Tissues Of Breast Cancer Patients Research Articles

Downregulation of MICA/B tumor surface expressions and augmented soluble MICA serum levels correlate with disease stage in breast cancer.

In this study, the profiling of the expression of major histocompatibility complex (MHC) class I-related chain A and B (MICA/B) in human breast cancer tumor tissue, saliva, and urine samples of breast cancer patients and control is carried out. MICA/B is ligand of NKG2D receptor expressed on malignant cells. The release of MICA/B from tumor tissue comprises an immune escape mechanism that impairs antitumor immunity. Based on this literature we explored the potential of soluble MICA (sMICA) as a marker in breast cancer (BC). The expression was profiled by using immunohistochemistry (MICA/B), western blot (MICA/B) and ELISA (MICA). The optical density of western blot of MICA/B in different stages of BC illustrated significant difference as per one way analysis of variance and significant difference with stage III and IV by Dunnett's multiple comparisons test respectively. Analysis of sMICA in serum, saliva and urine of BC patients revealed significantly higher levels (median 41.0 ± 4.1 pg/ml in pre-treatment sera, 181.9 ± 1.6 pg/ml in saliva and 90.7 ± 1.7 pg/ml in urine) than in control (median <1.2 pg/ml). The elevated levels of sMICA were related to the cancer stage. The elevated levels of sMICA were observed in patients with well differentiated cancer while the poor expression of sMICA was observed in patients with poorly differentiated tumors. Tumor immunity is impaired by the release of MICA in the biofluids and may be useful for detection and diagnosis of the stage of BC.

Inhibition of Galectin-9 sensitizes tumors to anthracycline treatment via inducing antitumor immunity.

Anthracyclines are a class of conventionally and routinely used first-line chemotherapy drugs for cancer treatment. In addition to the direct cytotoxic effects, increasing evidence indicates that the efficacy of the drugs also depends on immunomodulatory effects with unknown mechanisms. Galectin-9 (Gal-9), a member of the β-galactoside-binding protein family, has been demonstrated to induce T-cell death and promote immunosuppression in the tumor microenvironment. Here, we asked whether anthracycline-mediated immunomodulatory activity might be related to Gal-9. We found that combining doxorubicin with anti-Gal-9 therapy significantly inhibited tumor growth and prolonged overall survival in immune-competent syngeneic mouse models. Moreover, Gal-9 expression was increased in response to doxorubicin in various human and murine cancer cell lines. Mechanistically, doxorubicin induced tumoral Gal-9 by activating the STING/interferon β pathway. Clinically, Gal-9 and p-STING levels were elevated in the tumor tissues of breast cancer patients treated with anthracyclines. Our study demonstrates Gal-9 upregulation in response to anthracyclines as a novel mechanism mediating immune escape and suggests targeting Gal-9 in combination with anthracyclines as a promising therapeutic strategy for cancer treatment.

BAG2 drives chemoresistance of breast cancer by exacerbating mutant p53 aggregate.

Rationale: Chemoresistance is a major challenge in the clinical management of patients with breast cancer. Mutant p53 proteins tend to form aggregates that promote tumorigenesis in cancers. We here aimed to explore the mechanism for the generation of mutant p53 aggregates in breast cancer and assess its role in inducing chemoresistance. Methods: Expression of BCL2-associated athanogene 2 (BAG2) was evaluated by qRT-PCR, western blotting, and immunohistochemistry in breast cancer patient specimens. The significance of BAG2 expression in prognosis was assessed by Kaplan-Meier survival analysis and the Cox regression model. The roles of BAG2 in facilitating the formation of mutant p53 aggregates were analyzed by co-immunoprecipitation, immunofluorescence, and semi-denaturing detergent-agarose gel electrophoresis assays. The effects of BAG2 on the chemoresistance of breast cancer were demonstrated by cell function assays and mice tumor models. Results: In the present study, we found that BAG2 was significantly upregulated in relapse breast cancer patient tissues and high BAG2 was associated with a worse prognosis. BAG2 localized in mutant p53 aggregates and interacted with misfolded p53 mutants. BAG2 exacerbated the formation of the aggregates and recruited HSP90 to promote the propagation and maintenance of the aggregates. Consequently, BAG2-mediated mutant p53 aggregation inhibited the mitochondrial apoptosis pathway, leading to chemoresistance in breast cancer. Importantly, silencing of BAG2 or pharmacological targeting of HSP90 substantially reduced the aggregates and increased the sensitivity of chemotherapy in breast cancer. Conclusion: These findings reveal a significant role of BAG2 in the chemoresistance of breast cancer via exacerbating mutant p53 aggregates and suggest that BAG2 may serve as a potential therapeutic target for breast cancer patients with drug resistance.

