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AimsProgesterone receptor (PR) is a crucial prognostic marker in breast cancer. However, achieving consistent results in PR immunohistochemistry (IHC) remains challenging due to the lack of well-defined low-positive controls. This study aimed to identify benign tissues with consistent low-level PR expression to serve as ideal controls for IHC.MethodsWe evaluated PR expression in the squamous epithelium of the uterine cervix, nipple smooth muscle and pancreatic islets. QuPath digital image analysis was employed to compare the intensity and quantity of PR staining in target cells within a 2×2 mm area.ResultsThe squamous epithelium of the secretory phase cervix, nipple smooth muscle and pancreatic islets displayed appreciable weak PR expression, with mean values of 73, 55 and 60 cells, respectively. Notably, 62% (8/13) of the 2×2 mm areas in the atrophic cervix were completely negative for PR expression. The coefficients of variation for weak PR-expressing cells in pancreatic islets (57.4%) and nipple smooth muscle (65.0%) were lower than those observed in the cervix (96.2%–222.0%). The squamous epithelium of the cervix, especially during the secretory phase, exhibited weak positivity confined to the basal layers, providing another viable control option. However, variations in PR expression may be influenced by physiological factors, such as hormonal fluctuations.ConclusionsPancreatic islets and nipple smooth muscle, with their consistent low-level PR expression, offer a promising solution to the challenges associated with PR IHC. This approach may help minimise variations resulting from differing staining methods across laboratories.

BackgroundThe aim of this study was to investigate the role of IL-17A in the cancer microenvironment and the recurrence of triple negative breast cancer (TNBC).MethodsUsing human TNBC cell lines, the role of IL17-A was investigated by knocked down of IL-17A (ΔIL-17A) and by administration of IL-17A into the culture medium. Cell proliferation assays, migration assays, as well as Western blot analysis and real-time PCR, were used to evaluate IL-17A-related signaling. Three types of 4T1 cells were implanted into BALB/c mice, namely wild type (WT), ΔIL-17A, and WT + neutralizing IL-17 antibody (WT + Ab) cells. Tumor weight, necrosis area, and the number of circulating tumor cells (CTCs) were measured. Immunohistochemistry and Western blotting were used to analyze expression of CD34, CD8, and TGF-β1 as well as anoikis resistance. The Kaplan–Meier’s method was used to correlate IL-17A expression and patient outcome, including disease-free survival (DFS) and overall survival (OS).ResultsOur results demonstrated that IL-17A was able to stimulate the migratory activity, but not the growth rate, of MDA-MB-231/468 cells. In vivo, for the ΔIL-17A group, there was an increase in necrosis area, a decrease in tumor CD34 expression and a reduction in the number of CTCs. Furthermore, in WT + Ab group, there was a decreased in tumor expression of CD34, fewer CD8 ( +) cells, and fewer CTCs, but an increase in expression of TGF-β1 expression. Both of the above were compared to the WT group. Knockdown of IL-17A also decreased anoikis resistance in human TNBC and the murine 4T1 cell lines. Kaplan–Meier analysis disclosed a negative correlation between tumor expression of IL-17A and OS in TNBC patients.ConclusionWe conclude that IL-17A promotes migratory and angiogenic activity in tumors, enhances anoikis resistance, and modulates the immune landscape of the tumor microenvironment such changes favor cancer metastasis.

There is good evidence that the tumor microenvironment plays an important role in cancer metastasis and progression. Our previous studies have shown that brain-derived neurotrophic factor (BDNF) participates in the process of metastasis and in the migration of cancer cells. The aim of this study was to investigate the role of BDNF on the tumor cell microenvironment, namely, the cancer cell-endothelial cell interaction of TNBC cells. We conducted oligoneucleotide microarray analysis of potential biomarkers that are able to differentiate recurrent TNBC from non-recurrent TNBC. The MDA-MB-231 and human endothelial HUVEC lines were used for this study and our approaches included functional studies, such as migration assay, as well as Western blot and real-time PCR analysis of migration and angiogenic signaling. In addition, we analyzed the survival outcome of TNBC breast cancer patients according to their expression level of BDNF using clinical samples. The results demonstrated that BDNF was able to bring about autocrinal (MDA-MB-231) and paracrinal (HUVECs) regulation of BDNF-TrkB gene expression and this affected cell migratory activity. The BDNF-induced migratory activity was blocked by inhibitors of ERK, PI3K and TrkB when MDA-MB-231 cells were examined, but only an inhibitor of ERK blocked this activity when HUVEC cells were used. Furthermore, decreased migratory activity was found for △BDNF and △TrkB cell lines. Ingenuity pathway analysis (IPA) of MDA-MB-231 cells showed that BDNF is a key factor that is able to regulate a network made up of metalloproteases and calmodulin. Protein expression levels in a tissue array of tumor slices were found to be correlated with patient prognosis and the results showed that there was significant correlation of TrkB expression, but not of BDNF. expressionwith patient DFS and OS. Our study demonstrates that up-regulation of the BDNF signaling pathway seems tobe involved in the mechanism associated with early recurrence in triple negative breast cancer cell. In addition, BDNF can function in either an autocrine or a paracrine manner to increase the migration ability of both MDA-MB-231 cells and HUVEC cells. Finally, overexpression of TrkB, but not of BDNF, is significantly associated with a poor survival outcome for TNBC patients.

