Expression Of MKI67 Research Articles

Anacardium occidentale leaves extract and riboceine mitigate hyperglycemia through anti-oxidative effects and modulation of some selected genes associated with diabetes.

Diabetes mellitus (DM) is one of the leading causes of death globally and complications of DM have become a major health concern. Anacardium occidentale is a plant widely recognized for its hypoglycemic properties and traditionally used in developing nations as remedy for DM treatment. Riboceine is a supplement that enhances production of glutathione and known for its vital role in supporting cellular function. This study was designed to evaluate the antidiabetic and antioxidant potential of riboceine and ethanolic extract of A. occidentale leaves in streptozotocin (STZ)-induced diabetic rats. Twenty-nine adult male Wistar rats were induced with DM intraperitoneally using a single dose of STZ (70mg/kg). The STZ-induced rats were divided into groups and administered the same dose (100mg/kg) of A. occidentale leaves extract and riboceine via gastric gavage at the dose (100mg/kg) for seventeen days while metformin (40mg/kg) was used as positive control. Fasting blood glucose and weight of the model rats were examined periodically. Activities of total protein,creatinine, urea, antioxidants (SOD, GSH and GPX), and level of serum insulin were determined. Expression of diabetes related genes including pancreas (Insulin, pdx-1, P16NK4A, and Mki-67), Liver (FAS, ACC, and GFAT) and KIM-1 genes were also determined. Data showed that treatment of STZ-induced diabeticrats with A. occidentale and riboceine at the same dose significantly (p < 0.05) ameliorated hyperglycemic effects by improving hepatic and renal functions and antioxidants, preventing hepatic fat accumulation by downregulation of ACC, FAS and GFAT expression, improving β-cell functions through up-regulation of pancreatic insulin, P16NK4A, Mki-67 and pdx-1 expression. Induction of diabetes upregulated mRNA expression of KIM-1, which was ameliorated after treatment of the rats with A.occidentale and riboceine. The results obtained in this study demonstrate significant antidiabetic properties of ethanolic extract of A. occidentale and riboceine.

LBA1 Tissue-agnostic response predictor for immune checkpoint inhibitor therapy based on MKI67, FOXC1 and PDL1 expression

Immune checkpoint inhibitors (ICIs) have clinical efficacy in the neoadjuvant setting in multiple solid cancer types. However, suitable tissue-agnostic complementary diagnostics to help guide and tailor treatment recommendations are still lacking. Ki67, a routinely used proliferation marker predicts efficacy of chemotherapeutics but not ICIs. Forkhead Box C1 (FOXC1), a transcriptional driver of cell plasticity/partial EMT/metastasis was recently demonstrated to have potential value in predicting therapeutic efficacy of chemotherapy+immunotherapy in TNBC.

Lymphomagenic Roles of MYC, E2F and Mir-150-5p in Plasmablastic Lymphoma: Therapeutic and Prognostic Implications

Introduction: Plasmablastic lymphoma (PBL) is an uncommon non-Hodgkin lymphoma which is characterized by an aggressive clinical behavior, very poor prognosis and which mostly affects immunosuppressed individuals. Although different studies have described genetic alterations in PBL, its transcriptome and epigenome have been scarcely studied. Our goal was to describe the gene and miRNA expression profiles and to identify new therapeutic targets and new prognostic factors in PBL. Methods: Formalin fixed paraffin embedded (FFPE) samples from 66 patients with PBL and samples from 14 reactive adenopathies, as a control group, were included for miRNA and gene expression studies (microarrays and RNA-seq, respectively). Enrichment analyses of miRNAs and genes differentially expressed, using hallmark data set, were carried out. The role of miR-150-5p in PBL lymphomagenesis was studied in vitro upon introduction of specific miRNA mimic in the PBL-1 cell line (Mine S, Sci Rep. 2017). Expression of the miRNA and its target genes was analyzed by RT-qPCR and Western Blot. Its role in cell proliferation, cell cycle progression and apoptosis induction, was assessed respectively by MTT assay and flow cytometry. Results: Patients with PBL showed 36 differentially expressed miRNAs compared to controls. Two expression profiles of these miRNAs were identified in PBL, related with the presence of MYC-translocation. The functional enrichment analysis of genes targeted by deregulated miRNAs revealed an enrichment of MYC and E2F targets, genes involved in G2M checkpoint and mTORC1 signaling pathway. This latter observation was confirmed by gene set enrichment analysis (GSEA) of genes differentially expressed in PBL (Fig.1). Different genes are shared among the top 3 enriched hallmark gene sets (Fig.2), this data suggests that both MYC and E2F are involved in the deregulation of cell cycle in PBL. Additionally, the upregulation of E2F1, E2F3 and E2F8 correlates significantly with an increased expression of MKI67 (Ki67), indicating that E2F exerts a crucial role in the abnormal proliferation in PBL. To identify deregulated miRNA-target interactions related with the top 3 enriched hallmark gene sets, we studied the correlation between the most differentially expressed miRNAs (miR-148a-3p, -4417, -150-5p, -342-5p, -342-3p, -3609) and their differentially expressed target genes. Only miR-150-5p showed a significant negative correlation with the anti-apoptotic gene BIRC5, coding for survivin, in PBL patients (p. 0.0063) (Fig.2). Of interest, high expression of BIRC5 and low expression of miR-150-5p correlated with high expression of MKI67 (p. 0.0006 and p. 0.04, respectively) indicating that the deregulation of miR-150-5p and BIRC5 may favor the proliferation of PBL cells. Low expression of miR-150-5p (lower than median= 6.56) was associated with shorter overall survival (p <0.0001), suggesting that this miRNA may represent a potential prognostic factor in PBL. We further studied the role of miR-150-5p in PBL lymphomagenesis by mimicking this miRNA in PBL-1 cell line. Upregulation of miR-150-5p decreased the expression of survivin at both mRNA and protein level, promoted apoptosis and reduced viability and proliferation, pointing out a pro-apoptotic role of miR-150-5p via survivin inhibition. Consistent with the known role of survivin in the control of mitosis, the upregulation of miR-150-5p and the consequent reduction in survivin levels produced a slight arrest of cells in G1 cell cycle phase and lower numbers of cells in G2-M phase. Finally, a positive tendency between BIRC5 and E2F3 expression was observed (p. 0.06), being this latter gene negatively correlated with miR-150-5p (p. 0.017). Indeed, a greater expression of E2F3 was encountered in those patients with low expression of miR-150-5p. Thus, these results suggest that E2F3 could repress miR-150-5p and mediate apoptosis via BIRC5 induction. Conclusions: We identified miR-150-5p as a new promising prognostic factor, and MYC and E2F as important regulators of global gene expression in PBL. Among these genes, E2F3 could represent a lymphomagenic-driver in PBL by repressing miR-150-5p and inducing BIRC5 expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of a novel BRCAness score that predicts response to PARP inhibitors

