Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s10549-024-07421-8

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Publishing type：Research paper (scientific journal)   Publisher：Ovid Technologies (Wolters Kluwer Health)  

Objective:

To investigate the clinical relevance of common myeloid progenitor (CMP) cells in breast tumor microenvironment (TME).

Background:

The role of rare cells in TME is less studied. In Silico transcriptomic analyses of real-world data enable us to detect and quantify rare cells, including CMP cells.

Methods:

A total of 5176 breast cancer (BC) patients from SCAN-B, METABRIC, and 5 single-cell sequence cohorts were analyzed using the xCell algorithm. The high group was defined as more than two-thirds of the CMP scores in each cohort.

Results:

CMP cells consist of 0.07% to 0.25% of bulk breast tumor cells, more in estrogen receptor-positive (ER+) compared with triple-negative (TN) subtype (0.1% to 0.75%, 0.18% to 0.33% of immune cells, respectively). CMP cells did not correlate with any of the myeloid lineages or stem cells in TME. CMP infiltration was higher in smaller tumors, with lower Nottingham grade, and in ER+/HER2− than in TNBC consistently in both SCAN-B and METABRIC cohorts. High CMP was significantly associated with a lower risk of brain metastasis and with better survival, particularly in ER+/HER2−. High CMP enriched epithelial-to-mesenchymal transition and angiogenesis pathways, and less cell proliferation and DNA repair gene sets. High CMP ER+/HER2- was associated with less immune cell infiltration and cytolytic activity (P<0.001). CMP infiltration correlated with neoadjuvant chemoimmunotherapy response for both ER+/HER2- and TNBC in the ISPY-2 cohort (AUC=0.69 and 0.74, respectively).

Conclusions:

CMP in BC is inversely associated with cell proliferation and brain metastasis, better response to immunotherapy, and survival. This is the first to report the clinical relevance of CMP infiltration in BC.

DOI： 10.1097/sla.0000000000006428

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s12072-024-10678-2

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Language：English   Publishing type：Research paper (scientific journal)  

Tumor-infiltrating lymphocytes are a general term for lymphocytes or immune cells infiltrating the tumor microenvironment. Numerous studies have demonstrated tumor-infiltrating lymphocytes to be robust prognostic and predictive biomarkers in breast cancer. Recently, immune checkpoint inhibitors, which directly target tumor-infiltrating lymphocytes, have become part of standard of care treatment for triple-negative breast cancer. Surprisingly, tumor-infiltrating lymphocytes quantified by conventional methods do not predict response to immune checkpoint inhibitors, which highlights the heterogeneity of tumor-infiltrating lymphocytes and the complexity of the immune network in the tumor microenvironment. Tumor-infiltrating lymphocytes are composed of diverse immune cell populations, including cytotoxic CD8-positive T lymphocytes, B cells and myeloid cells. Traditionally, tumor-infiltrating lymphocytes in tumor stroma have been evaluated by histology. However, the standardization of this approach is limited, necessitating the use of various novel technologies to elucidate the heterogeneity in the tumor microenvironment. This review outlines the evaluation methods for tumor-infiltrating lymphocytes from conventional pathological approaches that evaluate intratumoral and stromal tumor-infiltrating lymphocytes such as immunohistochemistry, to the more recent advancements in computer tissue imaging using artificial intelligence, flow cytometry sorting and multi-omics analyses using high-throughput assays to estimate tumor-infiltrating lymphocytes from bulk tumor using immune signatures or deconvolution tools. We also discuss higher resolution technologies that enable the analysis of tumor-infiltrating lymphocytes heterogeneity such as single-cell analysis and spatial transcriptomics. As we approach the era of personalized medicine, it is important for clinicians to understand these technologies.

DOI： 10.1093/jjco/hyae033

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Language：Japanese   Publisher：(一社)日本外科学会  

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Elmer Press, Inc.  

DOI： 10.14740/wjon1732

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Elmer Press, Inc.  

DOI： 10.14740/wjon1751

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Publishing type：Research paper (scientific journal)   Publisher：Elmer Press, Inc.  

DOI： 10.14740/wjon1691

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Publishing type：Research paper (scientific journal)   Publisher：Elmer Press, Inc.  

DOI： 10.14740/wjon1694

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Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVE: To investigate the clinical relevance of intratumoral tumor infiltrating lymphocytes (TILs) in breast cancer as measured by computational deconvolution of bulk tumor transcriptomes. SUMMARY BACKGROUND DATA: Commonly assessed TILs, located in tumor stroma without direct contact with cancer cells (stromal TILs), correlate with breast cancer treatment response and survival. The clinical relevance of intratumoral TILs has been less studied partly due to their rarity; however, they may have nonnegligible effects given their direct contact with cancer cells. METHODS: In all, 5870 breast cancer patients from TCGA, METABRIC, GSE96058, GSE25066, GSE163882, GSE123845, and GSE20271 cohorts were analyzed and validated. RESULTS: The intratumoral TIL score was established by the sum of all types of lymphocytes using the xCell algorithm. This score was the highest in triple-negative breast cancer (TNBC) and the lowest in the ER-positive/HER2-negative subtype. It correlated with cytolytic activity and infiltrations of dendritic cells, macrophages, and monocytes, and uniformly enriched immune-related gene sets regardless of subtype. Intratumoral TIL-high tumors correlated with higher mutation rates and significant cell proliferation on biological, pathological, and molecular analyses only in the ER-positive/HER2-negative subtype. It was significantly associated with pathological complete response after anthracycline- and taxane-based neoadjuvant chemotherapy in about half of the cohorts, regardless of the subtype. Intratumoral TIL-high tumors correlated with better overall survival in HER2-positive and TNBC subtypes consistently in 3 cohorts. CONCLUSIONS: Intratumoral TILs estimated by transcriptome computation were associated with increased immune response and cell proliferation in ER-positive/HER2-negative and better survival in HER2-positive and TNBC subtypes, but not always with pathological complete response after neoadjuvant chemotherapy.

DOI： 10.1097/SLA.0000000000005954

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s10549-023-07111-x

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Language：English  

Since the popularization of cancer screening and an improvement in treatment over the last two decades, multiple primary malignant neoplasms (MPMNs) have been increasingly reported. We report a patient who developed metachronous MPMNs in the breast, the endometrium, and the pancreas over a period of 13 years. A 42-year-old woman was first diagnosed with breast cancer and underwent breast-conserving surgery with adjuvant radiation therapy and endocrine therapy. Four years after breast surgery, she was diagnosed with endometrial cancer and underwent a laparoscopic modified radical hysterectomy with bilateral oophorectomy with pelvic lymph node dissection followed by adjuvant chemotherapy. However, there was peritoneal dissemination of endometrial cancer 1 year after surgery, which could be removed laparoscopically followed by adjuvant chemotherapy. Ten years after breast cancer surgery, pleural metastasis of breast cancer was diagnosed and treated by endocrine therapy. Thirteen years after breast cancer surgery, a pancreatic tumor with multiple liver masses emerged. It was difficult to diagnose whether primary or metastasis cancer by the results of the pathological analysis. Finally, we diagnosed primary pancreatic cancer with liver metastasis by clinical examination with the BRCA2-pathogenic variant. These tumors were well responded to chemotherapy and the patient survived during a follow-up period of 8 months. According to MPMNs, breast cancer patients should be followed-up carefully for the possibility of BRCA pathogenic variant and development of different primary malignant neoplasms.

DOI： 10.14740/wjon1658

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It is well established that genetic information differs amongst the adolescent and young adult population (AYA) and older patients. Although several studies on genetic information have been conducted, no current prognostic biomarker exists to help differentiate survival outcomes amongst AYA patients. The GALNT family of genes have been associated with several cancer etiologies, such as the Tn antigen and epithelial-mesenchymal transition (EMT); however, the clinical significance of GALNT1 expression in breast cancer (BC) remains unclear. We investigated the clinical relevance of GALNT1 expression in BC using two large independent cohorts. We found that, although triple-negative BC (TNBC) had the highest GALNT1 expression compared to ER-positive/HER2-negative BC, GALNT1 levels in BC were not associated with clinical aggressiveness, including histological grade, AJCC stage and N-category, and patient survival, consistently in both the METABRIC and GSE96058 cohorts. There was also no biological difference between low- and high-GALNT1 expression BC, as analyzed by hallmark gene sets via gene set enrichment analysis (GSEA). Further, no significant difference was found in GALNT1 expression levels among AYAs and older patients. However, high GALNT1 expression was associated with significantly worse survival in AYA patients, in both cohorts. Furthermore, high GALNT1 expression was found to be an independent factor among several clinical features, including subtype, histological grade, AJCC T and N-category, in AYA patients. In both cohorts, BC with high GALNT1 expression demonstrated low levels of CD8+ T-cell infiltration, but not other anti-cancerous or pro-cancerous immune cells. Finally, high levels of GALNT1 BC demonstrated increased EMT, angiogenesis, and protein secretion in the AYA population, but not in older patients. In conclusion, our findings demonstrate that GALNT1 expression was found to be associated with angiogenesis and EMT, and may have potential as prognostic biomarker, specifically in AYA patients.

DOI： 10.3390/cancers15133489

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Language：Japanese   Publisher：日本外科系連合学会  

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Language：English  

Granulomatous mastitis (GM) is a rare disease, particularly among men. Herein, we present a case of GM diagnosed in a 63-year-old male patient who showed reduction in the tumor size during 3 months of observation.

DOI： 10.1002/ccr3.7048

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Metabolic reprogramming to sustain immortality is a hallmark of cancer and glycolysis is an important way to attain this. Thus, we investigate the association of glycolysis and associated pathways in the survival of breast cancer. A total of 5,176 breast cancer patients from multiple independent cohorts were analyzed. We determined the glycolytic signaling score by the degree of enrichment by Gene Set Variant Analysis and the median was used to divide each cohort into high vs low score groups. Glycolysis high breast cancer significantly enriched the hallmark cell proliferation-related gene sets (E2F targets, G2M checkpoint, and MYC targets v1 and v2) and was associated with high MKI67 expression. In all cohorts, triple-negative breast cancer (TNBC) was associated with the highest glycolysis score. It was found that in TNBC, glycolysis high breast cancer was associated with worse survival but in ER-positive/HER2-negative breast cancer this was not observed consistently. The glycolysis high TNBC enriched multiple pro-cancerous gene sets and was infiltrated with a low level of B-cells and anti-cancerous immune cells, and significantly associated with a decreased level of cytolytic activity. It was also observed that the glycolysis was higher in the metastatic sites than in the primary breast cancer and the survival was not affected by the metastatic sites. In conclusion, accelerated glycolysis is associated with cancer cell proliferation and worse survival in TNBC.