MST4: A Potential Oncogene and Therapeutic Target in Breast Cancer.

The mammalian STE 20-like protein kinase 4 (MST4) gene is highly expressed in several cancer types, but little is known about the role of MST4 in breast cancer, and the function of MST4 during epithelial-mesenchymal transition (EMT) has not been fully elucidated. Here we report that overexpression of MST4 in breast cancer results in enhanced cell growth, migration, and invasion, whereas inhibition of MST4 expression significantly attenuates these properties. Further study shows that MST4 promotes EMT by activating Akt and its downstream signaling molecules such as E-cadherin/N-cadherin, Snail, and Slug. MST4 also activates AKT and its downstream pro-survival pathway. Furthermore, by analyzing breast cancer patient tissue microarray and silicon datasets, we found that MST4 expression is much higher in breast tumor tissue compared to normal tissue, and significantly correlates with cancer stage, lymph node metastasis and a poor overall survival rate (p < 0.05). Taken together, our findings demonstrate the oncogenic potential of MST4 in breast cancer, highlighting its role in cancer cell proliferation, migration/invasion, survival, and EMT, suggesting a possibility that MST4 may serve as a novel therapeutic target for breast cancer.

Ionizing Radiation and Estrogen Affecting Growth Factor Genes in an Experimental Breast Cancer Model.

Genes associated with growth factors were previously analyzed in a radiation- and estrogen-induced experimental breast cancer model. Such in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line, MCF-10F, to low doses of high linear energy transfer (LET) α particle radiation (150 keV/μm) and subsequent growth in the presence or absence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation after being irradiated with either a single 60 cGy dose or 60/60 cGy doses of alpha particles. In the present report, the profiling of differentially expressed genes associated with growth factors was analyzed in their relationship with clinical parameters. Thus, the results indicated that Fibroblast growth factor2 gene expression levels were higher in cells transformed by radiation or in the presence of ionizing radiation; whereas the fibroblast growth factor-binding protein 1gene expression was higher in the tumor cell line derived from this model. Such expressions were coincident with higher values in normal than malignant tissues and with estrogen receptor (ER) negative samples for both gene types. The results also showed that transforming growth factor alpha gene expression was higher in the tumor cell line than the tumorigenic A5 and the transformed A3 cell line, whereas the transforming growth factor beta receptor 3 gene expression was higher in A3 and A5 than in Tumor2 cell lines and the untreated controls and the E cell lines. Such gene expression was accompanied by results indicating negative and positive receptors for transforming growth factor alpha and the transforming growth factor beta receptor 3, respectively. Such expressions were low in malignant tissues when compared with benign ones. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expressions were found to be present in all BRCA patients that are BRCA-Basal, BRCA-LumA, and BRCA-LumB, except in BRCA-Her2 patients. The results also indicated that the insulin growth factor receptor gene expression was higher in the tumor cell line Tumor2 than in Alpha3 cells transformed by ionizing radiation only; then, the insulin growth factor receptor was higher in the A5 than E cell line. The insulin growth factor receptor gene expression was higher in breast cancer than in normal tissues in breast cancer patients. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expression levels were in stages 3 and 4 of breast cancer patients. It can be concluded that, by using gene technology and molecular information, it is possible to improve therapy and reduce the side effects of therapeutic radiation use. Knowing the different genes involved in breast cancer will make possible the improvement of clinical chemotherapy.

