantly more often to a decrease of HER2 expression, compared to neoadjuvant endocrine therapy or no upfront therapy. Conclusions: Our results imply the high frequency of intrinsic subtype changes after neoadjuvant therapy. Subtype changes should be taken into account for an optimal and individual treatment. Further research needs to be conducted to investigate whether an individual treatment decision based on the receptor profile after primary treatment might improve clinical outcome of breast cancer patients. Citation Format: Laura Weydandt, Anne Kreklau, Ivonne Nel, Lars-Christian Horn, Bahriye Aktas. Heterogeneity between core needle biopsy and primary tumor tissue in early breast cancer patients: Comparison of intrinsic subtypes after different treatment regimes [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 P1-03-01.

MiR-93 is thought to be an onco-miRNA for its capabilities of enhancing tumor growth. The objective of this study was to evaluate the potential predictive value of miR-93 expression in formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. The expression of miR-93 was examined in 101 breast cancer patients and 40 controls using real-time quantitative PCR. We found that miR-93 was markedly upregulated in breast cancer patients compared with controls (p<0.01). The expression level of miR-93 was significantly correlated with miR-24/378 in breast cancer patients. MiR-93 exhibited great capability of discriminating between cancer patients and cancer-free controls by receiver-operator characteristic (ROC) curve analysis. MiR-93 showed 0.866 AUC (the area under the ROC curve) values. The MiR-93 level was found significantly correlated with breast cancer by univariable logistic regression. These results suggest that overexpression of miR-93 in FFPE tissues may serve as an indispensable source for biomarker discovery and validation in breast cancer patients.

Background: MicroRNAs (miRNAs) have important regulatory functions in breast cancer tumorigenesis. We previously found that let-7 miRNAs were significantly downregulated in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. Material and Methods: QRT-PCR was used to check let-7 family miRNAs in FFPE tissues and breast cancer cell lines as well as expression of estrogen receptor (ER)-≥36, a variant of ER-≥66, after let-7 miRNA transfecton. Immunoblot analysis was employed to check protein expression in FFPE tissue and breast cancer cell lines. Luciferase reporter assay was used to detect direct regulation of let-7 miRNA on ER-α expression. MTT assay was applied for cell proliferation after transfection of let-7 miRNAs. Results: We found that there was an inverse correlation between the expression of ER-≥36 and several members of let-7 family miRNAs in the FFPE tissue set. Let-7 miRNA sequences match sequence in the 3’ untranslated region (3’ UTR) of ER-≥36, indicating ER-≥36 may be a target of let-7. Cotransfection of let-7 mimics with ER-≥36 3’ UTR luciferase construct decreased the activity of reporter gene. Conversely, let-7 inhibitors enhanced the reporter gene activity. Transfection of let-7 mimics inhibited both the mRNA and protein levels of ER-≥36 and further inhibited the non-genomic estrogen pathway mediated by ER-≥36 in MDA-MB-231 cells. On the contrary, transfection of let-7 inhibitors enhanced the ER-≥36 expression at both mRNA and protein levels in 184A1 cells. The high expression of ER-≥36 in tamoxifen resistant MCF7 cells can be inhibited by transfection of let-7 mimics and sensitivity toward tamoxifen is enhanced. Conclusion: Let-7 miRNAs regulate the expression of ER-≥36, which is involved in non-genomic estrogen pathway and tamoxifen resistance. Let-7 could be therapeutic target for breast cancer treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-05.

Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues

Cancer is one of the leading causes of human death worldwide. One of the most common types of malignancy among women is breast cancer, which is the third most common cancer in the world after lung and stomach cancer. This study aimed to evaluate the expression level of Tissue Differentiation-Inducing Non-Protein Coding RNA (TINCR) in adjacent tumor and non-tumor tissues of 60 women with invasive ductal breast cancer. The relationship between TINCR expression and the clinical characteristics of patients has also been studied. For this purpose, total RNA was isolated from breast cancer patients' adjacent tumor and non-tumor tissue. RT Prime Script reagent was then used to convert total RNA to cDNA. The qRT-PCR quantified the TINCR expression level and analyzed the results by paired t-test. In addition, ROC curve analysis was used to evaluate the biomarker power of TINCR in breast cancer tumor tissues. According to the results, a decrease in the level of TINCR was obtained in the tumor tissue of breast cancer patients compared to the adjacent non-tumor tissue (P<0.001). TINCR expression was negatively correlated with tumor size and lymph node metastasis in breast cancer tumor tissue. In general, the decrease in the expression level of TINCR in the tumor tissue of breast cancer patients shows that its expression level can differentiate the adjacent tumor and non-tumor tissue from each other. In addition, TINCR has a lower expression level in breast cancer patients with large tumors, lymph node metastasis, and luminal subgroups A and B.