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BACKGROUND: BRCAness 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: We 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.

DOI： 10.1186/s40364-022-00427-8

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PURPOSE: Although the DNA repair mechanism is important in preventing carcinogenesis, its activation in established cancer cells may support their proliferation and aggravate cancer progression. RAD51 cooperates with BRCA2 and is essential in the homologous recombination of DNA repair. To this end, we hypothesized that RAD51 gene expression is associated with cancer cell proliferation and poor prognosis of breast cancer (BC) patients. METHODS: A total of 8515 primary BC patients with transcriptome and clinical data from 17 independent cohorts were analyzed. The median value was used to divide each cohort into high and low RAD51 expression groups. RESULTS: High RAD51 expression enriched the DNA repair gene set and was correlated with DNA repair-related genes. Nottingham histological grade, Ki67 expression and cell proliferation-related gene sets (E2F Targets, G2M Checkpoint and Myc Targets) were all significantly associated with the high RAD51 BC group. RAD51 expression was positively correlated with Homologous Recombination Deficiency, as well as both mutational burden and neoantigens that accompanied a higher infiltration of immune cells. Primary BC with lymph node metastases was associated with high expression of RAD51 in two cohorts. There was no strong correlation between RAD51 expression and drug sensitivity in cell lines, and RAD51 expression was lower after the neoadjuvant chemotherapy compared to before the treatment. High RAD51 BC was associated with poor prognosis consistently in three independent cohorts. CONCLUSION: RAD51 gene expression is associated with aggressive cancer biology, cancer cell proliferation, and poor survival in breast cancer.

DOI： 10.1007/s10549-022-06552-0

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PURPOSE: Sentinel lymph node biopsy is omitted in older women (≥ 70 years old) with clinical lymph node (LN)-negative hormone receptor-positive breast cancer as it does not influence adjuvant treatment decision-making. However, older women are heterogeneous in frailty while the chance of recurrence increase with improving longevity. Therefore, a biomarker that identifies LN metastasis may facilitate treatment decision-making. RUFY3 is associated with cancer progression. We evaluated RUFY3 expression level as a biomarker for LN-positive breast cancer in older women. METHODS: Clinical and transcriptomic data of breast cancer patients were obtained from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n = 1903) and The Cancer Genome Atlas (TCGA, n = 1046) Pan-cancer study cohorts. RESULTS: A total of 510 (METABRIC) and 211 (TCGA) older women were identified. LN-positive breast cancer, which represented 51.4% (METABRIC) and 48.4% (TCGA), demonstrated worse disease-free, disease-specific, and overall survival. RUFY3 levels were significantly lower in LN-positive tumors regardless of age. The area under the curve for the receiver operator characteristic (AUC-ROC) curves showed RUFY3-predicted LN metastasis. Low RUFY3 enriched oxidative phosphorylation, DNA repair, MYC targets, unfolded protein response, and mtorc1 signaling gene sets, was associated with T helper type 1 cell infiltration, and with intratumor heterogeneity and fraction altered. Low RUFY3 expression was associated with LN-positive breast cancer and with worse disease-specific survival among older women. CONCLUSION: Older women with breast cancers who had low expression level of RUFY3 were more frequently diagnosed with LN-positive tumors, which translated into worse prognosis.

DOI： 10.1007/s10549-021-06482-3

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The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) causes a point mutation from cytidine to uracil in DNA and/or RNA. The role of APOBEC3A and APOBEC3B in breast cancer has been well described, whereas that of APOBEC3F remains unknown. To investigate the clinical relevance of APOBEC3F expression, we analyzed a total of 3000 breast cancer cases from multiple independent large patient cohorts including METABRIC, TCGA, GSE75688, and GSE114725. High expression of APOBEC3F was associated with improved disease-specific and overall survival in triple negative breast cancer (TNBC). APOBEC3F is not usually a reflection of cancer cell biology in TNBC or luminal breast cancer, except for homologous recombination deficiency in TNBC. In the TNBC homologous recombination deficiency group, APOBEC3F expression was not consistently associated with intratumor heterogeneity, mutation rates, or neoantigens. APOBEC3F expression did not correlate with response to any of the drugs tested in breast cancer cell lines in vitro. However, high APOBEC3F expression was associated with enrichment of several immune-related gene sets and immune activity. High APOBEC3F expression also accompanied higher infiltration of anti-cancer immune cell infiltration in TNBC. However, in luminal breast cancer, high APOBEC3F tumor significantly enriched not only immune-related gene sets, but also cell proliferation-, metastasis-, and apoptosis-related gene sets. Analysis of single-cell transcriptomes showed APOBEC3F exclusively expressed in immune cells and significantly associated with cytolytic activity of the immune cells, immune response, and immune cell proliferation. Expression of immune checkpoint genes was uniformly elevated in APOBEC3F-high tumors. We conclude that APOBEC3F is exclusively expressed in immune cells and this expression is associated with enhanced anti-cancer immune response as well as improved survival in TNBC.

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Regulatory T cells (Tregs) are a subset of CD4+ T lymphocytes known to dampen the host immune response against cancer cells. Within the tumor microenvironment, Tregs are potent facilitators of immune tolerance, and a higher proportion of Tregs compared to cytotoxic T cells predicts a worse outcome in most solid tumors. We studied the association between Treg density, and cancer biology and clinical outcome in colorectal cancer (CRC). We used xCell to estimate intratumoral Tregs in total of 898 CRC patients in the Cancer Genome Atlas (TCGA) and GCE39582 cohorts. High-Treg CRCs enriched immune response-related gene sets; inflammatory response, IFN-γ and IFN-α response, IL2/IL6 signaling, and allograft rejection, and had significantly high infiltration of CD8, CD4, M1 and M2 macrophage, and dendritic cells in both cohorts. While high-Treg CRCs enriched multiple pro-cancer signaling pathways compared to low-Treg CRCs, such as Epithelial Mesenchymal Transition, K-ras, Hypoxia, TGF-β, TNF-α, and angiogenesis, Treg infiltration was surprisingly associated with earlier CRC stage in TCGA. Notably, in two separate cohorts a higher proportion of Tregs predicted an improved response to chemotherapy. In the GSE28702 cohort, metastatic CRCs with more Tregs showed a significantly better response to mFOLFOX6 versus low-Treg CRC metastases (88.9% response vs. 16.7%, P<0.001). In the GSE72970 cohort, high-Treg CRCs were found to have a 68.8% response to FOLFOX/FOLFIRI without bevacizumab, compared to 44% response in the low-Treg CRCs. Additionally, high-Treg CRCs were associated with increased expression of immune checkpoint molecules PD-L1/PD-L2, CTLA4, TIGIT and BTLA, implying susceptibility to immunotherapy. We also found that CRCs with higher proportions of Tregs were associated with lower amounts of three microorganisms in the tumor: Lachnoclostridium, flavivirus, and Ornithobacterium. In conclusion, we show that amount of Treg in the tumor is a predictor of host immune response and chemotherapy response in CRC.

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Lymphangiogenesis, the generation of new lymphatic vessels from existing ones, results from the dynamic interactions of lymphatic endothelial cells and the tumor microenvironment (TME). It is well known that lymphangiogenesis occurs during the initial stage of metastasis in various types of malignant tumors. However, it is currently not used as a biomarker partially because gold standard method to quantify it is labor and cost intensive. We hypothesized that the quantity of intratumoral lymphatic endothelial cells (iLECs) in the TME is an indicator of lymphangiogenesis and a predictor of metastatic potential and overall survival in breast cancer. We analyzed a total of 4145 breast cancer patients from the Cancer Genome Atlas (TCGA) and GSE96058 by quantifying their iLECs using the xCell algorithm, and correlated these scores with patient survival, tumor grade, and cancer stage. We also assessed various pro- and anti-cancer gene sets for each tumor to characterize tumor behavior and aggressiveness. As we expected, high-iLEC breast cancer demonstrated enriched lymphoangiogenesis and angiogenesis gene sets and was associated with increased expressions of related genes. Also enriched were inflammatory response and immune response-related gene sets; IL2/STAT5 pathway, IL6/JAK/STAT3 pathway, TNFα pathway, allograft rejection, and complement as well as cancer stemness related gene sets like Notch signaling, Hedgehog signaling, epithelial mesenchymal transition, and Wnt beta-catenin signaling. Tumors with high-iLEC showed higher proportions of stromal cells and fewer anti-cancer immune cells. On the other hand, iLEC score did not correlate with patient survival or lymph node metastasis. Surprisingly, breast cancers with fewer iLECs demonstrated enriched E2F Targets, G2M Checkpoint, MYC Targets v1, and MTORC1 signaling which are cancer cell proliferation-related gene sets and exhibited an abundance of pro-cancer immune cells. The amount of iLEC correlated inversely with Ki67 expression and histological grade, which is in agreement that low-iLEC breast cancer was associated with enhanced cancer cell proliferation. In conclusion, while iLECs can be used as a surrogate for lymphangiogenesis in breast cancer, low-iLEC tumors also exhibit features which correspond to aggressive tumor biology, which may explain why the amount of iLECs was not associated with patient survival in our cohorts.

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AIM: To examine the role of the Modified Glasgow Prognostic Score (mGPS) and Prognostic Nutritional Index (PNI) as prognostic markers for patients with metastatic breast cancer (MBC). PATIENTS AND METHODS: We investigated the associations of clinico-pathological factors with time-to-treatment failure (TTF) and overall survival (OS) in 110 patients with MBC treated with eribulin. RESULTS: C-Reactive protein >1 mg/dl, albumin <3.5 g/dl, mGPS=2, and PNI <40 were significant predictors of shorter TTF in univariate analyses. PNI <40 remained a significant and independent predictor of shorter TTF in multivariate analyses. De novo tumor, visceral metastases, C-reactive protein >1 mg/dl, albumin <3.5 g/dl, mGPS=2, and PNI <40 were significant predictors of poor OS at the univariate level. A PNI <40 was a significant and independent predictor of poor OS in multivariate analyses. CONCLUSION: PNI is a reliable predictor of TTF and OS in patients with MBC treated with eribulin.