PurposeDeleterious germline BRCA1/2 mutations are among the most highly pathogenic variants in hereditary breast and ovarian cancer syndrome. Recently, genes implicated in homologous recombination repair (HRR) pathways have been investigated extensively. Defective HRR genes may indicate potential clinical benefits from PARP (poly ADP ribose polymerase) inhibitors beyond BRCA1/2 mutations. MethodsWe evaluated the prevalence of BRCA1/2 mutations as well as alterations in HRR genes with targeted sequencing. A total of 648 consecutive breast cancer samples were assayed, and HRR genes were evaluated for prevalence in breast cancer tissues. ResultsAmong 648 breast cancers, there were 17 truncating and 2 missense mutations in BRCA1 and 45 truncating and 1 missense mutation in BRCA2, impacting 3% and 5% of the study population (collectively altered in 6%) with cooccurrence of BRCA1/2 in 7 breast cancers. On the other hand, HRR genes were altered in 122 (19%) breast cancers, while TBB (Talazoparib Beyond BRCA) trial-interrogated genes (excluding BRCA1/2) were mutated in 107 (17%) patients. Beyond BRCA1/2, the most prevalent HRR mutant genes came from ARID1A (7%), PALB2 (7%), and PTEN (6%). Collectively, 164 (25%) of the 648 Taiwanese breast cancer samples harbored at least one mutation among HRR genes. ConclusionsThe prevalence of BRCA1/2 mutations was far below one tenth, while the prevalence of HRR mutations was much higher and approached one-fourth among Taiwanese breast cancers. Further opportunities to take advantage of defective HRR genes for breast cancer treatment should be sought for the realization of precision medicine.

Background The homologous recombination (HR) repair pathway for DNA damage, particularly the BRCA1 and BRCA2 genes, has become a target for cancer therapy, with poly ADP-ribose polymerase (PARP) inhibitors showing significant outcomes in treating germline BRCA1/2 (g BRCA1/2 ) mutated breast cancer. Recent studies suggest that some patients with somatic BRCA1/2 (s BRCA1/2 ) mutation or mutations in HR-related genes other than BRCA1/2 may benefit from PARP inhibitors as well, particularly those with PALB2 mutations. The current analysis aims to evaluate the prevalence of genetic alterations specific to BRCA1 , BRCA2 , and PALB2 in a large cohort of Taiwanese breast cancer patients through tumor-targeted sequencing. Methods A total of 924 consecutive assays from 879 Taiwanese breast cancer patients underwent tumor-targeted sequencing (Thermo Fisher Oncomine Comprehensive Assay v3). We evaluated BRCA1 , BRCA2 , and PALB2 mutational profiles, with variants annotated and curated by the ClinVAR, the Oncomine™ Knowledgebase Reporter, and the OncoKB™. We also conducted reflex germline testing using either whole exome sequencing (WES) or whole genome sequencing (WGS), which is ongoing. Results Among the 879 patients analyzed (924 assays), 130 had positive mutations in BRCA1 (3.1%), BRCA2 (8.6%), and PALB2 (5.2%), with a total of 14.8% having genetic alterations. Co-occurrence was noted between BRCA1/BRCA2 , BRCA1/PALB2, and BRCA2/PALB2 mutations. In BRCA1 -mutated samples, only p.K654fs was observed in three patients, while other variants were observed no more than twice. For BRCA2 , p.N372H was the most common (26 patients), followed by p.S2186fs, p.V2466A, and p.X159_splice (5 times each). For PALB2 , p.I887fs was the most common mutation (30 patients). This study identified 176 amino acid changes; 60.2% (106) were not documented in either ClinVAR or the Oncomine™ Knowledgebase Reporter. Using the OncoKB™ for annotation, 171 (97.2%) were found to have clinical implications. For the result of reflex germline testing, three variants ( BRCA1 c.1969_1970del, BRCA1 c.3629_3630del, BRCA2 c.8755-1G > C) were annotated as Pathogenic/Likely pathogenic (P/LP) variants by ClinVar and as likely loss-of-function or likely oncogenic by OncoKB; while one variant ( PALB2 c.448C > T) was not found in ClinVar but was annotated as likely loss-of-function or likely oncogenic by OncoKB. Conclusion Our study depicted the mutational patterns of BRCA1 , BRCA2 , and PALB2 in Taiwanese breast cancer patients through tumor-only sequencing. This highlights the growing importance of BRCA1/2 and PALB2 alterations in breast cancer susceptibility risk and the treatment of index patients. We also emphasized the need to meticulously annotate variants in cancer-driver genes as well as actionable mutations across multiple databases.