Using a recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method, human breast cancer formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) are evaluated for N-linked glycan distribution in the tumor microenvironment. Tissue sections representing multiple human epidermal growth factor receptor 2 (HER2) receptor-positive and triple-negative breast cancers (TNBC) in both TMA and FFPE slide format are processed for high resolution N-glycan MALDI-IMS. An additional FFPE tissue cohort of primary and metastatic breast tumors from the same donors are also evaluated. The cumulative N-glycan MALDI-IMS analysis of breast cancer FFPE tissues and TMAs indicate the distribution of specific glycan structural classes to stromal, necrotic, and tumor regions. A series of high-mannose, branched and fucosylated glycans are detected predominantly within tumor regions. Additionally, a series of polylactosamine glycans are detected in advanced HER2+, TNBC, and metastatic breast cancer tissues. Comparison of tumor N-glycan species detected in paired primary and metastatic tissues indicate minimal changes between the two conditions. The prevalence of tumor-associated polylactosamine glycans in primary and metastatic breast cancer tissues indicates new mechanistic insights into the development and progression of breast cancers. The presence of these glycans could be targeted for therapeutic strategies and further evaluation as potential prognostic biomarkers.

The exact cause of breast cancer is unknown; it is a multifactorial disease. It is the most diagnosed and the second killer cancer among women. Breast cancer can be originated from tissues of breast or secondary from other organs via metastasis. Generally, cancer cells show aberrant metabolism and oxidative stress when compared to noncancerous tissues of breast cancer patients. The current study aims at evaluating glutamate and glucose metabolism through GDH and LDH enzyme activities, oxidant, and antioxidative status among breast cancer patients attending referral hospitals of Addis Ababa, Ethiopia. Result. Catalytic activities of glutamate dehydrogenase, lactate dehydrogenase, and oxidative stress index were significantly increased in both serum (4.2 mU/ml, 78.6 mU/ml, and 3.3 : 1, resp.) and cancerous tissues (1.4 mU/ml, 111.7 mU/ml, and 2.15 : 1, resp.) of breast cancer patients as compared to those in serum of control group (3.15 mU/ml, 30.4 mU/ml, and 2.05 : 1, resp.) and noncancerous tissues of breast cancer patients (0.92 mU/ml, 70.5 mU/ml, and 1.1 : 1, resp.) (P ≤ 0.05). Correspondingly, ratios of reduced to oxidized glutathione were significantly decreased in both serum (20 : 1) and cancerous tissues (23.5 : 1) of breast cancer patients when compared to those in serum of control group (104.5 : 1) and noncancerous tissues of breast cancer patients (70.9 : 1) (P ≤ 0.05). Conclusion. Catalytic activities of GDH and LDH, ratios of GSH to GSSG, and concentration of TOS among breast cancer patients were significantly higher than were those among control group and noncancerous tissues of breast cancer patients, while TAC of breast cancer patients is significantly lower than that of control group and normal tissues of breast cancer patients.

It is well established that carcinoma-associated fibroblasts (CAFs) differ phenotypically from fibroblasts associated with normal tissue, but the mechanisms underlying these differences remain controversial. Because CAFs can be propagated in vitro for extended periods and still maintain their cancer promoting phenotype, it has been proposed that they might have acquired somatic genetic alterations analogous to those observed in malignant epithelium. Whereas some investigators have reported frequent and profound genomic alterations in CAFs, other groups have found no such evidence. One striking common trait of those studies reporting frequent clonal somatic alterations in CAFs is the use of tissues and techniques which are well known to be highly prone to generating artefacts, such as limiting and poor quality DNA followed by highly multiplexed PCR-based analysis. We conclude that reported frequent clonal somatic mutations in CAFs are likely to be artefacts and are not the biological basis of the cancer promoting attributes of CAFs. [corrected]

Exploring the Potential of Breast Microbiota as Biomarker for Breast Cancer and Therapeutic Response