DOI： 10.21873/invivo.12903

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Purpose: Estrogen signals play an important role in the phenotype of estrogen receptor-positive breast cancer. However, comprehensive analyses of the effect of responsiveness to estrogen signals on the tumor microenvironment and survival in large cohorts of primary breast cancer patients have been lacking. We aimed to test the hypothesis that estrogen reactivity affects gene expression and immune cell infiltration profiles in the tumor microenvironment and survival. Methods: A total of 3,098 breast cancer cases were analyzed: 1,904 from the Molecular Taxonomy of Breast Cancer (METABRIC) cohort, 1,082 from The Cancer Genome Atlas (TCGA) cohort, and 112 from the Hokkaido University Hospital cohort. We divided the group into estrogen reactivity-high and estrogen reactivity-low groups utilizing the scores of ESTROGEN_RESPONSE_EARLY and ESTROGEN_RESPONSE_LATE in Gene Set Variation Analysis. Results: Breast cancer with high estrogen reactivity was related to Myc targets, metabolism-related signaling, cell stress response, TGF-beta signaling, androgen response, and MTORC1 signaling gene sets in the tumor microenvironment. Low estrogen reactivity was related to immune-related proteins, IL2-STAT5 signaling, IL6-JAK-STAT3 signaling, KRAS signaling, cell cycle-related gene sets, and EMT. In addition, breast cancer with high levels of estrogen reactivity had low immune cytolytic activity and low levels of immunostimulatory cells. It also had low levels of stimulatory and inhibitory factors of the cancer immunity cycle. Patients with high estrogen reactivity were also associated with a better prognosis. Conclusion: We demonstrated the relationship between estrogen reactivity and the profiles of immune cells and gene expression, as well as survival.

DOI： 10.3389/fonc.2022.865024

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Although miR-99b is a known suppressive microRNA (miRNA) in several cancers, its role in breast cancer has not been elucidated. In this study, we examined the clinical relevance of miR-99b expression in breast cancer. We analyzed miRNA and mRNA expression and their relationships with clinical parameters in 1,961 breast cancer samples from two independent large cohorts, the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA). Several algorithms, including gene set enrichment analysis (GSEA) and xCell, have been used to investigate biological functions and the tumor microenvironment. High miR-99b expression significantly enriched the mTORC1 signaling gene set in breast cancer (NES = 1.63, FDR = 0.03, and NES = 1.58, FDR = 0.10, in METABRIC and TCGA, respectively). No other mechanisms, including the epithelial mesenchymal transition, NFκB, and TGF-β signaling, were consistently enriched in both cohorts. MiR-99b-high breast cancer was associated with high homologous recombination deficiencies, intratumor heterogeneity, and high rates of mutation and neoantigens. In agreement, miR-99b-high breast cancer was associated with increased cell proliferation, correlating with Nottingham histological grade, and significant enrichment of E2F targets, G2/M checkpoint, and mitotic spindle gene sets consistently in both cohorts (P = 0.01, P < 0.001). High miR-99b levels were also associated with low stromal cell fractions in the tumor microenvironment, including adipocytes, keratinocytes, and lymphatic endothelial cells (P < 0.001). However, in both cohorts, miR-99b expression was not associated with significant infiltration of immune cells, except dendritic cells (P = 0.006, 0.020). Finally, in both cohorts, breast cancer with high miR-99b expression was significantly associated with worse disease-free survival (DSS) and overall survival (OS), particularly in estrogen receptor (ER)-positive/human epidermal growth factor (HER)2-negative breast cancer (DSS hazard ratio (HR) 1.29, 95% confidence interval (CI) 1.10-1.51, P < 0.001 in the METABRIC cohort and HR 1.82, 95% CI 1.12-2.98, P = 0.017 in the TCGA cohort). In conclusion, breast cancer with high miR-99b expression was significantly associated with mTORC1 signaling, cell proliferation, and decreased patient survival, particularly in the ER-positive/HER2-negative subtype.

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Background: Mutations of BRCA genes are the most studied in breast cancer, but the clinical relevance of BRCA2 gene expression has been less well studied. Given that BRCA2 is a DNA repair gene, we hypothesized that high BRCA2 expression is associated with highly proliferative and aggressive biology in breast cancer. Materials and Methods: A total of 4342 breast cancer patients were analyzed from The Cancer Genome Atlas (TCGA, n = 1069) as the testing cohort and Gene Expression Omnibus (GEO) dataset GSE96058 (n = 3273) as a validation cohort. Results: There was no relationship between BRCA2 mutation and BRCA2 gene expression. BRCA2 high expression breast cancer was associated with higher Nottingham grade (p &lt
0.001), with high proliferation (MKI-67, p &lt
0.001), and with higher intratumor heterogeneity, homologous recombination deficiency, mutation rate, fraction altered, and neoantigens (all p &lt
0.001). BRCA2 high expression was associated with E2F1, RB1, PALB2, and PARP, as well as cell proliferation-related gene sets, E2F targets, G2M checkpoints, and mitotic spindle, and with less ESR1 and ER response early and late gene sets. They were associated with significantly reduced number of stroma cells and with high infiltration of both favorable and unfavorable immune cells. BRCA2 high expression significantly correlated with response to olaparib, a PARP inhibitor, and inversely with cyclophosphamide in ER-positive/HER2-negative tumors, but not in TNBC. Conclusions: BRCA2 high gene expression was associated with highly proliferative and aggressive breast cancer that was highly immunogenic with better response to PARP inhibitors in ER-positive patients. BRCA2 gene expression may become a candidate marker for aggressive biology and to tailor specific treatment strategies in the future.

DOI： 10.1245/s10434-021-10063-5

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BACKGROUND: Mutations of BRCA genes are the most studied in breast cancer, but the clinical relevance of BRCA2 gene expression has been less well studied. Given that BRCA2 is a DNA repair gene, we hypothesized that high BRCA2 expression is associated with highly proliferative and aggressive biology in breast cancer. MATERIALS AND METHODS: A total of 4342 breast cancer patients were analyzed from The Cancer Genome Atlas (TCGA, n = 1069) as the testing cohort and Gene Expression Omnibus (GEO) dataset GSE96058 (n = 3273) as a validation cohort. RESULTS: There was no relationship between BRCA2 mutation and BRCA2 gene expression. BRCA2 high expression breast cancer was associated with higher Nottingham grade (p < 0.001), with high proliferation (MKI-67, p < 0.001), and with higher intratumor heterogeneity, homologous recombination deficiency, mutation rate, fraction altered, and neoantigens (all p < 0.001). BRCA2 high expression was associated with E2F1, RB1, PALB2, and PARP, as well as cell proliferation-related gene sets, E2F targets, G2M checkpoints, and mitotic spindle, and with less ESR1 and ER response early and late gene sets. They were associated with significantly reduced number of stroma cells and with high infiltration of both favorable and unfavorable immune cells. BRCA2 high expression significantly correlated with response to olaparib, a PARP inhibitor, and inversely with cyclophosphamide in ER-positive/HER2-negative tumors, but not in TNBC. CONCLUSIONS: BRCA2 high gene expression was associated with highly proliferative and aggressive breast cancer that was highly immunogenic with better response to PARP inhibitors in ER-positive patients. BRCA2 gene expression may become a candidate marker for aggressive biology and to tailor specific treatment strategies in the future.

DOI： 10.1245/s10434-021-10063-5

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DOI： 10.1245/s10434-021-10135-6

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Hepatocellular carcinoma is a leading cause of cancer death worldwide. The unfolded protein response (UPR) has been revealed to confer tumorigenic capacity in cancer cells. We hypothesized that a quantifiable score representative of the UPR could be used as a biomarker for cancer progression in HCC. In this study, a total of 655 HCC patients from 4 independent HCC cohorts were studied to examine the relationships between enhancement of the UPR and cancer biology and patient survival in HCC utilizing an UPR score. The UPR correlated with carcinogenic sequence and progression of HCC consistently in two cohorts. Enhanced UPR was associated with the clinical parameters of HCC progression, such as cancer stage and multiple parameters of cell proliferation, including histological grade, mKI67 gene expression, and enrichment of cell proliferation-related gene sets. The UPR was significantly associated with increased mutational load, but not with immune cell infiltration or angiogeneis across independent cohorts. The UPR was consistently associated with worse survival across independent cohorts of HCC. In conclusion, the UPR score may be useful as a biomarker to predict prognosis and to better understand HCC.

DOI： 10.3390/cancers13174443

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Advanced gastric cancer (GC) is one of the most lethal cancer types, thus a better understanding of its biology in patients is urgently needed. MicroRNA (miR)-29a is a known tumor suppressive miR that is related to metastasis, but its clinical relevance in GC remains ambiguous. Here, using a large GC patient cohort we hypothesized that low expression of miR-29a in GC is associated with aggressive cancer biology and worse survival. We demonstrated that low miR-29a GC enriched cell proliferation, apoptosis, metastasis, and angiogenesis related gene sets, as well as the higher expression of related genes. Low miR-29a GC was associated with less anti-cancer immune cell infiltration as well as immune related scoring. Low miR-29a GC demonstrated a worse overall survival (OS) as well as disease specific survival (DSS) compared with high expressing miR-29a GC. Notably, low miR-29a expression was the only factor, other than residual tumor status, to be an independent prognostic biomarker of worse OS and DSS. In conclusion, low miR-29a GC was associated with aggressive cancer biology and worse OS as well as DSS. Additionally, low expression of miR-29a was an independent prognostic biomarker of OS and DSS in gastric cancer patients.

DOI： 10.1038/s41598-021-93681-z

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AIM: To evaluate the association between bromodomain-containing protein 4 (BRD4) expression and clinicopathological factors and prognosis in human breast cancer specimens. PATIENTS AND METHODS: We used tissue microarrays constructed from samples of patients (n=183) who underwent surgery. We validated the association between BRD4 expression and prognosis in solid tumours, including breast cancer, using The Cancer Genome Atlas (TCGA) database. RESULTS: Immunohistochemical staining showed that BRD4 was widely distributed in breast cancer tissues. BRD4 was strongly expressed in 19.7% of patients but BRD4 staining intensity was not correlated with other clinicopathological factors. Most importantly, patients with a strong BRD4 expression had a significantly longer disease-specific survival than those with a weak BRD4 expression (100.0% vs. 91.3% at 5 years, p=0.027). mRNA expression analysis showed similar results (91.2% vs. 80.2% at 6 years, p=0.047). CONCLUSION: Strong BRD4 expression was associated with a significantly better prognosis in breast cancer tumours.

DOI： 10.21873/anticanres.15148

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Cancer-associated adipocytes are known to cause inflammation; however, the role of adipogenesis, the formation of adipocytes, in breast cancer is unclear. We hypothesized that intra-tumoral adipogenesis reflects a different cancer biology than abundance of intra-tumoral adipocytes. The Molecular Signatures Database Hallmark adipogenesis gene set of gene set variant analysis was used to quantify adipogenesis. Total of 5,098 breast cancer patients in multiple cohorts (training; GSE96058 (n = 3273), validation; TCGA (n = 1069), treatment response; GSE25066 (n = 508) and GSE20194 (n = 248)) were analyzed. Adipogenesis did not correlate with abundance of adipocytes. Adipogenesis was significantly lower in triple negative breast cancer (TNBC). Elevated adipogenesis was significantly associated with worse survival in TNBC, but not in the other subtypes. High adipogenesis TNBC was significantly associated with low homologous recombination deficiency, but not with mutation load. High adipogenesis TNBC enriched metabolism-related gene sets, but neither of cell proliferation- nor inflammation-related gene sets, which were enriched to adipocytes. High adipogenesis TNBC was infiltrated with low CD8+ T cells and high M2 macrophages. Although adipogenesis was not associated with neoadjuvant chemotherapy response, high adipogenesis TNBC was significantly associated with low expression of PD-L1 and PD-L2 genes, and immune checkpoint molecules index. In conclusion, adipogenesis in TNBC was associated with cancer metabolism and unfavorable tumor immune microenvironment, which is different from abundance of adipocytes.