Background Breast cancer is one of the leading causes of cancer-related deaths in women, and there is a demand in developing an Asian-based genetic profiling database for breast cancer in improving the treatment response. This study aimed to determine molecular alternations and identify potential therapeutic targets by analyzing the genetic profiling from a cohort of Taiwanese breast cancers using a commercialized next-generation sequencing (NGS) targeted panel. Methods The study population comprised a broad spectrum of breast cancer patients in Taiwan, including Group 1: planned to receive first-line surgery and followed by adjuvant therapy, or early relapse within three years, Group 2: planned to receive first-line neoadjuvant therapy and followed by surgery, and Group 3: de novo stage IV, or stage IV with recurrence beyond three years. Molecular profiles were determined using Thermo Fisher™ Oncomine™ Comprehensive Assay version 3 (TMO comprehensive assay) from Formalin-Fixed Paraffin-Embedded (FFPE) tissues. Level of actionability was evaluated with the ESMO Scale of clinical actionability of molecular targets (ESCAT). Results A total of 380 TMO comprehensive assays were conducted on 372 patients, and we presented targeted sequencing analyses of Tier I: alteration-drug match associated with improved outcome in clinical trials including ERBB2 amplification, BRCA1/2 germline mutation, PIK3CA mutation, and NTRK translocation, and Tier II: antitumor activity associated with the matched alteration-drug but lack of prospective outcome data including PTEN loss, ESR1 mutation, AKT1 mutation, and ERBB2 mutation, and Tier III: matched drug-alteration that led to clinical benefit in another tumor type including MDM2 amplification, and ERBB3 mutation. Among them, 249 (66%) showed at least one actionable alternation based on the ESCAT criteria. The most frequent impacted genes (all variants combined within each sample) were PIK3CA (38%), followed by ERBB2 (23%), ESR1 (10%), AKT1 (6%), and BRCA2 (5%), and the remaining rare variants (less than 5% of assayed cohort) were BRCA1 (3%), MDM2 (2.2%), and ERBB3 (1.1%). Conclusion Targeted sequencing of actionable genes is believed to provide clinical applicability and substantial benefits for Taiwanese breast cancer patients. A valid scale of clinical actionability should be adopted for precision medicine practice under multidisciplinary molecular tumor board.

The purpose of present study was to longitudinally investigate the alterations in infrapatellar fat pad (IPFP) vascularity in 5/6 nephrectomized rats by using dynamic contrast enhanced (DCE) MRI and IPFP degeneration by using MRI T2* relaxation time. Twelve male Sprague–Dawley rats were assigned to a control group and a 5/6 nephrectomy CKD group. The right knees of all rats were longitudinally scanned by 4.7 T MRI, and serial changes in the IPFP were assessed at 0, 8, 16, 30, and 44 weeks by DCE-MRI (parameters A, k el and k ep ) and MRI T2* mapping. After MRI measurements, knee specimens were obtained and evaluated histologically. The CKD group had IPFPs with lower blood volume A and lower permeability k ep values from 16 weeks ( p  < 0.05), lower venous washout k el value from 30 weeks ( p  < 0.001), and significantly higher T2 * values reflecting adipocyte degeneration beginning at 16 weeks ( p  < 0.05). The histopathological results confirmed the MRI findings. Hypoperfusion and adipocytes degeneration related to CKD were demonstrated in a rodent 5/6 nephrectomy model. DCE parameters and MRI T2* can serve as imaging biomarkers of fat pad degeneration during CKD progression.