Background Obesity and the insulin resistance syndrome are risk factors for breast cancer and might also affect breast cancer progression. The anti-diabetic drug Metformin (METF) reduces the breast cancer risk in diabetic women. Insulin like growth factor 1 (IGF1) and insulin are involved in breast cancer tumorigenesis and progression. We tested the effect of METF on the IGF1/insulin pathway and its involvement in breast cancer progression. Methods We developed a prognostic signature based on IGF1/insulin pathway genes using the Stockholm breast cancer microarray dataset of 149 cases for training and primary validation and the Uppsala dataset of 249 for external validation. The effect of METF on the prognostic gene set identified was tested in vitro on a panel of breast cancer cell lines. METF effects on proliferation and glucose metabolism were analyzed in vitro and in vivo. The insulin receptor substrate 2 (IRS2) was silenced by transfection with shRNA-lentiviral vectors. Xenograft growth, in the presence and absence of METF, was studied and 18FDG-uptake was measured in vitro and in vivo. Results A 15-gene signature (Insulin sensitivity score, ISS) was developed and predicted breast cancer metastasis with an accuracy similar to the Recurrence Score. ISS genes were expressed at variable levels in a breast cancer cell line panel and showed variable responsiveness to METF. The high expression correlation among the ISS genes observed in untreated breast cancer cell lines was lost upon treatment with METF. METF reduced breast cancer cell growth in vitro with IC50 values ranging from 1mM to 25mM. Growth of MDA-MB-231 cells and hyper-invasive subpopulations derived therefrom was reduced in vivo by oral administration of METF to xenografted nude mice. Response to METF in terms of IC50 values correlated with basal expression of the 15 ISS genes with the strongest inverse correlation observed for IRS2. Stable silencing of IRS2 reduced the MDA-231 cell responsiveness to METF in vitro. Discussion METF acts on the insulin/IGF1 axis by disturbing a network of breast cancer progression related genes and appears to depend in its action on the expression of IRS2 that inversely correlates with the sensitivity of cell lines to the drug. The disruption of the ISS gene network is expected to correlate with an effect on breast cancer growth and progression and in fact, mouse xenografts show reduced growth upon treatment with METF. IRS2 appears to be a major mediator of METF effects. Citation Format: Alessia I. Esposito, Adriana Amaro, Giovanna Angelini, Laura Emionite, Alessandra Gennari, Stefano Indraccolo, Davide Maggi, Cecilia Marini, Barabara Salani, Gianmario Sambuceti, Maria Pia Sormani, Ulrich Pfeffer. Metformin affects breast cancer cell growth and disturbs an IGF1/insulin related gene network that correlates with breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1182. doi:10.1158/1538-7445.AM2015-1182

Comparison Of Single Nucleotide Mutations (SNVs) and Copy Number Variants (CNVs) Detection In Formalin Fixed Paraffin Embedded (FFPE) and Paired Frozen Tumor Tissues Using Target Capture and Sequencing Approach

Background:Cancer is a global public health problem affecting human health. Early stage of cancer diagnosis, when it is not too large and has not spread is important for successful treatment. Many researchers have proposed that the let-7 microRNA family can be used as a biomarker for cancer diagnosis. The aim of this meta-analysis is to evaluate whether let-7 family can be used as a diagnostic tool for cancer patients.Methods:We conducted a comprehensive literature search on PubMed, EMBASE, Web of Science, Cochrane Library, Google Scholar, China National Knowledge Infrastructure (CNKI) and Wanfang database, updated to October 23, 2020. A random effects model was used to pool the sensitivity and specificity. Besides, we measured the diagnostic value using positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and area under the curve (AUC) were pooled. In addition, meta-regression and subgroup analysis were performed to explore the possible sources of heterogeneity, and Deeks’ funnel chart was used to assess whether there was publication bias.Results:31 studies from 15 articles were included in the current meta-analysis. The overall sensitivity, specificity, PLR, NLR, DOR and AUC were 0.80 (95% CI: 0.75-0.85), 0.81 (95% CI: 0.74-0.86), 4.2 (95% CI: 2.9-5.9), 0.24 (95% CI: 0.19-0.32), 17 (95% CI: 10-29) and 0.87 (95% CI: 0.84-0.90), respectively. Subgroup analysis shows that the let-7 family cluster of serum type showed a better diagnostic accuracy of cancer, especially the breast cancer. Although there is no publication bias, it still has some limitations.Conclusions:let-7 family can be considered as a promising non-invasive diagnostic biomarker for cancer.