BackgroundBRCAness is a characteristic feature of homologous recombination deficiency (HRD) mimicking BRCA gene mutation in breast cancer. We hypothesized that a measure to quantify BRCAness that causes synthetic lethality in BRCA mutated tumors will identify responders to PARP inhibitors.MethodsA total of 6753 breast cancer patients from 3 large independent cohorts were analyzed. A score was generated by transcriptomic profiling using gene set variation analysis algorithm on 34 BRCA1-mutation related genes selected by high AUC levels in ROC curve between BRCA1 mutation and wildtype breast cancer.ResultsThe score was significantly associated with BRCA1 mutation, high mutation load and intratumoral heterogeneity as expected, as well as with high HRD, DNA repair and MKi67 expression regardless of BRCA mutations. High BRCAness tumors enriched not only DNA repair, but also all five Hallmark cell proliferation-related gene sets. High BRCAness tumors were significantly associated with higher cytolytic activity and with higher anti-cancerous immune cell infiltration. Not only did the breast cancer cell lines with BRCA-mutation show high score, but even the other cells in human breast cancer tumor microenvironment were contributing to the score. The BRCAness score was the highest in triple-negative breast cancer consistently in all 3 cohorts. BRCAness was associated with response to chemotherapy and correlated strongly with response to PARP inhibitor in both triple-negative and ER-positive/HER2-negative breast cancer.ConclusionsWe established a novel BRCAness score using BRCA-mutation-related gene expressions and found that it associates with DNA repair and predicts response to PARP inhibitors regardless of BRCA mutation.

Revised International Staging System (R-ISS) stage-dependent analysis uncovers oncogenes and potential immunotherapeutic targets in multiple myeloma (MM).

Multiple myeloma (MM) accounts for ~10% of all haematologic malignancies. Little is known about high intratumour heterogeneities in patients stratified by the Revised International Staging System (R-ISS). Herein, we constructed a single-cell transcriptome atlas to compare differential expression patterns among stages. We found that a novel cytotoxic plasma cell (PC) population exhibited with NKG7 positive was obviously enriched in stage II patients. Additionally, a malignant PC population with significantly elevated expression of MKI67 and PCNA was associated with unfavourable prognosis and Epstein-Barr virus (EBV) infection in our collected samples. Moreover, ribonucleotide reductase regulatory subunit M2 (RRM2) was found and verified to promote proliferation of MM cell lines, suggesting RRM2 may serve as a detrimental marker in MM. The percentages of CD8+ T cells and NKT cells decreased along with R-ISS stages, reflecting the plasticity of the tumour immune microenvironment. Importantly, their crosstalks with myeloid cells and PC identified several potential immunotargets such as SIRPA-CD47 and CD74-MIF, respectively. Collectively, this study provided an R-ISS-related single-cell MM atlas and revealed the clinical significance of novel PC clusters, as well as potential immunotargets in MM progression.

Tripeptidyl Peptidase 1 Regulates Human Trophoblast Cell Proliferation Implying a Role in Placentation.

Proper placentation in the first trimester is essential for a healthy pregnancy in humans. A recent proteomics study of human placental tissue has identified that tripeptidyl peptidase 1 (TPP1) production is reduced in the placenta in early-onset preeclampsia compared to uncomplicated pregnancy. However, it remains to be investigated if TPP1 plays a role in regulating trophoblast cell function during early pregnancy. In this study, immunohistochemistry was used to determine the production and localization of TPP1 in human placenta throughout gestation and the first-trimester decidua/implantation sites. TPP1 siRNA (20 nM) was transfected into a human trophoblast cell line (HTR8/SVneo) to knock down TPP1, and functional consequences on cell adhesion, proliferation, migration, and invasion were analyzed via xCELLigence real-time monitoring. The expression of TPP1 downstream targets was examined by qPCR. Our data show that TPP1 localized to the discrete foci in the cytoplasm in syncytiotrophoblast, cytotrophoblast, and decidual cells across all trimesters of pregnancy. In the first-trimester human decidua, TPP1 exhibited similar staining patterns in the cytotrophoblast cells based at the cell columns. However, minimal/no staining was identified in the HLA-G positive extravillous trophoblast cells (EVTs), especially in the EVTs that invaded in the decidua. Knockdown of TPP1 in HTR8/SVneo cells by 95% significantly impaired cell adhesion and proliferation without affecting cell migration and invasion. qPCR revealed that the expression of cell proliferation markers P21 and MKI67 and TPP1-related genes MRE11, CLN3, and CLN8 was significantly changed after TPP1 knockdown in HTR8/SVneo cells compared to control. Overall, our data demonstrate that TPP1 alters trophoblast cell line function suggesting that it may be involved in regulating human placentation in the first trimester via controlling trophoblast cell adhesion and proliferation.