Reduced expression of FRG1 facilitates breast cancer progression via GM-CSF/MEK-ERK axis by abating FRG1 mediated transcriptional repression of GM-CSF

Multiple molecular subtypes and distinct clinical outcomes in breast cancer, necessitate specific therapy. Moreover, despite the improvements in breast cancer therapy, it remains the fifth cause of cancer-related deaths, indicating the involvement of unknown genes. To identify novel contributors and molecular subtype independent therapeutic options, we report reduced expression of FRG1 in breast cancer patients, which regulates GM-CSF expression via direct binding to its promoter. Reduction in FRG1 expression enhanced EMT and increased cell proliferation, migration, and invasion, in breast cancer cell lines. Loss of FRG1 increased GM-CSF levels which activated MEK/ERK axis and prevented apoptosis by inhibiting p53 in an ERK-dependent manner. FRG1 depletion in the mouse model increased tumor volume, phospho-ERK, and EMT marker levels. The therapeutic potential of anti-GM-CSF therapy was evident by reduced tumor size, when tumors with decreased FRG1 were treated with anti-GM-CSF mAb. We found an inverse expression pattern of FRG1 and phospho-ERK levels in breast cancer patient tissues, corroborating the in vitro and mouse model-based findings. Our findings first time elucidate the role of FRG1 as a metastatic suppressor of breast cancer by regulating the GM-CSF/MEK-ERK axis.

The expression and clinical significance of STAMBP in breast cancer.

Breast cancer is the leading cause of death from cancer in women worldwide. STAMBP functions as a JAMM family deubiquitinating enzyme that modulates the stability of substrate proteins in cells by cleaving ubiquitin moieties. The expression of STAMBP and its clinical significance in breast cancer remain unclear. The level of the STAMBP protein in noncancerous and tumor tissues of breast cancer patients was examined by immunohistochemical staining. The expression of STAMBP mRNA in tissues based on healthy individual and breast cancer patient data in the TCGA database was evaluated. The association between the expression of STAMBP mRNA and clinical features and prognosis was evaluated using TCGA database. Cell growth was assessed by Cell Counting Kit-8 (CCK-8) assay, and cell migration and invasion were assessed by wound healing and Transwell assays. Activation of the ERK signaling was detected by Western blotting. The expression of STAMBP was markedly upregulated in the cytoplasm of tumor cells from breast cancer patients. The level of STAMBP was closely associated with the tumor subtype and size and the TNM stage of the breast cancer patients. Importantly, high expression of STAMBP predicted poor overall survival (OS) for breast cancer patients. Furthermore, knockdown of STAMBP expression reduced cell mobility and invasion of breast cancer cells. Notably, the phosphorylation of EGFR and ERK was markedly reduced in STAMBP-knockdown cells. STAMBP plays a critical role in the progression of breast cancer and may serve as a biomarker to monitor the progression of the disease.

Single-Vesicle Infrared Nanoscopy for Noninvasive Tumor Malignancy Diagnosis.

Protein heterogeneity in molecular expression and structures determines tumorigenesis and is the diagnostic and therapeutic cancer biomarker. Small extracellular vesicles (sEVs) are cell-released nanoscaled membrane-bound vesicles transferring bioactive molecules for intercellular communication and playing essential roles in tumor progression and metastasis. Therefore, protein heterogeneity in tumor-derived sEVs indicates the degree of malignant transformation, providing a noninvasive biomarker for cancer diagnosis and malignancy evaluation. We employ near-field infrared (nano-FTIR) spectroscopy to investigate malignancy-related protein heterogeneity in a single sEV and demonstrate the discriminability of sEV protein heterogeneity to evaluate tumor malignancy and metastasis. We found that the amide I/II adsorption ratio of the sEVs increases with tumor malignancy, the proportion of α-helix + random coil (α-helix and random coil) in sEV proteins decreases with tumor malignancy, and the proportion of β-sheet + β-turn (β-sheet and β-turn) increases with tumor malignancy. These nano-FTIR spectral signatures of the sEVs from the primary tumor tissue of breast cancer patients show high sensitivity and specificity in evaluating tumor metastasis. This study shows the advantages of nano-FTIR in single sEV characterization and demonstrates the significance of sEV protein heterogeneity in cancer diagnosis. It provides a noninvasive solution to elucidate cancer development and facilitates the exploitation of potential cancer biomarkers.