Promoter methylation is the most significant mechanism to regulate O6-methylguanine-DNA-methyltransferase (MGMT) expression. Single-nucleotide polymorphisms (SNPs) in the MGMT promoter region may also play a role. The aim of this study was to evaluate the clinical significance of SNPs in the MGMT promoter region of glioblastoma. Genomic DNAs from 118 glioblastomas were collected for polymerase chain reaction (PCR) amplification. Sanger sequencing was used to sequence the MGMT promoter region to detect SNPs. The results were correlated with MGMT status and patient survival. Rs1625649 was the only polymorphic SNP located at the MGMT promoter region in 37.5% of glioblastomas. Homozygous rs1625649 (AA genotype) was correlated with a higher MGMT methylation level and a lower protein expression, but the result was not statistically significant. In patients with MGMT methylated glioblastoma, cases with homozygous rs1625649 (AA genotype) were significantly associated with a lack of MGMT protein expression and a better progression-free survival (PFS) than the cases with wild type rs1625649 (CC genotype) or heterozygous rs1625649 (CA genotype). The survival impact was significant in multivariate analyses. In conclusion, the MGMT promoter homozygous rs1625649 (AA genotype) was found to correlate with a better PFS in patients with MGMT methylated glioblastoma.

Subependymal giant cell astrocytoma is a benign brain tumor mostly associated with tuberous sclerosis complex. However, it may be misinterpreted as other high-grade brain tumors due to the presence of large tumor cells with conspicuous pleomorphism and occasional atypical features, such as tumor necrosis and endothelial proliferation. In this study, we first investigated thyroid transcription factor-1 (TTF-1) expression in a large series of subependymal giant cell astrocytomas and other histologic and locational mimics to validate the diagnostic utility of this marker. We then examined TTF-1 expression in non-neoplastic brain tissue to determine the cell origin of subependymal giant cell astrocytoma. Twenty-four subependymal giant cell astrocytoma specimens were subjected to tissue microarray construction. For comparison, a selection of tumors, including histologic mimics (21 gemistocytic astrocytomas and 24 gangliogliomas), tumors predominantly occurring at the ventricular system (50 ependymomas, 19 neurocytomas, and 7 subependymomas), and 134 astrocytomas (3 pleomorphic xanthoastrocytomas, 45 diffuse astrocytomas, 46 anaplastic astrocytomas, and 40 glioblastomas) were used. Immunohistochemical stain for TTF-1 was positive in all 24 subependymal giant cell astrocytomas, whereas negative in all astrocytomas, gangliogliomas, ependymomas, and subependymomas. Neurocytomas were positive for TTF-1 in 4/19 (21%) of cases using clone 8G7G3/1 and in 9/19 (47%) of cases using clone SPT24. In the three fetal brains that we examined, TTF-1 expression was seen in the medial ganglionic eminence, a transient fetal structure between the caudate nucleus and the thalami. There was no BRAF V600E mutation identified by direct sequencing in the 20 subependymal giant cell astrocytomas that we studied. In conclusion, TTF-1 is a useful marker in distinguishing subependymal giant cell astrocytoma from its mimics. Expression of TTF-1 in the fetal medial ganglionic eminence indicates that subependymal giant cell astrocytoma may originate from the progenitor cells in this region.

xCT, also known as solute carrier family 7 member 11 (SLC7A11), the light chain of the cystine/glutamate antiporter, is positively correlated with cancer progression due to antioxidant function. During glucose deprivation, the overexpression of xCT does not protect cancer cells but instead promotes cell death. Further understanding the mechanism of glucose deprivation-induced cell death is important for developing anticancer treatments targeting the glucose metabolism. In this study, we found that breast cancer cells with a high expression of xCT demonstrated increased levels of reactive oxygen species (ROS) and were more sensitive to glucose deprivation than the cells with a low expression of xCT. However, AMP-activated protein kinase (AMPK) did not significantly affect glucose-deprivation-induced cell death. The antioxidant N-acetyl-cysteine prevented glucose-deprivation-induced cell death, and the glutathione biosynthesis inhibitor L-buthionine-S, R-sulfoximine enhanced glucose-deprivation-induced cell death. The inhibition of xCT by sulfasalazine or a knockdown of xCT reduced the glucose-deprivation-increased ROS levels and glucose-deprivation-induced cell death. Glucose deprivation reduced the intracellular glutamate, and supplementation with α-ketoglutarate prevented the glucose-deprivation-increased ROS levels and rescued cell death. The knockdown of sirtuin-3 (SIRT3) further enhanced the ROS levels, and promoted xCT-related cell death after glucose deprivation. In conclusion, our results suggested that ROS play a critical role in xCT-dependent cell death in breast cancer cells under glucose deprivation.