KDELR2-KIF20A axis facilitates bladder cancer growth and metastasis by enhancing Golgi-mediated secretion

BackgroundBladder cancer (BCa) is a fatal form of cancer worldwide associated with a poor prognosis. Identifying novel drivers of growth and metastasis hold therapeutic potential for the disease. Transport homeostasis between the endoplasmic reticulum and Golgi and the secretion of matrix metalloproteinases (MMPs) mediated by Golgi have been reported to be closely associated with tumor progression. However, to date, mechanistic studies remain limited.ResultsHere, we identified KDELR2 as a potential risk factor with prognostic value in patients with BCa, especially those harbouring the KDELR2 amplification. In addition, we found that KDELR2 is a regulator of BCa cell proliferation and tumorigenicity based on bioinformatic analysis with functional studies. Mechanistically, we revealed that KDELR2 could regulate the expression of KIF20A, thus stimulating the expression of MMP2, MMP9 and MKI67. Functionally, the overexpression of KDELR2 and KIF20A markedly promoted proliferation, migration, and invasion in vitro and enhanced tumor growth in vivo, while knockdown of KDELR2 and KIF20A exerted the opposite effects. And the overexpression of KDELR2 also enhanced lymph node metastasis in vivo.ConclusionsCollectively, our findings clarified a hitherto unexplored mechanism of KDELR2-KIF20A axis in increasing Golgi-mediated secretion of MMPs to drive tumor progression in BCa.

In silico and in vivo analysis of TIPE1 expression in diffuse large B cell lymphoma.

TIPE1 is a gene in the TNFAIP8 family involved in immune regulation and tumorigenesis. Although previous studies demonstrated that TIPE1 might play different roles in different tumors, its expression and role in lymphoma are unclear. Here we observed TIPE1 expression in diffuse large B cell lymphoma (DLBCL). Two microarrays containing 96 tumor tissue specimens were obtained from the Affiliated Hospital of Nantong University biobank. All specimens came from patients with a clear pathological diagnosis of lymphoma, lymphadenitis, breast cancer, or bladder cancer, and we performed immunohistochemical experiments on these tissue specimens. GEPIA and TIMER platforms were used for bioinformatic analyses. We found higher TIPE1 expression in tumor tissues from patients with lymphoma compared with those with lymphadenitis, breast cancer, or bladder cancer. The GEPIA and TIMER analyses revealed that TIPE1 was upregulated in DLBCL tissues but not in invasive breast carcinoma, urothelial bladder carcinoma, or liver hepatocellular carcinoma tissues. TIPE1 expression was irrelevant for pathological stage, overall survival, or DLBCL immune infiltration levels. However, TIPE1 expression was correlated with MKI67 expression in DLBCL. Overall, TIPE1’s high expression levels in DLBCL may contribute to tumor growth in DLBCL.

QS52. Mechanical Stimulation Reverses the Pro-Fibrotic Transcriptome of Senescent Dermal Fibroblasts

Purpose: Senescent fibroblasts accumulate with aging in nearly all connective tissues, contributing to fibrosis and impaired healing in multiple organs. It is thus of longstanding clinical interest to develop strategies for the reversal of this pro-fibrotic transcriptional state. Interestingly, senescence has been shown to downregulate mechanotransduction in fibroblasts and other cells, but the effects of this phenomenon on skin fibrosis and wound healing are unclear. We therefore utilized a stretchable collagen I hydrogel to investigate the ability of exogenous mechanical stimulation to reverse the pro-fibrotic transcriptome of senescent dermal fibroblasts. Methods: Dermal fibroblasts were harvested from dorsal skin of C57BL/6 mice by enzymatic digestion and flow cytometry, cultured in standard growth medium, and exposed to 0 or 125nM doxorubicin for 72h to induce non-senescent and senescent phenotypes, respectively. Senescence was quantified using β-galactosidase. Fibroblasts were encapsulated at 1,000,000 cells/mL in 1.92 mg/mL collagen I, 0.8x MEM, and 16mM HEPES. Hydrogels were formed in 20x20x5 mm cruciforms and cultured for 7 days (media replacement every 2-3 days) with 0 or 10% of equibiaxial strain to produce control or mechanically stimulated conditions, respectively. Cells were retrieved by collagenase II/IV digestion, with TRIzol RNA extraction. RNA sequencing (RNAseq) was performed with 40M paired-end reads, with reads aligned to the mm10 genome (HISAT2) and quantified (StringTie). Differential expression analysis and gene ontology (GO) analysis of the top 500 differentially expressed genes was performed (edgeR) (α=0.05). All experiments were performed with n=3 biological replicates. Results: β-galactosidase staining showed optimal 76.7% induction of senescence with 125nM doxorubicin. RNAseq showed increased expression of Glb1 (senescence) and decreased expression of Mki67 (non-senescence, proliferation) for senescent fibroblasts, both of which were effectively reversed by mechanical stimulation. Yap1 (mechanosensing) expression was also increased with mechanical stimulation of both non-senescent and senescent fibroblasts. Pdgfra and Acta2 (canonical pro-fibrotic markers) were highly enriched in senescent fibroblasts. Critically, these pro-fibrotic markers was reversed by mechanical stimulation only in senescent fibroblasts. Interestingly, Col1a1 was downregulated by both senescence and mechanical stimulation, while Col1a2 was uniquely downregulated by mechanical stimulation of senescent fibroblasts. GO analysis also revealed that mechanical stimulation differentially modified the transcriptomes of non-senescent and senescent fibroblasts. The top 5 enriched GO terms for senescent fibroblasts were largely characterized by systemic modifications to biological quality/development, while the top 5 enriched GO terms for non-senescent fibroblasts were dominated by superficial responses to the stretched extracellular matrix/hydrogel. Conclusions: Mechanical stimulation produced differential effects on the transcriptomes of normal and senescent fibroblasts, simply increasing pro-fibrotic gene expression for non-senescent fibroblasts but reversing pro-fibrotic transcription and systemically modifying biological qualities for senescent fibroblasts. Furthermore, mechanical stimulation may mitigate some inherent features of the senescent phenotype (Glb1+, Mki67-). Ultimately, our hydrogel model suggests that exogenous mechanical stimulation may be used to reverse the pro-fibrotic phenotype of senescent dermal fibroblasts that accumulate during aging.