The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

Mitophagy is a vital process that controls mitochondria quality, dysregulation of which can promote cancer. Oncoprotein mucin 1 (MUC1) targets mitochondria to attenuate drug-induced apoptosis. However, little is known about whether and how MUC1 contributes to mitochondrial homeostasis in cancer cells. We identified a novel role of MUC1 in promoting mitophagy. Increased mitophagy is coupled with the translocation of MUC1 to mitochondria, where MUC1 interacts with and induces degradation of ATPase family AAA domain-containing 3A (ATAD3A), resulting in protection of PTEN-induced kinase 1 (Pink1) from ATAD3A-mediated cleavage. Interestingly, MUC1-induced mitophagy is associated with increased oncogenicity of cancer cells. Similarly, inhibition of mitophagy significantly suppresses MUC1-induced cancer cell activity in vitro and in vivo. Consistently, MUC1 and ATAD3A protein levels present an inverse relationship in tumor tissues of breast cancer patients. Our data validate that MUC1/ATAD3A/Pink1 axis-mediated mitophagy constitutes a novel mechanism for maintaining the malignancy of cancer cells, providing a novel therapeutic approach for MUC1-positive cancers.

Regulation of KDM5C stability and enhancer reprogramming in breast cancer

Abnormality of enhancer regulation has emerged as one of the critical features for cancer cells. KDM5C is a histone H3K4 demethylase and frequently mutated in several types of cancer. It is critical for H3K4me3 and activity of enhancers, but its regulatory mechanisms remain elusive. Here, we identify TRIM11 as one ubiquitin E3 ligase for KDM5C. TRIM11 interacts with KDM5C, catalyzes K48-linked ubiquitin chain on KDM5C, and promotes KDM5C degradation through proteasome. TRIM11 deficiency in an animal model represses the growth of breast tumor and stabilizes KDM5C. In breast cancer patient tissues, TRIM11 is highly expressed and KDM5C is lower expressed, and their expression is negatively correlated. Mechanistically, TRIM11 regulates the enhancer activity of genes involved in cell migration and immune response by targeting KDM5C. TRIM11 and KDM5C regulate MCAM expression and cell migration through targeting H3K4me3 on MCAM enhancer. Taken together, our study reveals novel mechanisms for enhancer regulation during breast cancer tumorigenesis and development.

Gene expression responses reflecting 5-FU-induced toxicity: Comparison between patient colon tissue and 3D human colon organoids

Capecitabine is a chemotherapeutic drug that is widely used as a monotherapy option in advanced cancer patients. After administration, it is converted into its active metabolite 5-fluorouracil (5-FU), a cytotoxic compound that may also induce adverse side effects in the gastrointestinal (GI) tract. Although these side effects can interfere with the continuation of the chemotherapy, diagnostic tools to detect early onset and prevention strategies are not available. In this explorative case study, we aim to identify differentially expressed genes (DEGs) that provide insight into the molecular mechanisms of toxicity induced by 5-FU in healthy colon tissue of breast cancer patients receiving capecitabine. Gene expression responses observed in patients were compared with those established in an in vitro model of healthy colon organoids. Colon biopsies from two patients with advanced breast cancer were collected before and after the treatment with capecitabine and used for RNA sequencing to determine transcriptomic responses. Differential expression analysis resulted in 31 affected genes, showing that the most affected pathways were transport of small molecules, cellular responses to stress, folate metabolism, NF-kB signalling pathway and immune system responses. The most biologically relevant genes were haemoglobin subunits encoding genes, involved in several processes; ATP12A, SLC26A3 and AQP8, involved in the transport of ions and water; TRIM31, a regulator of NF-kB signalling pathway; MST1P2 and MST1L, stimulators of macrophages. Comparison of human in vitro and in vivo responses showed that the gene expression of TRIM31 was similarly altered in the colon organoids exposed to 5-FU. Therefore, this gene constitutes a potential biomarker of colon toxicity that might be used in future in vitro drug safety design and screening.