DOI： 10.1038/s41598-021-91897-7

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Elderly patients are known to have a worse prognosis for breast cancer. This is commonly blamed on their medical comorbidities and access to care. However, in addition to these social issues, we hypothesized that the extreme elderly (octogenarians-patients over 80 years old) have biologically worse cancer with unfavorable tumor immune microenvironment. The Cancer Genomic Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were analyzed. The control (aged 40-65) and octogenarians numbered 668 and 53 in TCGA and 979 and 118 in METABRIC, respectively. Octogenarians had significantly worse breast cancer-specific survival in both cohorts (p < 0.01). Octogenarians had a higher ER-positive subtype rate than controls in both cohorts. Regarding PAM50 classification, luminal-A and -B subtypes were significantly higher in octogenarians, whereas basal and claudin-low subtypes were significantly lower (p < 0.05) in octogenarians. There was no difference in tumor mutation load, intratumor heterogeneity, or cytolytic activity by age. However, the octogenarian cohort was significantly associated with high infiltration of pro-cancer immune cells, M2 macrophage, and regulatory T cells in both cohorts (p < 0.05). Our results demonstrate that octogenarians' breast cancer is associated with worse survival and with an unfavorable tumor immune microenvironment.

DOI： 10.3390/cancers13122933

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Tissue biopsy is the gold standard for diagnosis and morphological and immunohistochemical analyses to characterize cancer. However, tissue biopsy usually requires an invasive procedure, and it can be challenging depending on the condition of the patient and the location of the tumor. Even liquid biopsy analysis of body fluids such as blood, saliva, gastric juice, sweat, tears and cerebrospinal fluid may require invasive procedures to obtain samples. Liquid biopsy can be applied to circulating tumor cells (CTCs) or nucleic acids (NAs) in blood. Recently, urine has gained popularity due to its less invasive sampling, ability to easily repeat samples, and ability to follow tumor evolution in real-time, making it a powerful tool for diagnosis and treatment monitoring in cancer patients. With the development and advancements in extraction methods of urinary substances, urinary NAs have been found to be closely related to carcinogenesis, metastasis, and therapeutic response, not only in urological cancers but also in non-urological cancers. This review mainly highlights the components of urine liquid biopsy and their utility and limitations in oncology, especially in non-urological cancers.

DOI： 10.3390/cancers13112652

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Heterogeneity is the characteristic of breast tumors, making it difficult to understand the molecular mechanism. Alteration of gene expression in the primary tumor versus the metastatic lesion remains challenging for getting any specific targeted therapy. To better understand how gene expression profile changes during metastasis, we compare the primary tumor and distant metastatic tumor gene expression using primary breast tumors compared with its metastatic variant in animal models. Our RNA sequencing data from cells revealed that parental cell and the metastatic variant cell are different in gene expression while gene signature significantly altered during metastasis to distant organs than primary breast tumors. We found that secreted mediators encoding genes (ANGPTL7, MMP3, LCN2, S100A8, and ESM1) are correlated with poor prognosis in the clinical setting as divulged from METABRIC and TCGA-BRCA cohort data analysis.

DOI： 10.3390/cancers13112641

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Mild cold stress induced by housing mice with a 4T1 triple-negative breast cancer (TNBC) cell implantation model at 22 °C increases tumor growth rate with a pro-tumorigenic immune microenvironment (lower CD8 +T cells, higher myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs)). Since cold stress also activates thermogenesis, we hypothesized that enhanced thermogenesis is associated with more aggressive cancer biology and unfavorable tumor microenvironment (TME) in TNBC patients. A total of 6479 breast cancer patients from METABRIC, TCGA, GSE96058, GSE20194, and GSE25066 cohorts were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) thermogenesis score. High-thermogenesis TNBC was associated with a trend towards worse survival and with angiogenesis, adipogenesis, and fatty acid metabolism pathways. On the other hand, low-thermogenesis TNBC enriched most of the hallmark cell-proliferation-related gene sets (i.e., mitotic spindle, E2F targets, G2M checkpoint, MYC targets), as well as immune-related gene sets (i.e., IFN-α and IFN-γ response). Favorable cytotoxic T-cell-attracting chemokines CCL5, CXCL9, CXCL10, and CXCL11 were lower; while the MDSC- and Treg-attracting chemokine CXCL12 was higher. There were higher M2 but lower M1 macrophages and Tregs. In conclusion, high-thermogenesis TNBC is associated with pro-tumor immune microenvironment and may serve as biomarker for testing strategies to overcome this immunosuppression.

DOI： 10.3390/cancers13112559

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Although triple-negative breast cancer (TNBC) typically responds better to neoadjuvant chemotherapy (NAC) compared to the other subtypes, a pathological complete response (pCR) is achieved in less than half of the cases. We established a novel three-gene score using genes based on the E2F target gene set that identified pCR after NAC, which showed robust performance in both training and validation cohorts (total of n = 3862 breast cancer patients). We found that the three-gene score was elevated in TNBC compared to the other subtypes. A high score was associated with Nottingham histological grade 3 in TNBC. Across multiple cohorts, high-score TNBC enriched not only E2F targets but also G2M checkpoint and mitotic spindle, which are all cell proliferation-related gene sets. High-score TNBC was associated with homologous recombination deficiency, high mutation load, and high infiltration of Th1, Th2, and gamma-delta T cells. However, the score did not correlate with drug sensitivity for paclitaxel, 5-fluorouracil, cyclophosphamide, and doxorubicin in TNBC human cell lines. High-score TNBC was significantly associated with a high rate of pCR not only in the training cohort but also in the validation cohorts. High-score TNBC was significantly associated with better survival in patients who received chemotherapy but not in patients who did not receive chemotherapy. The three-gene score is associated with a high mutation rate, immune cell infiltration, and predicts response to NAC in TNBC.

DOI： 10.3390/cancers13102401

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BACKGROUND: Tumor-associated tissue eosinophilia (TATE) has been associated with outcomes in a variety of solid tumors; however, its role in breast cancer is not well defined. We hypothesized that tumor-associated tissue eosinophilia is associated with a high mutation and neoantigen load, and we assessed its correlation with cancer outcomes. METHODS: The Cancer Genome Atlas was analyzed for eosinophil signatures in breast cancer specimens. Descriptive analyses were performed, including the tumor-infiltrating cell composition using CIBERSORT, cytolytic activity score, and gene set enrichment analysis. Overall survival and disease-free survival were calculated using the Kaplan-Meier method. RESULTS: Out of 1069 cases analyzed, 40 (3.7%) had tissue eosinophils (the tumor-associated tissue eosinophilia group). Tumor-associated tissue eosinophilia was noted in 32.5% luminal, 5% HER2-positive, and 15% triple-negative breast cancer subtypes. The single nucleotide variant-neoantigen load was significantly higher in the tumor-associated tissue eosinophilia group (P = .005), with a higher nonsilent mutation rate (P = .01). The tumor-associated tissue eosinophilia group had lower cytolytic activity (P = .02) but had enriched MYC-targeted (P = .002), E2F-targeted (P = .04), deoxyribonucleic acid repair (P = .03), and unfolded protein response gene sets (P = .05). Tumor-associated tissue eosinophilia was associated with a trend toward improved disease-free survival (P = .06) but presented no differences in overall survival (P = .56). CONCLUSION: Tumor-associated tissue eosinophilia was noted in 3.7% of breast cancers and was associated with a higher single nucleotide variant-neoantigen load and nonsilent mutation rate, similar to that of tumor-infiltrating lymphocytes in the triple-negative subtype. However, a lower cytolytic activity score and enriched cell proliferation-related gene sets implicate different roles for tumor-associated tissue eosinophilia than for tumor-infiltrating lymphocytes.

DOI： 10.1016/j.surg.2020.07.052

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The generation of pathologic, immature, and dysfunctional vessels by angiogenesis is a mechanism of metastasis that has been a therapeutic target for colorectal cancer (CRC). In this study, we investigated the clinical relevance of intra-tumoral microvascular endothelial (mvE) cells in CRC using the xCell algorithm on transcriptome. A total of 1244 CRC patients in discovery and validation cohorts were analyzed. We found that an abundance of mvE cells did not mirror angiogenesis but reflected mature blood vessels because it was significantly associated with a high expression of vascular stability-related genes, including sphingosine-1-phosphate receptor genes and pericytes. Epithelial-mesenchymal transition and myogenesis gene sets were enriched in mvE cell abundant CRC, while mvE cell-less CRC enriched cell proliferation, oxidative phosphorylation, and protein secretion gene sets. mvE cell abundant CRC was associated with infiltration of M2 macrophages, dendritic cells, and less gamma-delta T cells (all p < 0.001), but not with the interferon-γ response. mvE cell abundant CRC was significantly associated with worse patient survival in CRC. Interestingly, mvE cell abundant CRC was significantly associated with a high response rate to chemotherapy (p = 0.012) and worse patient survival for those that did not receive chemotherapy. However, there was no survival difference in patients who underwent chemotherapy. In conclusion, we estimated the abundance of mvE cells using the xCell algorithm on tumor transcriptome finding its association with the number of mature blood vessels in a tumor microenvironment and its ability to predict response to chemotherapy, thereby patient survival in CRC.