NR2F1, a Tumor Dormancy Marker, Is Expressed Predominantly in Cancer-Associated Fibroblasts and Is Associated with Suppressed Breast Cancer Cell Proliferation.

Simple SummaryTumor dormancy is a state in which some cancer cells are dormant by less cell proliferation, and are resistant to treatments, thus leading to late recurrence. As nuclear receptor subfamily 2 group F member 1 (NR2F1) is a biomarker of dormancy shown in experimental settings, we studied its clinical relevance by analyzing comprehensive gene expression profiles of approximately 7000 breast cancer patients using computational biological approaches. NR2F1 gene expression of bulk tumors correlated with the suppression of cell proliferation and metastasis-related pathways, but not with metastasis or survival. Using single-cell sequence cohorts, we found that NR2F1 was predominantly expressed in cancer-associated fibroblasts, particularly in inflammatory type, rather than in cancer cells. Further, NR2F1 expression in breast cancer cells did not consistently correlate with stem cell-like traits. Our findings suggest that the NR2F1 gene expression signal from a patient’s bulk tumor is not from the cancer cells.Background: Tumor dormancy is a crucial mechanism responsible for the late recurrence of breast cancer. Thus, we investigated the clinical relevance of the expression of NR2F1, a known dormancy biomarker. Methods: A total of 6758 transcriptomes of bulk tumors from multiple breast cancer patient cohorts and two single-cell sequence cohorts were analyzed. Results: Breast cancer (BC) with high NR2F1 expression enriched TGFβ signaling, multiple metastases, and stem cell-related pathways. Cell proliferation-related gene sets were suppressed, and MKi67 expression was lower in high NR2F1 BC. In tumors with high Nottingham grade, NR2F1 expression was found to be lower. There was no consistent relationship between NR2F1 expression and metastasis or survival. Cancer mutation rates, immune responses, and immune cell infiltrations were lower in high NR2F1 tumors, whereas the infiltration of stromal cells including cancer-associated fibroblasts (CAFs) was higher. NR2F1 was predominantly expressed in CAFs, particularly inflammatory CAFs, rather than in cancer cells, consistently in the two single-cell sequence cohorts. Conclusions: NR2F1 expression in breast cancer is associated with tumor dormancy traits, and it is predominantly expressed in CAFs in the tumor microenvironment.

Establishment of a novel BRCAness score that predicts response to PARP inhibitors.

549 Background: BRCAness is a generic term used to describe characteristic features of homologous recombination deficiency (HRD) mimicking mutations in BRCA genes. Although clinical genetic testing has increased the detection of mutations in BRCA1 and BRCA2, we hypothesized that a measure to quantify BRCAness will identify the responders to PARP inhibitors that cause synthetic lethality in BRCA mutation tumors. Methods: The BRCAness score was established by using gene set variation analysis (GSVA) algorithm on 34 BRCA-mutation related genes. We investigated the clinical relevance of the score by performing silico analyses of 6245 breast cancer patients using multiple independent large cohorts in this study. Results: A score to quantify BRCAness was generated using gene set variation analysis algorithm on 34 BRCA1-mutation related genes selected by high AUC levels in ROC curve between BRCA1 mutation and wildtype breast cancer. The score was significantly associated with BRCA1 mutation, high overall mutation load and intratumoral heterogeneity as well as high HRD, DNA repair and MKI67 expression regardless of mutation in BRCA gene. High score tumor enriched not only DNA repair, but also five cell proliferation-related gene sets (E2F targets, G2M checkpoint, MYC targets v1 and v2, and MITOTIC signaling) in Hallmark collection (all false discovery rate &lt; 0.10). Breast cancers with high score were significantly associated with higher infiltration of anti-cancerous immune cells and higher cytolytic activity. Not all breast cancer cell lines with BRCA-mutation showed high score and the other cells in human breast cancer tumor microenvironment were contributing to the score. We found that the BRCAness score was the highest in triple-negative among subtypes consistently in all cohorts (all p &lt; 0.001). Finally, BRCAness was associated with response to chemotherapy and correlated strongly with response to PARP inhibitor in both triple-negative (AUC = 0.815) and ER-positive/HER2-negative breast cancer (AUC = 0.715). Conclusions: We established a novel BRCAness score using mRNA expression of BRCA-mutation-related genes and found that it associates with DNA repair and response to PARP inhibitor regardless of BRCA mutation.

Association of NR2F1, a tumor dormancy marker, with cancer cell proliferation and lymph node metastasis, and expression in cancer-associated fibroblasts in breast cancer.

e12550 Background: It is now well accepted that some cancer cells remain in quiescent state and survive through adjuvant systemic treatments, also known as tumor dormancy, which preserve the potential for late recurrence. NR2F1 is a known biomarker for dormant cancer cells, which suppress cell proliferation in experimental models. Here, we hypothesized that patient breast cancer (BC) with high NR2F1 gene expression is associated with less cell proliferation and worse survival. Methods: More than 7000 patients from 3 large-scale primary and 5 metastatic BC patient cohorts and 2 single-cell sequencing cohorts were analyzed. Results: NR2F1 expression was upregulated in dormant cell lines and in bone metastatic clone cells compared with mother 4T1 cells. High NR2F1 BC enriched multiple metastasis-related pathways such as TGFβ, EMT, angiogenesis, as well as cancer stem cell-related signaling (NOTCH, HEDGEHOG, and Wntβ-catenin). As expected, all 5 cell proliferation-related gene sets including E2F Targets and G2M Checkpoints were enriched in low NR2F1 BC, and so was MKI67 expression. Unexpectedly, there were no consistent association between NR2F1 expression and survival among the 3 cohorts. Three out of four cohorts demonstrated significantly high NR2F1 expression in primary BC with lymph node metastasis. There was no difference in NR2F1 expression in primary BC by the distant metastasis in the four cohorts, by the metastatic sites, or between the primary and metastatic BC neither. Given the discrepancy between the biology and clinical outcome of high NR2F1 BC, we performed the functional analysis exclusively . Surprisingly, there were no consistent enrichment of metastasis-related gene sets nor downregulation of cell proliferation-related gene sets to high NR2F1 expression cancer cells, unlike the analyses of bulk tumors. Intratumoral heterogeneity, HRD, proliferation score, and mutation load were all less in high NR2F1 BC, along with IFN-γ response and TIL fractions. Infiltration of immune cells such as Th1 and Th2 T-cells, M1 and M2 macrophages were all less, while stromal cells such as adipocytes, fibroblasts, stromal cells and endothelial cells were more abundant in high NR2F1 BC. Surprisingly, NR2F1 was barely expressed in cancer cells and was most highly expressed in cancer-associated fibroblasts consistently regardless of subtypes in two independent single-cell sequence cohorts. The expression of NR2F1 was higher in inflammatory than myofibroblastic CAFs, and that of CAFs, but not cancer cells, was associated with lymph node metastasis or advanced stage. Conclusions: The tumor dormancy marker NR2F1 expression in primary BC is associated with mechanism of metastasis and suppression of cancer growth, but is most expressed in cancer-associated fibroblasts in the tumor microenvironment.