A Pair of Prognostic Biomarkers in Triple-Negative Breast Cancer: KLK10 and KLK11 mRNA Expression

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor patient prognosis and limited therapeutic options. A lack of prognostic biomarkers and therapeutic targets fuels the need for new approaches to tackle this severe disease. Extracellular matrix degradation, release, and modulation of the activity of growth factors/cytokines/chemokines, and the initiation of signaling pathways by extracellular proteolytic networks, have been identified as major processes in the carcinogenesis of breast cancer. Members of the kallikrein-related peptidase (KLK) family contribute to these tumor-relevant processes, and are associated with breast cancer progression and metastasis. In this study, the clinical relevance of mRNA expression of two members of this family, KLK10 and KLK11, has been evaluated in TNBC. For this, their expression levels were quantified in tumor tissue of a large, well-characterized patient cohort (n = 123) via qPCR. Although, in general, the overall expression of both factors are lower in tumor tissue of breast cancer patients (encompassing all subtypes) compared to normal tissue of healthy donors, in the TNBC subtype, expression is even increased. In our cohort, a significant, positive correlation between the expression levels of both KLKs was detected, indicating a coordinate expression mode of these proteases. Elevated KLK10 and KLK11 mRNA levels were associated with poor patient prognosis. Moreover, both factors were found to be independent of other established clinical factors such as age, lymph node status, or residual tumor mass, as determined by multivariable Cox regression analysis. Thus, both proteases, KLK10 and KLK11, may represent unfavorable prognostic factors for TNBC patients and, furthermore, appear as promising potential targets for therapy in TNBC.

High prevalence of somatic PIK3CA and TP53 pathogenic variants in the normal mammary gland tissue of sporadic breast cancer patients revealed by duplex sequencing

The mammary gland undergoes hormonally stimulated cycles of proliferation, lactation, and involution. We hypothesized that these factors increase the mutational burden in glandular tissue and may explain high cancer incidence rate in the general population, and recurrent disease. Hence, we investigated the DNA sequence variants in the normal mammary gland, tumor, and peripheral blood from 52 reportedly sporadic breast cancer patients. Targeted resequencing of 542 cancer-associated genes revealed subclonal somatic pathogenic variants of: PIK3CA, TP53, AKT1, MAP3K1, CDH1, RB1, NCOR1, MED12, CBFB, TBX3, and TSHR in the normal mammary gland at considerable allelic frequencies (9 × 10−2– 5.2 × 10−1), indicating clonal expansion. Further evaluation of the frequently damaged PIK3CA and TP53 genes by ultra-sensitive duplex sequencing demonstrated a diversified picture of multiple low-level subclonal (in 10−2–10−4 alleles) hotspot pathogenic variants. Our results raise a question about the oncogenic potential in non-tumorous mammary gland tissue of breast-conserving surgery patients.

A polarization-resolved ECL strategy based on the surface plasmon coupling effect of orientational Au nanobipyramids patterned structures

Here, we developed a polarization-resolved surface plasmon coupling electrochemiluminescence (ECL) strategy based on orientational Au nanobipyramids (Au NBPs) patterned structures. As the surface plasmonic materials, the Au NBPs with large localized electromagnetic field enhancement were used to construct the different patterned structures through the droplet evaporation induced assembly. The distinct morphology of the horizontal and vertical Au NBPs patterned structures provided the different plasmonic properties reflecting on resonance peak positions and polarized absorbance. As a result, the variations of the polarized ECL emission of SnS2 QDs have been regulated by the Au NBPs patterned structures via the surface plasmon coupling progress. Because of the superior polarization-resolved ECL performances, the sensing system was used to analyze the miRNA-21 and miRNA-205 expression level in tumor tissues of triple negative breast cancer patients. The satisfactory results indicated this polarization-resolved sensing strategy possessed great potential for clinical analysis.