DOI： 10.3390/cancers13061477

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Annexin A1 (ANXA1) is a calcium-dependent phospholipid-binding protein overexpressed in pancreatic cancer (PC). ANXA1 expression has been shown to take part in a wide variety of cancer biology, including carcinogenesis, cell proliferation, invasion, apoptosis, and metastasis, in addition to the initially identified anti-inflammatory effect in experimental settings. We hypothesized that ANXA1 expression is associated with cell proliferation and survival in PC patients. To test this hypothesis, we analyzed 239 PC patients in The Cancer Genome Atlas (TCGA) and GSE57495 cohorts. ANXA1 expression correlated with epithelial-mesenchymal transition (EMT) but weakly with angiogenesis in PC patients. ANXA1-high PC was significantly associated with a high fraction of fibroblasts and keratinocytes in the tumor microenvironment. ANXA1 high PC enriched multiple malignant gene sets, including hypoxia, tumor necrosis factor (TNF)-α signaling via nuclear factor-kappa B (NF-kB), and MTORC1, as well as apoptosis, protein secretion, glycolysis, and the androgen response gene sets consistently in both cohorts. ANXA1 expression was associated with TP53 mutation alone but associated with all KRAS, p53, E2F, and transforming growth factor (TGF)-β signaling pathways and also associated with homologous recombination deficiency in the TCGA cohort. ANXA1 high PC was associated with a high infiltration of T-helper type 2 cells in the TME, with advanced histological grade and MKI67 expression, as well as with a worse prognosis regardless of the grade. ANXA1 expression correlated with a sensitivity to gemcitabine, doxorubicin, and 5-fluorouracil in PC cell lines. In conclusion, ANXA1 expression is associated with EMT, cell proliferation, survival, and the drug response in PC.

DOI： 10.3390/cells10030653

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The colon adenoma-carcinoma sequence is a multistep genomic-altering process that occurs during colorectal cancer (CRC) carcinogenesis. Organoids are now commonly used to model both non-cancerous and cancerous tissue. This study aims to investigate how well organoids mimic tissues in the adenoma-carcinoma sequence by comparing their transcriptomes. A total of 234 tissue samples (48 adenomas and 186 CRC) and 60 organoid samples (15 adenomas and 45 CRC) were analyzed. We found that cell-proliferation-related gene sets were consistently enriched in both CRC tissues and organoids compared to adenoma tissues and organoids by gene set enrichment analysis (GSEA). None of the known pathways in the colon adenoma-carcinoma sequence were consistently enriched in CRC organoids. There was no enrichment of the tumor microenvironment-related gene sets in CRC organoids. CRC tissues enriched immune-response-related gene sets, whereas CRC organoids did not. The proportions of infiltrating immune cells were different between tissues and organoids, whereas there was no difference between cancer and adenoma organoids. The amounts of cancer stem cells and progenitor cells were not different between CRC and adenoma organoids, whereas a difference was noted between CRC and adenoma tissues. In conclusion, we demonstrated that organoids model only part of the adenoma-carcinoma sequence and should be used with caution after considering their limitations.

DOI： 10.3390/cells10030488

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Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related deaths worldwide. In this study, a total of 749 HCC patients from 5 cohorts were studied to examine the relationships between enhancement of DNA repair and cancer aggressiveness, tumor immune microenvironment, and patient survival in HCC, utilizing a DNA repair pathway score. Our findings suggest that the DNA repair pathway was not only enhanced by the stepwise carcinogenic process of HCC, but also significantly enhanced in grade 3 HCC compared with grade 1 and 2 tumors. DNA repair high HCC was associated with worse survival, elevated intratumor heterogeneity, and mutation load, but not with the fraction of immune cell infiltration nor immune response. HCC tumors with a DNA repair high score enriched the cell proliferation- and other cancer aggressiveness-related gene sets. Interestingly, these features were more pronounced in grade 1 and 2 HCC compared to grade 3 HCC. To our knowledge, this is the first study to use DNA repair pathway-related gene set expression data to examine and validate the clinical relevance of DNA repair pathway activity in HCC. The DNA repair score may be used to better understand and predict prognosis in HCC.

DOI： 10.3390/cancers13020323

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BACKGROUND: High infiltration of Th2 is linked to breast cancer progression and metastasis through the induction of cytokine release and T-cell anergy. The estrogen receptor (ER)-positive subtype, which accounts for 70% of breast cancer, is known to respond less to neoadjuvant chemotherapy (NAC) due to its low potential for proliferation. We hypothesized that Th2 high tumors are highly proliferative, and thus more likely to respond to NAC in ER-positive breast cancer. METHODS: We obtained clinicopathological data and overall survival information on 1,069 breast cancer patients from The Cancer Genome Atlas (TCGA). Computational algorithms and CIBERSORT were used to estimate immune cell infiltration. Additionally, xCell was used for validation. RESULTS: Th2 high tumors did not consistently associate with an unfavorable immune cell composition and tumor immune microenvironment but were found to be significantly elevated in the cancer stage. Th2 high tumors also correlated with high Nottingham pathological grade, as well as with Ki-67 and proliferation score in ER-positive subtypes. High Th2 tumors achieved a pathological complete response (pCR) significantly higher in ER-positive breast cancer. CONCLUSIONS: In conclusion, high levels of Th2 are associated with aggressive features of breast cancer. Th2 levels may be a biomarker in patient selection for NAC in ER-positive breast cancer.

DOI： 10.21037/gs-20-571

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Some may think that prediction of metastasis is meaningless since metastatic breast cancer is currently incurable. We argue that effective identification of developing metastasis will enable us to design and conduct clinical trials specifically targeting those patients at high risk. The current study sought to generate the KAM score by 4 genes (BRSK2, EYA1, SIGLEC15, and AGTR1) overexpressed in primary breast cancer that developed metastasis to bone compared with matched controls without metastasis longer than 10 years. A high KAM score was prognostic of poor overall (OS), disease free survival (DFS), and disease specific survival (DSS) in the METABRIC, and OS in the GSE96058 cohorts. A high KAM score was significantly associated with clinical aggressiveness, such as high American Joint Committee Cancer (AJCC) stage, lymph node metastasis, Nottingham pathological grade, and triple negative breast cancer (TNBC). Subgroup analysis revealed that a high KAM score was associated with worse OS in ER-positive/HER2-negative breast cancer in both cohorts. A high KAM breast cancer enriched all 5 cell proliferation-related gene sets of the Hallmark collection and interferon (IFN)-γ response gene sets. Furthermore, a high KAM breast cancer was significantly infiltrated with a high fraction of not only anti-cancer but also pro-cancer immune cells and associated with high level of cytolytic activity. Finally, a high KAM breast cancer was significantly associated with lung metastasis. In conclusion, we developed KAM score using 4 gene expressions that predict lung metastasis and patient survival in breast cancer.

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MiR-195 is a tumor suppressive microRNA in breast cancer. Its clinical relevance remains debatable as it has only been studied via in vitro experiments or small cohort studies. We analyzed a total of 2,038 patients in the TCGA and METABRIC cohorts to assess whether low miR-195 expressing tumors are associated with aggressive cancer characteristics and poor prognostic outcomes. The median cutoff of miR-195 expression was used to split the groups into miR-195 high and low groups. Low miR-19 expressing tumors demonstrated high cell proliferating features by enriching the gene sets associated with cell proliferation, MKI67 expression and pathological grade. One-third of the top target miR-195 genes were related to cell proliferation. Low miR-195 expressing tumors were associated with both pro-cancerous and anti-cancerous immune cells. Low miR-195 expressing tumors were associated with enhanced glycolysis and poor survival in ER-positive tumors, but not other subtypes of breast cancer. In conclusion, low expression of miR-195 in ER-positive breast cancer was associated with enhanced cancer cell proliferation, glycolysis, and worse overall survival.

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Neoadjuvant Chemotherapy (NAC) is not frequently used in ER-positive/HER2-negative breast cancer (BC) because around 10% patients achieve pathological complete response (pCR). Since NAC can result in cancer downstaging both in the breast and axilla and prevent a morbid surgery, thus a score to predict pCR in this population will be crucial to identify patients who can benefit from this approach. A total of 4038 patients from cohorts; GSE25066, GSE20194, Hess, GSE20181, TCGA-BRCA and METBRIC were analyzed. The score was generated by the 5 most highly expressed genes in the Hallmark E2F targets gene set amongst patients in the GSE25066 cohort with ER-positive/HER2-negative BC who achieved pCR. The area under the curve was significantly higher in the score than that for the E2F targets score. High score ER-positive/HER2-negative BCs were significantly associated with higher Nottingham pathological grade, AJCC cancer stage, MKI67 expression levels, intratumor heterogeneity, homologous recombination defects, mutation burden, neoantigen load, and infiltration of anti-cancer immune cells (CD4+, T helper type1, plasmacytoid dendritic cells, M1 macrophages). They also expressed lower abundance of stromal cells including fibroblasts, lymphatic endothelial cells, pericytes and adipocytes consistently in GSE25066, TCGA and METABRIC cohorts. All cell proliferation-related gene sets, G2M checkpoint, E2F targets, MYC targets v1 and v2, Mitotic Spindle, were strongly enriched in high score BCs consistently in 3 cohorts. The gene score was significantly associated with high pCR rate consistently in the GSE25066 (38%, P < 0.001), GSE20194 (16%, P = 0.006), and Hess cohort (23%, P = 0.037). In conclusion, the 5-gene score reflects cancer cell proliferation and immune cell infiltration, and predicts pCR after NAC in ER-positive/HER2-negative breast cancer.

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Given the severe side effects of the treatments and poor survival, prognostic and predictive biomarkers to guide management of pancreatic cancer are in critical need. We hypothesized that cell proliferation-related pathways are associated with drug response and survival in pancreatic cancer. Six Hallmark cell proliferation-related gene sets (G2M Checkpoint, E2F Targets, MYC Targets V1 and V2, Mitotic Spindle, p53 pathway) defined by MSigDB in gene set variant analysis were evaluated in 3 independent cohorts- TCGA-PAAD (n = 176), GSE57495 (n = 63), and GSE62452 (n = 69). G2M and E2F, as well as Mitotic and p53 pathway correlated highly with other gene sets. All pathways were significantly correlated with MKI67 expression and its proliferation score, but none with cytolytic activity and the rate of pathologically complete resection (R0). All pathways were significantly associated with high alteration and expression of KRAS gene except for MYC v1. G2M, E2F, and p53 pathway were significantly associated with high alteration of TP53 gene. Interestingly, different pathways correlated with the AUC of different cancer therapeutics, such as Gemcitabine (Mitotic: r = 0.706 [P = 0.01]), Paclitaxel (MYC v2: r = -0.636 [P < 0.05]), Apatinib (Mitotic: r = -0.556 [P = 0.03]), Palbociclib (E2F: r = 0.675 [P < 0.01]), and Sorafenib (G2M: r = -0.593 [P = 0.03]). Among all six pathways, only G2M was consistently associated with worse patient survival in all three cohorts. In conclusion, each cell proliferation-related pathway was predictive of a unique agent, and the G2M score alone predicts survival in pancreatic cancer.