Unravelling the sex-specific diversity and functions of adrenal gland macrophages.

SummaryDespite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.

Differential expression of somatostatin receptor (SSTR) subtypes across a spectrum of neuroendocrine neoplasms (NENs).

3071 Background: Targeted therapy of NENs based on the presence of SSTRs fills a unique niche in tumor biology and clinical treatment of patients with solid tumors. SSTRs have multiple isoforms and are collectively expressed in the majority of NENs. However, subtypes are still not routinely tested and thus not assessed for clinical decision-making, especially for patients meriting consideration of targeted radionucleotide therapy. Clarifying the landscape of SSTR subtypes using molecular techniques more sensitive than immunohistochemistry (IHC)-the standard of testing, and identifying associated genomic biomarkers that differ between them, will pave the way for more sophisticated decision-making in the future. Additionally, leveraging transcriptomics to better assess mitotic markers such as Ki-67 to assess tumor grade, would increase diagnostic accuracy. Here we provide initial validation across a spectrum of NENs. Methods:1595 NENs were analyzed using Next Generation Sequencing (592 gene panel, NextSeq), Whole Exome and Transcriptome Sequencing (NovaSeq), and IHC at Caris Life Sciences (Phoenix, AZ). Significance was determined using chi-square, Fisher-Exact or Mann-Whitney U and p-adjusted for multiple comparisons (q&lt;0.05) where applicable. Results: In a subset of 492 NENs with accompanying tumor grading information, a median MKI67 (gene encoding Ki-67) TPM value of 2.27 for low-grade (LG-), and 38.7 for high-grade NENs (HG-NENs) was observed (q&lt;0.05). Using ROC curve analysis, a threshold of MKI67 expression (13.4375 TPM) differentiated LG- from HG-NENs, with a true positive rate of 86.84%, a false positive rate of 11.9% and an AUC of 95% and was subsequently applied to the entire cohort to infer HG/LG. Compared to HG-NENs (n = 862), LG-NENs(n = 733) expressed higher levels of SSTR 1(3.5-fold),2 (2.9-fold) and 5 (1.67-fold) and lower levels of SSTR4 (0.28-fold)(q&lt;0.05). Further, the expression of SSTRs 3 and 4 in HG-NEN (rs= 0.63) and SSTRs 1 and 2 in LG-NENs (rs= 0.64) were positively correlated. Overall, the prevalence of altered TP53, RB1, PIK3CA, APC, KRAS was higher and MEN1 was lower in HG-vs LG-NENs (q&lt;0.05). For each SSTR subtype, we established high and low cohorts based on median expressions. In LG-NENs, increased alterations in TP53 and RB1 were associated with increased expression of SSTRs 1 and 2 and reduced expression of SSTRs 3 and 4. In HG-NENs, increased alterations in APC were associated with increased expression of SSTR 1 and 4 and reduced expression of SSTRs 3 and 5. Additional subtype- and grade-specific alterations were also observed. Conclusions: This study provides evidence that WTS and NGS can be leveraged to predict grade of NENs and define characteristic differences in the genomic landscape across SSTR subtypes in HG and LG NENs. Incorporating the molecular profiling of NENs can thus aid in advancing the development of more tailored therapeutic strategies.

Single-cell RNA sequencing reveals immunological rewiring at the maternal-fetal interface following asymptomatic/mild SARS-CoV-2 infection.

While severe coronavirus 2019 (COVID-19) is associated with immune activation at the maternal-fetal interface, responses to asymptomatic/mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy remain unknown. Here, we assess immunological adaptations in blood and term decidua in response to asymptomatic/mild disease in pregnant women. We report attenuated antigen presentation and type I interferon (IFN) signaling pathways, loss of tissue-resident decidual macrophages, and upregulated cytokine/chemokine signaling in monocyte-derived decidual macrophages. Furthermore, we describe increased frequencies of activated tissue-resident T cells and decreased abundance of regulatory T cells with infection while frequencies of cytotoxic CD4/CD8 T cells are increased in the blood. In contrast to decidual macrophages, type I IFN signaling is higher in decidual T cells. Finally, infection leads to a narrowing of T cell receptor diversity in both blood and decidua. Collectively, these observations indicate that asymptomatic/mild COVID-19 during pregnancy results in remodeling of the immunological landscape of the maternal-fetal interface, with a potential for long-term adverse outcomes for the offspring.