Differences in tumor microenvironment between HER2-positive and HER2-negative breast cancer.

e12562 Background: The tumor microenvironment of breast cancer is extremely complex, which containing different types of lymphocytes, macrophages, NK cells and other immune cell subtypes. At present, the relationship between immune cell subtypes and molecular subtypes of breast cancer is still unclear. Methods: Tumor tissue samples of 20 breast cancer patients were collected from January 2020. Then the density (number of positive cells/mm2) and ratio of immune cells in intratumoral area and stromal area were detected by multiple fluorescence immunohistochemistry (mIHC), the detection range was included CD3+T cells, CD4+T cells,CD8+T cells,PD-1+CD8+T cells,Tregs,M1/M2 tumor associated macrophages (TAM), PD-L1+CD68+TAM,B cells,CD56bright NK cells and CD56dim NK cells and so on. The immune cell subtypes of all area were included both intratumoral area and stromal area. Graphpad prism (version 7.01) was used for t test and the statistical significance was set at p=0.05. Results: These 20 patients were divided into two groups, HER2 positive (n=8) and HER2 negative group (n=12). The density of CD3+T cells in all area (including intratumoral area and stromal area) of tumor tissue in HER2 positive breast cancer patients were significantly higher than that of HER2 negative breast cancer patients (median density, HER2 positive group vs. HER2 negative group=2998 vs. 1520/mm², p=0.031) as well as the CD8+T cells (median density, HER2 positive group vs. HER2 negative group=536 vs. 175/mm², p=0.015). Meanwhile, the density of Tregs in all area of tumor tissue in HER2 positive breast cancer patients was also significantly higher (median density, HER2 positive group vs. HER2 negative group=119 vs. 27/mm², p=0.022).However, there was no significant difference in the other subtypes of immune cells infiltrated in tumor tissue between HER2 positive and HER2 negative samples, such as CD4+T cells, M1/M2 macrophages, B cells and so on (p＞0.05). Conclusions: Our research indicates that the patterns of immune cell subtypes in different types of early breast cancer are different. Further exploration of the immune microenvironment in different molecular types of breast cancer may provide additional targets for immunotherapy.

The impact of tumor-infiltrating lymphocytes on tumor features and pathological characteristics in breast cancer patients: the role of cytotoxic T lymphocytes and regulatory T cells.

Though tumor-infiltrating lymphocytes (TILs) have a predictive impact in cancer patients, their association with presentation and prognosis in breast cancer is less consistent. This study aimed to assess the level of infiltrating cytotoxic T lymphocytes (CTLs) and regulatory T lymphocytes (Tregs) and their association with the clinicopathological features of breast cancer. Tissue samples from female patients (n=153) diagnosed with primary invasive breast cancer were stained with CD8 (a CTL marker) and Foxp3 (a Treg marker) using immunohistochemistry. CTLs were distributed between tumor bed and stroma whereas Treg cells were mainly located in the stroma. The level of intratumoral CTLs correlated positively with Tregs in both tumor and stroma (rho=0.312, p<0.001 and rho=0.176, p=0.031; respectively). Stromal CTLs correlated positively with stromal Tregs (rho=0.319, p=0.005). Tumor size correlated inversely with the number of Treg cells in the tumor bed (rho= - 0.179, p=0.028). Tregs were associated with lymphovascular invasion status in the tumor bed (p=0.042). The ratio of intratumoral CTLs to Tregs was associated with estrogen receptor positivity and luminal subtype (p=0.029 and p=0.045, respectively). The median number of CTLs was significantly lower in patients using aspirin or antihypertensive medications compared to nonusers (p=0.024 and p=0.03, respectively). TILs were distributed differently in tumor tissues of breast cancer patients. CTLs infiltrates were found in both tumor bed and stroma while Tregs were dominant in the stroma. TILs were also distinctly associated with tumor features. The impact of TILs on prognosis and treatment outcomes in Jordanian breast cancer patients needs further investigation.