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Angiogenesis is a cornerstone of cancer as it allows tumors to receive oxygen and nutrients. A high level of angiogenesis within a tumor may therefore be indicative of its aggressiveness. In this study, we examined this hypothesis in gastric cancer. Gene set variation analysis was used to measure the level of angiogenesis in tumors in 1,348 gastric cancer patients using the Hallmark_angiogenesis gene set to score tumor transcriptomes. As we predicted, there was a significant correlation between angiogenesis score and expression of angiogenesis-related genes. The score moderately correlated with abundance of vessel-related stromal cells, fibroblasts and chondrocytes in the tumor microenvironment (TME). Tumors with high score had low infiltration of T helper type 1 and 2 cells but a greater infiltration of M1 macrophages and dendritic cells. They also had enriched expression of gene sets for coagulation, hypoxia, epithelial mesenchymal transition (EMT), and TGF-β signaling. High angiogenesis score was significantly associated with advanced AJCC stage and higher T- but not N-parameters in the TNM staging system. Patients with a high score also had shorter survival. In conclusion, bulk tumor transcriptome-based quantification of tumor angiogenesis using a computational algorithm may serve to identify patients with worse survival in gastric cancer.

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Patients with triple negative breast cancer (TNBC) have a poor prognosis. A novel prognostic biomarker may guide management by appropriately selecting patients for particular treatments. Peripheral blood neutrophil-to-lymphocyte ratio (NLR) was reported to associate with cancer progression, thus we hypothesized that intratumor genetic NLR will reflect tumor immune microenvironment (TIME) and breast cancer biology. The intratumoral genetic NLR previously defined as the ratio of CD66b (CEACAM8) and CD8 (CD8A) gene expressions was utilized to analyze total of 2,994 patients from METABRIC, TCGA, GSE21094, GSE22358, GSE25088, GSE32646, and GSE2603 cohorts. Intratumoral genetic NLR did not correlate with cancer stage nor clinical parameters of cancer cell proliferation such as Nottingham histological grade or MKI67 expression levels in neither the METABRIC or TCGA cohorts. Intratumoral genetic NLR-high breast cancer was not associated with pathologic complete response (pCR) after neoadjuvant chemotherapy in 5 independent cohorts with different regimens. Despite these results, intratumoral genetic NLR-high TNBC demonstrated worse disease-free, disease-specific, and overall survival. Intratumoral genetic NLR-low TNBC enriched multiple immune-related gene sets, was associated with higher favorable immune-related scores and with a favorable TIME, whereas no gene sets enriched to NLR-high TNBC. In conclusion, intratumoral genetic NLR-low TNBC was associated with favorable TIME and with better survival.

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Evaluation of the functional aspects if the tumor immune microenvironment (TIME), such as the recently introduced cytolytic activity score (CYT) index have been under the spotlight in cancer research; however, clinical relevance of immune cell killing activity in breast cancer has never been analyzed in large patient cohorts. We hypothesized that CYT reflects the immune activity of TIME and can predict patient survival. A total of 7533 breast cancer patients were analyzed as both discovery and validation cohorts. We found that high CYT was associated with advanced histological grade and triple-negative breast cancer (TNBC). High CYT in tumors was significantly associated with better survival in TNBC, but unexpectedly, not in other breast cancer subtypes. High CYT TNBC included both favorable immune-related, as well as unfavorable (suppressive) inflammation-related gene sets, and characterized by high infiltration with T cells and B cells. High CYT TNBC was associated with high homologous recombination deficiency and low somatic copy number alteration score and less mutant allele tumor heterogeneity, but not with tumor mutation burden (TMB). Although CYT was not associated with pathological complete response after neoadjuvant chemotherapy, it was significantly associated with high expression of multiple immune checkpoint molecules. In conclusion, CYT of TNBC is associated with enhanced anti-cancer immunity, less intra-tumoral heterogeneity, and with better survival.

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Tumor infiltrating immune cells plays a critical role in cancer progression. Apoptosis is an autonomous cell death that counteracts tumor growth. To this end, we hypothesized that increased apoptosis in breast cancer is associated with immune cell killing. Apoptosis score of MSigDB Hallmark collection was used to analyze METABRIC cohort (n=1904) and TCGA (n=1069) as validation cohort. High apoptosis tumors enriched cancer promoting signaling pathways; hypoxia, KRAS, TGF-β, PI3K signaling, and was associated with low MKI67 expression and less cell proliferation gene sets, less homologous recombination defects, and less altered fraction. High apoptosis tumors also enriched angiogenesis and high infiltration of vascular endothelial cells, pericytes and stromal cells and significantly enriched inflammation and immune response-related gene sets and high infiltration of CD8, CD4 memory, dendritic cells, M1 and M2 macrophages and significant elevation of cytolytic activity and immune checkpoint molecules, consistently in both cohorts. In conclusion, breast cancer patients with high apoptosis are associated with angiogenesis, immune response, high immune cell infiltration and cytolytic activity. To the best of our knowledge, this is the first study to utilize in silico translational approach to demonstrate the clinical relevance of apoptosis in breast cancer patients in large cohorts.

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Sphingosine-1-Phosphate (S1P) is produced by Sphingosine Kinase 1 (SphK1) in the cell and is transported out of the cells by ABCC1 transporter. S1P induces inflammation, angiogenesis and modulates tumor immune microenvironment (TIME) in autocrine and paracrine manner. We hypothesized that high S1P export is associated with hepatocellular carcinoma (HCC) progression and worse survival. Transcriptome linked with clinical data were obtained from a total of 533 patients from TCGA (The Cancer Genome Atlas)-HCC (n = 350), GSE6764 (n = 75), and GSE89377 (n = 108) cohorts. Both SphK1 and ABCC1 were expressed higher in aggressive HCC than normal liver or cirrhosis and correlated with MKi67 expression. High S1P export by high expression of both SphK1 and ABCC1 enriched gene sets related with cell proliferation (E2F targets, G2M checkpoint, MYC targets), inflammation (Inflammatory response, TNFα, IL6), angiogenesis, metastasis (TGF-β, epithelial-mesenchymal transition), and immune response (allograft rejection, complement, interferon-gamma) in gene set enrichment analysis. High S1P export was associated with elevation of HGF, HSP90AA1, TRAF2, and AKR1B10. It was also associated with high intratumor heterogeneity, leucocyte fraction, macrophage regulation and lymphocyte infiltration, as well as T helper type2 cells, macrophages, dendritic cells, CD4+ T memory activated cells, B-cells and cytolytic activity score in TIME. High S1P export was associated with significantly worse disease specific survival (P = 0.034) and overall survival (P = 0.004) compared to low S1P export group. In conclusion, simultaneous high expression of SphK1 and ABCC1 that reflect S1P export is associated with enhancement of both HCC progression and immune response. Given that S1P export was also associated with worse survival, we cannot help but speculate that pro-cancer pathways activated by S1P may overwhelm the anti-cancer immune response mediated by S1P.

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Metaplastic breast cancer (MBC) constitutes a rare but unique histologic entity with poor prognosis. We hypothesized that MBC possesses unique genetic profile and tumor immune microenvironment. MBC cases were identified from a total of 10827 breast cancer entries in the Cancer Genome Atlas Data Set (TCGA) and the AACR-GENIE (Genomics Evidence Neoplasia Information Exchange) cohorts. Tumor infiltrated immune cells were estimated by xCell. Baseline clinical characteristics were compared, and gene set enrichment analysis (GSEA) was performed. MBC comprised 0.66% of the cohorts (1.2% of TCGA and 0.6% of GENIE). MBC cases were predominantly triple-negative (TNBC) (8 (61.5%) vs 151 (14.4%), P<0.001), and high Nottingham histological grade (8 (61.5%) vs 222 (21.1%), P=0.02) compared to non-MBC in the TCGA cohort. Increased infiltration of M1 macrophages (P=0.012), dendritic cells (P<0.001) and eosinophils (P=0.036) was noted in the MBC cohort however there was no difference in cytolytic activity (P=0.806), CD4 memory (P=0.297) or CD8 T-cells (P=0.864). Tumor mutation burden was lower in the MBC compared to the non-MBC, median: 0.4 vs 1.6/Mb in the TCGA-TNBC cohort (P=0.67) and 3.0 vs 4.0/Mb (P=0.1) in the GENIE-cohort. MBC had increased intratumor heterogeneity (P<0.001), macrophage regulation (P=0.008) and TGF-beta response (P<0.001). Disease-specific survival was decreased in MBC (P=0.018). Angiogenesis and epithelial-to-mesenchymal transition pathways were enriched in triple-negative MBC by GSEA (P=0.004 and P<0.001, respectively). Our results suggest that high intratumor heterogeneity, enriched angiogenesis and EMT pathway expression represent possible mechanisms leading to worse disease-specific survival found in metaplastic breast cancer.

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In experimental settings, maternal embryonic leucine zipper kinase (MELK), an apical member of the snf1/AMPK serine-threonine kinases family, plays a role in tumor growth. We investigated the clinical relevance of MELK expression by performing silico analyses of 7,135 breast cancer patients using multiple independent large cohorts. In triple negative breast cancer (TNBC) found that elevated MELK expression significantly correlates with Nottingham histologic grade and tumor growth according to American Joint Committee Cancer (AJCC) stage. High MELK tumor enriched cell proliferation-related gene sets as well as DNA repair, unfolded protein response, and MTORC signaling gene sets. In two independent cohorts a high mutation rate and worse survival was significantly associated with high MELK tumor. In immune-related gene sets including, allograft rejection, interferon (IFN)-α response, and IFN-γ response, high MELK tumor significantly enriched. Pro-cancer regulatory T cells, T helper type 2 cells and anti-cancer immune cells including CD4+ memory T cells, T helper type1 cells, CD8+ T cells, M1 macrophages, gamma-delta T cells, and dendritic cells with high levels of cytolytic activity (CYT) were highly infiltrated. MELK expression did not correlate with the responses to any of the drugs tested in cell lines. However, pathologic complete response was significantly associated with high MELK following NAC in both TNBC and ER-positive plus HER2-negative breast cancer. In conclusion, cell proliferation, immune response, and NAC breast cancer response was associated with MELK expression.

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Epidermal growth factor receptor (EGFR) is one of the receptors that belong to the epidermal growth factor family of receptor tyrosine kinases (ErbBs). Several malignancies including breast cancer that express EGFR have poor prognosis. Our study examined the EGFR expression among 5176 breast cancer patients from GSE96058 and METABRIC cohorts and the contribution of tumor immune microenvironment in different subtypes. We found that among different breast cancer subtypes, EGFR expression in TNBC was the highest compared to other subtypes. EGFR high ER-positive/HER2-negative breast cancer had significantly higher survival compared to EGFR low ER-positive/HER2-negative breast cancer. It was also associated with high level of intratumor heterogeneity and homologous recombination defects (HRD). This group was also enriched in immune-related gene sets. On the other hand, low EGFR tumor was enriched in cell proliferation-related gene sets. However, these findings were not observed in TNBC. Interestingly, there was a greater infiltration of anti-cancer immune cells in high EGFR ER-positive/HER2-negative breast cancers, while, TNBC with higher EGFR expression had lower fraction of immune cells along with low level of cytolytic activity. Tumor cells have significantly higher EGFR expression compared to immune cells in single cell sequencing data. There was higher expression of immune checkpoint molecules in high EGFR ER-positive/HER2-negative breast cancer but lower expression in TNBC. High EGFR metastatic tumor was significantly associated with worse survival, but no association with infiltrating immune cells was observed. Our study shows that higher EGFR expression in ER-positive/HER2-negative breast cancer is associated with improved outcomes and an anti-cancer immune microenvironment.