Abstract P1-08-17: Melk expression is associated with immune cell infiltration and pathological compete response (pcr) after neoadjuvant chemotherapy in breast cancer

Abstract In experimental settings, maternal embryonic leucine zipper kinase (MELK), an apical member of the snf1/AMPK serine-threonine kinases family, is highly expressed in several malignancies, and plays a role in cell cycle and proliferation in cell culture settings. However, there is no clear insight on the underlying mechanism or association of MELK expression with several key players in the tumor microenvironment (TME) in regulating cancer progression and response to several drugs in the human tumor. We investigated the clinical relevance of MELK expression by performing silico analyses of 7,135 breast cancer patients using multiple independent large cohorts in this study. We found that MELK expression was significantly correlated with tumor growth assessed by American Joint Committee Cancer (AJCC) stage (p &amp;lt; 0.001), Nottingham histological grade (both p &amp;lt; 0.001), MKI67 expression (spearman rank correlation (r) = 0.704 and 0.888, respectively, both p &amp;lt; 0.001), triple-negative breast cancer (TNBC) subtype (both p &amp;lt; 0.001) and also with cell proliferation-related gene sets (all normalized enrichment score (NES) &amp;gt; 1.70, all false discovery rate (FDR) &amp;lt; 0.01) using gene set enrichment analysis (GSEA), in two large cohorts METABRIC and GSE96058. Furthermore, we observed worse patient survival (both p &amp;lt; 0.001), high mutation rate (all p &amp;lt; 0.03), and enhanced cancer cell survival pathways, including MTORC1 signaling, DNA repair and unfolded protein response (all NES &amp;gt; 1.50) in high MELK expression breast cancer. Additionally, breast cancer with high MELK expression was significantly enriched in immune-related gene sets, including allograft rejection, interferon (IFN)-α response and IFN-γ response (all NES &amp;gt; 1.30). Furthermore, infiltration of anti-cancer immune cells (CD4+ memory T cells, T helper type1 cells, CD8+ T cells, M1 macrophages, gamma-delta T cells, and dendritic cells) and pro-cancer (T helper type 2 cells and regulatory T cells), calculated by xCell algorithm, was associated with high MELK expression. High immune cell killing activity (CYT) was also significantly associated with high MELK expression. Although MELK expression did not correlate with sensitivity of any drug tested in cell lines, high MELK was significantly associated with high pathological complete response (pCR) rate after neoadjuvant chemotherapy (NAC) not only in TNBC (area under the curve (AUC) = 0.78, 0.81, and 0.93, respectively), the aggressive breast cancer subtype known to be associated with around 30-40% pCR, but also in ER-positive plus HER2-negative breast cancer (AUC = 0.62, 0.75, and 0.80, respectively), a subtype of breast cancer where pCR rates are very low, in three cohorts GSE25066, GSE20194 and HESS cohorts. In conclusion, our study shows that high MELK expression is significantly associated with cell proliferation, immune cell infiltration, and higher incidence of response to NAC both in ER-positive/HER2-negative and TNBC. Citation Format: Masanori Oshi, Shipra Gandhi, Michelle R Huyser, Yoshihisa Tokumaru, Li Yan, Rongrong Wu, Akimitsu Yamada, Itaru Endo, Kazuaki Takabe. Melk expression is associated with immune cell infiltration and pathological compete response (pcr) after neoadjuvant chemotherapy in breast cancer [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 P1-08-17.

Abstract P1-05-06: Establishment of novel BRCA1ness score to quantify BRCAness in breast cancer

Abstract Germline mutations in the loci of BRCA1 or BRCA2 genes with loss of heterozygosity or additional somatic truncation at the WT allele in breast cancer result in characteristic clinicopathological features of homologous recombination deficiency, also known as “BRCAness.”. Although clinical genetic testing for mutations of BRCA1 and BRCA2 has increased the chances of identifying pathogenic variants, we speculated that there may be a difference in cancer biology by the degree of BRCAness. To address this question, we sought to develop a means to estimate the amount of BRCAness using a score consists of BRCA1-mutation-related genes. Thirty-four BRCA1-mutation related genes were selected by high area under the curve (AUC) levels in receiver operating characteristic (ROC) curve between BRCA1 mutation and non-mutation breast cancer, consistently in two large breast cancer cohorts, TCGA and METABRIC. The score was established by using gene set variation analysis (GSVA) algorithm on 34 genes. We found that the BRCA1ness score was the highest in triple negative breast cancer (TNBC) among subtypes consistently in both cohorts (both p &amp;lt; 0.001). Although BRCA1ness score did not correlate with mRNA expression of BRCA1 nor BRCA2 at all (spearman’s rank correlation coefficient (r) = 0.191 and 0.093, respectively), high BRCA1ness score was significantly associated with high rate of BRCA1 mutation (AUC = 0.735 and 0.709, respectively) in both cohorts. This level of AUC for BRCA1ness mutation score was highest compared to other factors, including BRCA1 expression (AUC = 0.323), DNA repair gene set score (AUC = 0.553 and 0.543), nor homologous recombination deficient (HRD) (AUC = 0.659), which are known as the characteristics of BRCAness. Furthermore, the score was significantly correlated with DNA repair gene set score (r = 0.593 and 0.323, both p &amp;lt; 0.01) as well as MKI67 expression (r = 0.655 and 0.626, both p &amp;lt; 0.01) in both cohorts. HRD score was also strongly correlated with BRCA1ness score in the TCGA cohort (r = 0.633). Interestingly, high BRCA1ness was significantly associated with worse disease-free survival (DFS) (p = 0.037 and p &amp;lt; 0.001, respectively) and disease-specific survival (DSS) (p = 0.012 and p &amp;lt; 0.001, respectively) in both cohorts. In conclusion, we established novel BRCA1ness score using mRNA expression of BRCA1-mutation-related genes, and found that it associate with DNA repair and worse survival. Citation Format: Masanori Oshi, Rongrong Wu, Akimitsu Yamada, Li Yan, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. Establishment of novel BRCA1ness score to quantify BRCAness in breast cancer [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 P1-05-06.