Tumor-infiltrating lymphocytes (TILs) dynamics in breast cancer patients receiving neoadjuvant therapy: A systematic review and meta-analysis.

e12620 Background: Increased baseline tumor-infiltrating lymphocytes (TILs) are associated with improved pathological complete response rates and better prognosis in HER2+ and triple negative breast cancer (TNBC) patients receiving neoadjuvant therapy (NAT). However, the role of TILs dynamics/change (ΔTILs) at the neoadjuvant setting remains unclear, thus a meta-analysis of the published studies was carried out. Methods: Medline, Embase, Cochrane Library and Web of Science Core Collection were searched for studies reporting on TILs expression in paired invasive breast cancer patient tissue samples before and after NAT. Data were extracted by two investigators (Y.Z., E.T.) and discordances were resolved by a third (I.Z.). Outcomes included pooled TILs rates pre- &amp; post-treatment (also per subtype), pooled rates of ΔTILs and direction of change after NAT as well as correlation of ΔTILs with survival outcomes. Heterogeneity was assessed using the I2 statistic. Results: Of 1569 identified entries, 22 studies fulfilled the criteria and provided adequate data for the outcomes of interest. Overall, a significantly decreased level of TILs was observed after NAT in paired samples (pooled OR = 1.60, 95% CI: 1.12-2.30, p = 0.01; TILs as categorical variable). Regarding pooled rates of ΔTILs, a change was observed after NAT, irrespective of BC subtype. Among the different subtypes, the effect of NAT on TILs was most prominent in HER2+ disease with a direction towards decreased TILs to be more common (pooled ΔTILs rates: 14.4% increased vs 46.2%, decreased). In TNBC, bi-directional TIL kinetics were noted (pooled ΔTILs rates: 41.6% increased vs 37.1% decreased). An increase in ΔTILs in TNBC was associated with better disease-free/relapse-free survival in univariate analysis (HR = 0.59, 95% CI: 0.37–0.95, p = 0.03). Substantial between-study heterogeneity was observed in most analyses. Conclusions: The first to our knowledge meta-analysis on TILs dynamics during NAT in BC informs about differences in matched pre- and post-treatment patient samples and the prognostic implications of ΔTILs in TNBC. The potential clinical utility of the longitudinal assessment of immune response during neoadjuvant therapy warrants further investigation in prospective trials.

Detection of hypermethylated BCAT1 and IKZF1 DNA in blood and tissues of colorectal, breast and prostate cancer patients.

Detection of circulating cell-free DNA (ccfDNA) methylated in BCAT1 and IKZF1 is sensitive for detection of colorectal cancer (CRC), but it is not known if these biomarkers are present in other common adenocarcinomas. Compare methylation levels of BCAT1 and IKZF1 in tissue and plasma from breast, prostate, and colorectal cancer patients. Blood was collected from 290 CRC, 32 breast and 101 prostate cancer patients, and 606 cancer-free controls. Tumor and matched normal tissues were collected at surgery: 26 breast, 9 prostate and 15 CRC. DNA methylation in BCAT1 and IKZF1 was measured in blood and tissues. Either biomarker was detected in blood from 175/290 (60.3%) of CRC patients. The detection rate was higher than that measured in controls (48/606 (8.1%), OR = 18.2, 95%CI: 11.1-29.0). The test positivity rates in breast and prostate cancer patients were 9.4% (3/32) and 6.9% (7/101), respectively, and not significantly different to that measured in gender-matched controls (8.0% (33/382) females (OR = 0.84, 95%CI: 0.23-3.1) and 7.6% (26/318) males (OR = 0.86, 95%CI: 0.65-2.1). In tumor and non-neoplastic tissues, 93.5% (14/15) of CRC tumors were methylated in BCAT1 and/or IKZF1 (p< 0.004). Only 11.5% (3/26) and 44.4% (4/9) (p= 0.083) of breast and prostate tumors were hypermethylated in these two genes. Detection of circulating DNA methylated in BCAT1 and IKZF1 is sensitive and specific for CRC but not breast or prostate cancer.