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Colon cancer stem cells (CSC) identified by cell surface markers CD133, CD24, and CD44, have been shown to be involved with tumor formation, chemotherapy resistance, and the progression of metastatic disease. Using an in silico translational approach, we hypothesize that a combination of these CSC markers has prognostic value in a large cohort of patients with colorectal cancer. Clinicopathologic and RNA expression data from a total of 594 colorectal cancer (CRC) patients from TCGA were analyzed. The expression of CD133, CD24, and CD44 was individually defined as "high" or "low" based on the median expression. Disease specific survival (DSS) and overall survival (OS) were not associated with tumors that are CD133-high or CD44-high alone. Patients with CD24-high tumors have significantly better DSS (P<0.001) and OS (P = 0.043). CD24-high, CD44-high and CD133-high tumors were associated with significantly greater EGFR, KRAS and Ki67 expression (all P<0.001). CD133, CD24 and CD44-high tumors were independently enriched for conventional stemness-related signaling pathways such as Wnt/β-catenin and Hedgehog signaling pathways. There was no survival difference linked to CD133-high/CD44-low patients, but CD44-high/CD24-low patients have worse DSS (P = 0.005) compared with CD44-low/CD24-high patients. CD133-high/CD24-low tumors show significant negative enrichment of MYC targets, E2F targets, G2M checkpoint and mitotic spindle gene sets, suggesting less cell proliferation in these tumors. Patients with CD133-high/CD24-low tumors have worse DSS (P = 0.004) and OS (P = 0.044), and are more likely to have early and late recurrences. In conclusion, we demonstrated that CD133-high/CD24-low tumors may predict colorectal cancer prognosis.

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Nuclear factor erythroid 2-related factor 2 (NRF2) is a key modifier in breast cancer. It is unclear whether NRF2 suppresses or promotes breast cancer progression. We studied the clinical relevance of NRF2 expression by conducting in silico analyses in 5443 breast cancer patients from several large patient cohorts (METABRIC, GSE96058, GSE25066, GSE20194, and GSE75688). NRF2 expression was significantly associated with better survival, low Nottingham pathological grade, and ER-positive/HER2-negative and triple negative breast cancer (TNBC). High NRF2 ER-positive/HER2-negative breast cancer enriched inflammation- and immune-related gene sets by GSEA. NRF2 expression was elevated in immune, stromal, and cancer cells. High NRF2 tumors were associated with high infiltration of immune cells (CD8+, CD4+, and dendritic cells (DC)) and stromal cells (adipocyte, fibroblasts, and keratinocytes), and with low fraction of Th1 cells. NRF2 expression significantly correlated with area under the curve (AUC) of several drug response in multiple ER-positive breast cancer cell lines, however, there was no significant association between NRF2 and pathologic complete response (pCR) rate after neoadjuvant chemotherapy in human samples. Finally, high NRF2 breast cancer was associated with high expression of immune checkpoint molecules. In conclusion, NRF2 expression was associated with enhanced tumor-infiltrating lymphocytes in ER-positive/HER2-negative breast cancer.

DOI： 10.3390/cancers12123856

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Pathologically complete (R0) resection is essential for prolonged survival in pancreatic cancer. Survival depends not only on surgical technique, but also on cancer biology. A biomarker to predict survival is a critical need in pancreatic treatment. We hypothesized that this 4-gene score, which was reported to reflect cell proliferation, is a translatable predictive biomarker for pancreatic cancer. A total of 954 pancreatic cancer patients from multiple cohorts were analyzed and validated. Pancreatic cancer had the 10th highest median score of 32 cancers in The Cancer Genome Atlas (TCGA) cohort. The four-gene score significantly correlated with pathological grade and MKI67 expression. The high four-gene score enriched cell proliferation-related and cancer aggressiveness-related gene sets. The high score was associated with activation of KRAS, p53, transforming growth factor (TGF)-β, and E2F pathways, and with high alteration rate of KRAS and CDKN2A genes. The high score was also significantly associated with reduced CD8+ T cell infiltration of tumors, but with high levels of interferon-γ and cytolytic activity in tumors. The four-gene score correlated with the area under the curve of irinotecan and sorafenib in primary pancreatic cancer, and with paclitaxel and doxorubicin in metastatic pancreatic cancer. The high four-gene score was associated with significantly fewer R0 resections and worse survival. The novelty of the study is in the application of the four-gene score to pancreatic cancer, rather than the bioinformatics technique itself. Future analyses of inoperable lesions are expected to clarify the utility of our score as a predictive biomarker of systemic treatments.

DOI： 10.3390/cancers12123635

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BACKGROUND/AIM: Aldehyde dehydrogenase 1 (ALDH1) is known as a breast cancer stem cell (CSC) marker. This study aimed to identify genes associated with ALDH1. MATERIALS AND METHODS: ALDH1-positive and -negative breast cancer cells were isolated using laser capture microdissection from five tissue samples of ALDH1-positive breast cancer patients. Messenger RNA expression levels were compared between ALDH1-positive and -negative cells. RESULTS: We found 104 differentially expressed genes between ALDH1-positive and -negative cells. Gene ontology and pathway analysis revealed that these genes were correlated with CSC functions and pathways. Network analyses identified 10 genes that were closely associated with ALDH1. We validated these 10 genes utilizing The Cancer Genome Atlas and the Molecular Taxonomy of Breast Cancer International Consortium cohort, and found that they were associated with ALDH1 expression and correlated with Wnt pathway signaling. CONCLUSION: The 10 genes we identified could be potential targets for CSC therapy of breast cancer.

DOI： 10.21873/anticanres.14696

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Endocrine therapy is the gold-standard treatment for ER-positive/HER2-negative breast cancer. Although its clear benefit, patient compliance is poor (50-80%) due to its long administration period and adverse effects. Therefore, a predictive biomarker that can predict whether endocrine therapy is truly beneficial may improve patient compliance. In this study, we use estrogen response early gene sets of gene set enrichment assay algorithm as the score. We hypothesize that the score could predict the response to endocrine therapy and survival of breast cancer patients. A total of 6549 breast cancer from multiple patient cohorts were analyzed. The score was highest in ER-positive/HER2-negative compared to the other subtypes. Earlier AJCC stage, as well as lower Nottingham pathological grade, were associated with a high score. Low score tumors enriched only allograft rejection gene set, and was significantly infiltrated with immune cells, and high cytolytic activity score. A low score was significantly associated with a worse response to endocrine therapy and worse survival in both primary and metastatic breast cancer patients. The hazard ratio was double that of ESR1 expression. In conclusion, the estrogen response early score predicts response to endocrine therapy and is associated with survival in primary and metastatic breast cancer.

DOI： 10.3390/cancers12123557

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Dendritic cells (DC) represent a major antigen-presenting cell type in the tumor immune microenvironment (TIME) and play an essential role in cancer immunity. Conventional DC (cDC) and plasmacytoid DC (pDC) were defined by the xCell algorithm and a total of 2968 breast cancer patients (TCGA and METABRIC) were analyzed. We found that triple-negative breast cancer (TNBC) had a high fraction of cDC and pDC compared to the other subtypes. In contrast to cDC, high pDC in TNBC was significantly associated with better disease-specific and disease-free survival consistently in both cohorts. High cDC TNBC tumors enriched not only inflammation and immune-related, but also metastasis-related gene sets in Gene Set Enrichment Analysis, whereas high pDC TNBC enriched inflammation and immune -related gene sets including IFN-γ signaling more strongly than cDC. pDC TNBC correlated with CD8+, CD4+ memory, IFN-γ score, and cytolytic activity stronger than cDC TNBC. High pDC TNBC were associated with a high fraction of anti-cancer immune cells and high expression of all the immune check point molecules examined. In conclusion, pDC levels correlated with the infiltration of immune cells and patient survival in TNBC more strongly than cDC; this is the first study suggesting the clinical relevance of pDC infiltration in TNBC.

DOI： 10.3390/cancers12113342

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MYC is one of the most studied oncogenes that is known to promote cell proliferation. We utilized MYC targets v1 and MYC targets v2 scores of gene set variation analysis and hypothesized that these scores correlate with tumor aggressiveness and survival outcomes. We examined a total of 3109 breast cancer patients from TCGA, METABRIC, and GSE124647 cohorts. In each cohort, the patients were divided into high- and low-score groups using the upper third value as the cut off. As expected, higher scores were related to increased cell proliferation and worse clinical and pathologic features. High MYC targets scores were associated with worse survival, specifically in primary ER-positive breast cancer, consistently in both TCGA and METABRIC cohorts. In ER-positive breast cancer, high MYC targets v1, but not v2 score, was associated with high mutation load, and high MYC targets v1 and v2 scores were both associated with increased infiltration of pro- and anti-cancerous immune cells. We found that high MYC scores were associated with worse survival in metastatic breast cancer. Our findings show that the MYC targets v1 and v2 scores are associated with tumor aggressiveness and poor prognosis in ER-positive primary tumors, as well as in metastatic breast cancer.

DOI： 10.3390/ijms21218127

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Regulatory CD4+ T cell (Treg), a subset of tumor-infiltrating lymphocytes (TILs), are known to suppress anticancer immunity but its clinical relevance in human breast cancer remains unclear. In this study, we estimated the relative abundance of Tregs in breast cancer of multiple patient cohorts by using the xCell algorithm on bulk tumor gene expression data. In total, 5177 breast cancer patients from five independent cohorts (TCGA-BRCA, GSE96058, GSE25066, GSE20194, and GSE110590) were analyzed. Treg abundance was not associated with cancer aggressiveness, patient survival, or immune activity markers, but it was lower in metastatic tumors when compared to matched primary tumors. Treg was associated with a high mutation rate of TP53 genes and copy number mutations as well as with increased tumor infiltration of M2 macrophages and decreased infiltration of T helper type 1 (Th1) cells. Pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) was significantly associated with low Treg abundance in triple negative breast cancer (TNBC) but not in ER-positive/Her2-negative subtype. High Treg abundance was significantly associated with high tumor expression of multiple immune checkpoint inhibitor genes. In conclusion, Treg abundance may have potential as a predictive biomarker of pCR after NAC in TNBC.