Abstract P1-01-15: Clinical relevance of TRIM37 gene expression in breast cancer

Abstract Background: Tripartite motif-containing protein 37 (TRIM37) is an oncogenic histone H2A ubiquitin ligase that is overexpressed in a subset of breast cancers. TRIM37 was suggested to be associated with chemotherapy resistance and metastasis of triple-negative breast cancer (TNBC) in in vivo and in vitro studies. Breast cancer with TRIM37 amplification is sensitive to polo-like kinase 4 (PLK4) inhibition. On the other hand, clinical relevance of TRIM37 in breast cancer was never investigated. Material and Methods: Total of 6836 breast cancer patients from three large patient cohorts (Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), The Cancer Genome Atlas (TCGA), GSE96058, GSE25066 and GSE20194. Gene set enrichment analysis (GSEA) was performed. The high and low expression of TRIM37 gene were divided by median. Results: TRIM37 expression was significantly associated with worse survival (disease-free (DFS), disease-specific (DSS), and overall survival (OS)) in ER-positive/HER2-negative breast cancer (p=0.012, p=0.011, and p=0.003, respectively), but not in the other subtypes. TRIM37 expression was higher in HER2-positive among the subtypes. TRIM37 expression correlated with Nottingham histological grade (p&amp;lt;0.001), and with MKI-67 expression (p&amp;lt;0.001) consistently in METABRIC and GSE96058 cohorts. TRIM37 high expression enriched cell proliferation-related gene sets; E2F targets, G2M checkpoints, mitotic spindle, and MYC targets v1 and v2, as well as DNA repair gene sets regardless of subtypes. TRIM37 high expression was also associated with RAD51C and/or PLK4 expression, which are DNA repair related genes. In agreement, silent and non-silent mutation rate and altered fraction were all significantly elevated in TRIM37 high tumors regardless of subtypes. TRIM37 expression correlated with response to Cisplatin, Paclitaxel, and Tamoxifen in breast cancer cell line study (r=0.655, r=0.446, and r=0.9, respectively). TRIM37 high tumors demonstrated significantly worse pathological complete response after neoadjuvant chemotherapy in ER-positive/HER2-negative patients (p=0.002), but the opposite in TNBC (p=0.025) in GSE25066 cohort, but this result was not validated by GSE20194, a cohort with very small samples size. Interestingly, TRIM37 low expression enriched immune related gene sets; inflammatory response, IL2 signaling, IL6 signaling, TNF-a signaling, and allograft rejection in ER-positive/HER2-negative patients, but not in TNBC. Indeed, CD8 central memory T cells and CD4 effector memory T cells are highly infiltrated in TRIM37 low tumors consistently in both METABRIC and GSE96058 in ER-positive/HER2-negative patients, but not in TNBC. Taken together, TRIM37 high expression was associated with cell proliferation regardless of subtypes, but TRIM37 low expression was associated with high tumor infiltrating lymphocytes and immune response that may have contributed to the survival difference in ER-positive/HER2-negative patients, but not in TNBC. Conclusions: In conclusion, TRIM37 expression is associated with cell proliferation and DNA repair, less tumor infiltrating lymphocytes and immune response, and with worse survival in ER-positive/HER2-negative breast cancer. Citation Format: Junko Tsuchida, Rongrong Wu, Maiko Endo, Kazuki Moro, Chie Toshikawa, Yu Koyama, Hiroshi Ichikawa, Takaaki Hanyu, Yamato Takabe, Kazuyasu Takizawa, Yoshifumi Shimada, Takashi Kobayashi, Takashi Ishikawa, Jun Sakata, Toshifumi Wakai, Kazuaki Takabe. Clinical relevance of TRIM37 gene expression in breast cancer [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 P1-01-15.

Abstract P4-07-03: Development of prognostic models based on clinical, immune-related and proliferation factors in early breast cancer patients treated with neoadjuvant chemotherapy

Abstract Introduction and objectives Up to 30% of early breast cancer (BC) patients treated with neoadjuvant chemotherapy will have systemic relapse during the follow-up. The integration of clinical, analytical and molecular parameters associated to tumor biology or host immune response could help to better stratify the prognosis of these patients. The aim of this study was to analyze the prognostic ability of immune-related and proliferation markers in combination with clinical parameters in patients with early BC treated with neoadjuvant chemotherapy. Methods and materials Retrospective and single-center cohort of BC patients treated with neoadjuvant chemotherapy between 2001 and 2010. We analyzed the following pre-treatment biological markers: neutrophil-to-lymphocyte ratio (NLR) in peripheral blood and CD3+-tumor-infiltrating lymphocytes (TIL), interferon-gamma and interleukin-10 in tumor samples by using a tissue microarray. Gene expression of AURKA, MYBL2, MKI67 and CTNNB1 in RNA from tumor samples was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Survival analysis was performed using Cox regression. The predictive capacity of the regression models was evaluated using AIC (Akaike Information Criterion) index, ROC curves and Harrell's C statistics. Results A total of 121 patients were included. Median age: 56 years. Cancer stage at diagnosis: 16% IIA, 28% IIB, 33% IIIA, 7% IIIB and 16% IIIC. Molecular subtype: 64% hormone receptor-positive (12% HER2-positive), 11% HER2-positive and 22% triple-negative. Pathological complete response (pCR): 16.5%. Median follow-up: 12 years. In the univariate analysis, NLR (HR 1.23, 95% CI 1.11-1.36; p&amp;lt;0.001), TIL (HR 0.89, CI95% 0.81-0.98; p=0, 02), AURKA (HR 1.02, 95% CI 1.01-1.04; p&amp;lt;0.001) and MYBL2 (HR 1.10, 95% CI 1.03-1.19; p=0.007) showed prognostic value for overall survival (OS). In the multivariate analysis, including staging after neoadjuvant therapy (HR 6.54, 95% CI 1.36-31.49; p=0.02), NLR (HR 1.33, 95% CI 1.08-1.64; p=0.008), TIL (HR 0.84, 95% CI 0.73-0.97; p=0.21), AURKA (HR 1.05, 95% CI 0.99-1.10; p=0.055) and MYBL2 (HR 1.14, 95% CI 1.00-1.31; p=0.04) remained as independent predictive variables in a regression analysis. Consecutive addition of these biomarkers to a regression model based on staging after neoadjuvant treatment progressively increased the discrimination accuracy of the models. These differences were more marked for the predictive model that included the four biological parameters identified in the multivariate analysis: NLR, TIL, AURKA and MYBL2 (Table 1). Conclusion In our cohort, the creation of a prognostic model integrated by clinical factors together with proliferation and both tissue and circulating immune biomarkers demonstrated high predictive capacity for OS. The validation of these findings in independent cohorts could impact in patient’s management. Table 1.Analysis of prognostic capacity with the consecutive addition of biomarkersAICAUC ROC (95% CI)pHarrell's C-index (95% CI)pModel 1: ypTN2740,76 (0,58-0,94)Ref*0,74 (0,57-0,91)Ref*Model 2: ypTN + NLR1870,78 (0,61-0,95)0,830,80 (0,67-0,93)0,53Model 3: ypTN + NLR + TIL850,85 (0,73-0,98)0,300,83 (0,73-0,94)0,30Model 4: ypTN + NLR + TIL + + AURKA + MYBL2570,91 (0,80-1)0,110,89 (0,81-0,97)0,13*Ref: model 1 was the reference category that was used for comparison of AUC and C-index.AIC: Akaike Information Criterion. AUC ROC: Area Under the ROC Curve. ROC: Receiver Operating Characteristic. Citation Format: Esmeralda Garcia-Torralba, Beatriz Álvarez-Abril, Carlos Bravo-Pérez, Esther Navarro Manzano, Pilar de la Morena Barrio, Alejandra Ivars Rubio, Elisa García-Garre, Gema Marín Zafra, Francisco Ayala de la Peña, Elena García-Martínez. Development of prognostic models based on clinical, immune-related and proliferation factors in early breast cancer patients treated with neoadjuvant chemotherapy [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 P4-07-03.