Clinical implications of lipid peroxides levels in plasma and tumor tissue in breast cancer patients

Oxidative stress can promote the oxidation of lipoproteins and polyunsaturated fatty acids present in cell membranes; an event known as lipid peroxidation (LPO). LPO has been associated with carcinogenesis and cancer progression, however, its meaning concerning the clinicopathological aspects of human breast cancer is not clear. This study investigated LPO profiles in tumor and plasma samples from breast cancer patients (n = 140) considering their clinicopathological features (age at diagnosis, menopausal status, body mass index, tumor histological grade, tumor size, ki-67 proliferation index, presence of metastasis, chemotherapy response, the molecular subtype of cancer and overall survival status). LPO levels were estimated by tert-butyl hydroperoxide-initiated chemiluminescence. High LPO levels were found regarding poor prognosis parameters as young age at diagnosis (p = 0.006 in tissue), premenopausal patients (p = 0.012 in tissue), high-grade tumors (p = 0.010 in tissue and p = 0.002 in plasma), metastatic disease (p = 0.046 in tissue), chemoresistant tumors (p = 0.041 in tissue), disease relapse (p = 0.018 in tissue and p = 0.009 in plasma) and overall survival status (p = 0.001 in plasma). Our findings point out the clinical meaning of LPO and highlight it as an oxidative stress event linked to poor prognosis and disease aggressiveness in breast cancer patients.

Chemotherapy, Microvascular Function, and Angiogenesis ‐ a Longitudinal Study

RationaleAnti‐cancer therapies (CTx), such as anthracyclines (e.g., doxorubicin, Dox), have known anti‐angiogenic effects in vitro, and inhibition of angiogenesis has known cardiotoxic effects. Cardiovascular (CV) toxicities that result from CTx significantly contribute to the morbidity and mortality of breast cancer survivors. While the negative impact of CTx, including Dox, on isolated cardiomyocytes is well‐established, significantly less is known about the effects of CTx on microvascular (MV) function and angiogenic potential in patients. Our goal was to understand how CTx damages the microcirculation in order to further the understanding of mechanisms that contribute to systemic cardiovascular toxicity. Utilizing a longitudinal study design of neoadjuvant breast cancer patients, adipose tissue was collected before, during, and after CTx treatment.MethodsAngiogenic potential was evaluated using a matrigel angiogenesis assay of patient adipose. Data are presented as a percentage of wells with capillary sprouts and area of capillary sprouting ± standard error. In addition, we investigated the expression of a subset of genes involved in angiogenesis regulation (initiation/sprouting, migration, ECM remodeling, maturation, and permeability). Freshly isolated microvessels were evaluated for dilation to flow (FMD) and acetylcholine (Ach). Cardiac function was monitored via standard clinical echocardiogram. Significance was determined using paired t‐test (p ≤ 0.05*).ResultsPercentage of wells with capillary sprouts was impaired in adipose samples collected during CTx (26.3% ± 13.6%, N=4) and one month post CTx (44.3% ± 9.1%*, N=9) compared to pre‐CTx (67.1% ± 4.7%*, N=13). However, sprouting area only changed during CTx (15.0 mm2± 8.5*, N=4) compared to pre‐CTx (68.3 mm2± 10.2, N=13). One month post CTx, the sprouting area (56.8 mm2± 19.1, N=9) was not significantly different than pre‐CTx. Supporting this change in angiogenesis function, we observe changes in expression of genes involved in initiation of angiogenesis during CTx (VEGFA, VEGFB, FLT1) that show recovery of expression post‐CTx. We also observe changes in expression of genes involved in maturation of angiogenesis post CTx (KLF4, VCAM1, MMP8). Some clinical signs of reduced cardiac function were observed post CTx after microvascular and angiogenic changes were observed.ConclusionOur data show pathological impairment in angiogenic potential and gene expression in adipose tissue of breast cancer patients in a longitudinal study. These data support our observed defects in MV function (response to FMD and Ach) during and after CTx treatment. We observed significant reductions in MV function and capillary sprouting during treatment with CTx in the absence of a clinically relevant reduction in cardiac function. Our findings suggest that CTx‐induced MV dysfunction precedes and may contribute to future adverse CV events.