Abstract P3-10-04: microRNA-125a high expressing tumors enrich cell proliferation associated gene sets and associated with poor prognosis in estrogen receptor positive breast cancer patients

Abstract Background: MicroRNA-125a (miR-125a) has been shown to function as tumor suppressive miRNA by pre-clinical in vitro studies. However, to the best of our knowledge, there is no study that investigated the clinical relevance of miR-125a in breast cancer patients. We hypothesized that miR-125a high expressing breast cancer associate with less aggressive cancer characteristics and with favorable survival outcome. Material and Methods: The clinicopathological and survival information associated with comprehensive transcriptomic data was analyzed in 2042 breast cancer patients from large publicly available databases, The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC) The Cancer Genome Atlas (TCGA), and GSE57897. The survival analysis, gene set enrichment analysis (GSEA) were conducted comparing miR-125a high expressing and low expressing tumors, divided by the median cutoff. The association between the miR-125a expression and tumor immune microenvironment was assessed by utilizing xCell. Results: The expression levels of miR-125a were lower in tumors compared with normal breast tissues in both TCGA and GSE57897 cohorts, which is in agreement with the notion that it is a tumor suppressive miRNA (p&amp;lt;0.001 and p&amp;lt;0.001, respectively). ER-positive/HER2-negative (ER+/HER2-) showed the highest miR-125a expression among the subtypes in TCGA (p&amp;lt;0.001). MiR-125a expression was not associated with cancer staging in any of the subtypes in neither TCGA nor METABRIC cohorts. Surprisingly, miR-125a high expressing tumors demonstrated worse disease free (DFS), disease specific (DSS), and overall survival (OS) compared with low expressing in ER+/HER2- breast cancer patients (p=0.008, p=0.005, and p=0.037) which was not the case for the other subtypes in METABRIC cohort. Interestingly, we found that miR-125a expression significantly correlated with Nottingham histological grade only in ER+/HER2- among the subtypes (p&amp;lt;0.001). miR-125a high tumors significantly demonstrated higher expression of MKI67, one of the most commonly used parameters for cell proliferation, correlated with miR-125a expression in ER+/HER2- and Her2-positive patients (both &amp;lt; p=0.02), but not in triple negative breast cancer (TNBC). Furthermore, miR-125a high expressing tumors enriched four out of five cell proliferation related gene sets in Hallmark collection, such as E2F Targets, G2M Checkpoint, Mitotic Spindle, and MYC Targets V2 in ER+/HER2- subtype, but not in TNBC. This was also the case in immune-related gene sets; interferon-alpha response and interferon-gamma response that enriched to miR-125a high tumors in ER+/HER2-, but not in TNBC. Infiltration of CD8 cells, T-helper type 1 cells, T-helper type 2 cells and M1 macrophages were all elevated in MiR-125a high ER+/HER2- subtype (all p&amp;lt;0.03), but none in TNBC. We found that tumor suppressive miR-125a was highly expressed in ER+/HER2- subtype, where its expression was associated with multiple clinical and molecular biological parameters of cell proliferation, as well as with both favorable and unfavorable immune response. Given its association with the survival outcome, we cannot help but speculate that miR-125a expression parallels with highly proliferative ER+/HER2- breast cancer, but its tumor suppressive effect is not enough to improve survival outcome. This study is hypothesis generating, and our results need mechanistic analyses by experimental investigations. Conclusion: MiR-125a high tumors were associated with aggressive characteristics in ER+/HER2- patients which may contribute to the worse survival outcomes. The current result support the importance of analyses of large patient cohorts to clarify the role of a miRNAs in patients. Citation Format: Yoshihisa Tokumaru, Manabu Futamura, Kohei Taniguchi, Masanori Oshi, Junichi Mase, Yoshimi Asano, Ryutaro Mori, Kazuaki Takabe, Kazuhiro Yoshida. microRNA-125a high expressing tumors enrich cell proliferation associated gene sets and associated with poor prognosis in estrogen receptor positive breast cancer patients [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 P3-10-04.