DOI： 10.3390/cancers12103038

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Tumor associated macrophages (TAMs) play a critical role in biology of various cancers, including breast cancer. In the current study, we defined "M1" macrophage and "M1"/"M2" ratio by transcriptomic signatures using xCell. We investigated the association between high level of "M1" macrophage or "M1"/"M2" ratio and the tumor immune microenvironment by analyzing the transcriptome of publicly available cohorts, TCGA and METABRIC. We found that "M1" high tumors were not associated with prolonged survival compared with "M1" low tumors, or with the response to neoadjuvant chemotherapy. "M1" high tumors were associated with clinically aggressive features and "M1" high tumors enriched the cell proliferation and cell cycle related gene sets in GSEA. At the same time, "M1" high tumors were associated with high immune activity and favorable tumor immune microenvironment, as well as high expression of immune check point molecules. Strikingly, all these results were mirrored in "M1"/"M2" ratio high tumors. In conclusion, transcriptomically defined "M1" or "M1"/"M2" high tumors were associated with aggressive cancer biology and favorable tumor immune microenvironment but not with survival benefit, which resembled only part of their conventional clinical characteristics.

DOI： 10.1038/s41598-020-73624-w

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Pancreatic cancer is highly mortal due to uncontrolled cell proliferation. The G2M checkpoint pathway is an essential part of the cell cycle. We hypothesized that a high G2M pathway score is associated with cell proliferation and worse survival in pancreatic cancer patients. Gene set variation analysis using the Hallmark G2M checkpoint gene set was used as a score to analyze a total of 390 human pancreatic cancer patients from 3 cohorts (TCGA, GSE62452, GSE57495). High G2M score tumors enriched other cell proliferation genes sets as well as MKI67 expression, pathological grade, and proliferation score. Independent of other prognostic factors, G2M score was predictive of disease-specific survival in pancreatic cancer. High G2M tumor was associated with high mutation rate of KRAS and TP53 and significantly enriched these pathway gene sets, as well as high infiltration of Th2 cells. High G2M score consistently associated with worse overall survival in 3 cohorts, particularly in R1/2 resection, but not in R0. High G2M tumor in R1/2 highly enriched metabolic and cellular components' gene sets compared to R0. To our knowledge, this is the first study to use gene set variation analysis as a score to examine the clinical relevancy of the G2M pathway in pancreatic cancer.

DOI： 10.3390/cancers12102871

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INTRODUCTION: Cancer biology dominates the behavior and prognosis of a tumor. Although Nottingham histological grade is a subjective pathological determination, it has been accepted as a surrogate model for cancer biology. As such, histologic grade was incorporated into the latest 8th edition of the American Joint Committee on Cancer breast cancer staging system. In this study, we hypothesized that grade 3 breast cancers demonstrate aggressive molecular biological profiles, reflecting worse biology and possible underlying immunogenicity. METHODS: Transcriptomic and clinical data were obtained from the Molecular Taxonomy of Breast Cancer International Consortium, and the findings were validated by The Cancer Genome Atlas breast cancer cohort and GSE25066. RESULTS: Overall, 2876 patients were analyzed in this study. Grade 3 tumors were more common in estrogen receptor (ER)-negative, advanced-stage patients, and were associated with human epidermal growth factor receptor 2 and basal subtypes by the PAM50 classifier, as well as with increased MKI67 expression (all p <0.001). Disease-free survival was significantly worse in grade 3 tumors (all cohorts). Gene set enrichment analysis demonstrated that grade 3 tumors were significantly enriched with not only cell proliferation and cell cycle-related gene sets but also immune activity-related gene sets. CIBERSORT confirmed that grade 3 tumors were infiltrated with macrophage M1, follicular helper T cells, and activated natural killer cells (all p <0.001). Furthermore, grade 3 tumors were associated with more diverse T cell receptors (p =0.001) and increased cytolytic activity (p <0.001). Lastly, major T-cell exhaustion markers were significantly elevated in grade 3 breast cancers (p <0.001). CONCLUSION: Grade 3 breast cancers demonstrated aggressive transcriptomic features with enhanced immunogenicity and elevated T-cell exhaustion markers.

DOI： 10.1245/s10434-020-08608-1

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DOI： 10.1245/s10434-020-08680-7

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Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with higher mortality than the others. Pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) is considered as a surrogate to predict survival. Inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) is a negative regulator of T cell activation, and reduction in ITPKC function is known to promote Kawasaki disease. Given the role of tumor infiltrating lymphocytes in NAC and since TNBC has the most abundant immune cell infiltration in breast cancer, we hypothesized that the ITPKC expression level is associated with NAC response and prognosis in TNBC. The ITPKC gene was expressed in the mammary gland, but its expression was highest in breast cancer cells among other stromal cells in a bulk tumor. ITPKC expression was highest in TNBC, associated with its survival, and was its independent prognostic factor. Although high ITPKC was not associated with immune function nor with any immune cell fraction, low ITPKC significantly enriched cell proliferation-related gene sets in TNBC. TNBC with low ITPKC achieved a significantly higher pCR rate after NAC. To the best of our knowledge, this is the first report to demonstrate that ITPKC gene expression may be useful as a prognostic and predictive biomarker in TNBC.

DOI： 10.3390/cancers12102758

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CD8 T cell is an essential component of tumor-infiltrating lymphocytes (TIL) and tumor immune microenvironment (TIME). Using the xCell CD8 T cell score of whole tumor gene expression data, we estimated these cells in total of 3837 breast cancer patients from TCGA, METABRIC and various GEO cohorts. The CD8 score correlated strongly with expression of CD8 genes. The score was highest for triple-negative breast cancer (TNBC), and a high score was associated with high tumor immune cytolytic activity and better survival in TNBC but not other breast cancer subtypes. In TNBC, tumors with a high CD8 score had enriched expression of interferon (IFN)-α and IFN-γ response and allograft rejection gene sets, and greater infiltration of anti-cancerous immune cells. The score strongly correlated with CD4 memory T cells in TNBC, and tumors with both a high CD8 score and high CD4 memory T cell abundance had significantly better survival. Finally, a high CD8 score was significantly associated with high expression of multiple immune checkpoint molecules. In conclusion, a high CD8 T cell score is associated with better survival in TNBC, particularly when tumor CD4 memory T cells were elevated. Our findings also suggest a possible use of the score as a predictive biomarker for response to immune checkpoint therapy.

DOI： 10.3390/ijms21186968

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Angiogenesis is one of the hallmarks of cancer. We hypothesized that intra-tumoral angiogenesis correlates with inflammation and metastasis in breast cancer patients. To test this hypothesis, we generated an angiogenesis pathway score using gene set variation analysis and analyzed the tumor transcriptome of 3999 breast cancer patients from The Cancer Genome Atlas Breast Cancer (TCGA-BRCA), Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), GSE20194, GSE25066, GSE32646, and GSE2034 cohorts. We found that the score correlated with expression of various angiogenesis-, vascular stability-, and sphingosine-1-phosphate (S1P)-related genes. Surprisingly, the angiogenesis score was not associated with breast cancer subtype, Nottingham pathological grade, clinical stage, response to neoadjuvant chemotherapy, or patient survival. However, a high score was associated with a low fraction of both favorable and unfavorable immune cell infiltrations except for dendritic cell and M2 macrophage, and with Leukocyte Fraction, Tumor Infiltrating Lymphocyte Regional Fraction and Lymphocyte Infiltration Signature scores. High-score tumors had significant enrichment for unfavorable inflammation-related gene sets (interleukin (IL)6, and tumor necrosis factor (TNF)α- and TGFβ-signaling), as well as metastasis-related gene sets (epithelial mesenchymal transition, and Hedgehog-, Notch-, and WNT-signaling). High score was significantly associated with metastatic recurrence particularly to brain and bone. In conclusion, using the angiogenesis pathway score, we found that intra-tumoral angiogenesis is associated with immune reaction, inflammation and metastasis-related pathways, and metastatic recurrence in breast cancer.

DOI： 10.3390/ijms21186708

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Cancer-associated adipocytes are known to cause inflammation, leading to cancer progression and metastasis. The clinicopathological and transcriptomic data from 2256 patients with breast cancer were obtained based on three cohorts: The Cancer Genome Atlas (TCGA), GSE25066, and a study by Yau et al. For the current study, we defined the adipocyte, which is calculated by utilizing a computational algorithm, xCell, as "intratumoral adipocyte". These intratumoral adipocytes appropriately reflected mature adipocytes in a bulk tumor. The amount of intratumoral adipocytes demonstrated no relationship with survival. Intratumoral adipocyte-high tumors significantly enriched for metastasis and inflammation-related gene sets and are associated with a favorable tumor immune microenvironment, especially in the ER+/HER2- subtype. On the other hand, intratumoral adipocyte-low tumors significantly enriched for cell cycle and cell proliferation-related gene sets. Correspondingly, intratumoral adipocyte-low tumors are associated with advanced pathological grades and inversely correlated with MKI67 expression. In conclusion, a high amount of intratumoral adipocytes in breast cancer was associated with inflammation, metastatic pathways, cancer stemness, and favorable tumor immune microenvironment. However, a low amount of adipocytes was associated with a highly proliferative tumor in ER-positive breast cancer. This cancer biology may explain the reason why patient survival did not differ by the amount of adipocytes.

DOI： 10.3390/ijms21165744

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E2F transcription factors play critical roles in the cell cycle. Therefore, their activity is expected to reflect tumor aggressiveness and responsiveness to therapy. We scored 3905 tumors of nine breast cancer cohorts for this activity based on their gene expression for the Hallmark E2F targets gene set. As expected, tumors with a high score had an increased expression of cell proliferation-related genes. A high score was significantly associated with shorter patient survival, greater MKI67 expression, histological grade, stage, and genomic aberrations. Furthermore, metastatic tumors had higher E2F scores than the primary tumors from which they arose. Although tumors with a high score had greater infiltration by both pro- and anti-cancerous immune cells, they had an increased expression of immune checkpoint genes. Estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative cancer with a high E2F score achieved a significantly higher pathological complete response (pCR) rate to neoadjuvant chemotherapy. The E2F score was significantly associated with the expression of cyclin-dependent kinase (CDK)-related genes and strongly correlated with sensitivity to CDK inhibition in cell lines. In conclusion, the E2F score is a marker of breast cancer aggressiveness and predicts the responsiveness of ER-positive/HER2-negative patients to neoadjuvant chemotherapy and possibly to CDK and immune checkpoint inhibitors.

DOI： 10.3390/cells9071643

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Breast cancer (BC) patients experience increased stress with elevated cortisol levels, increasing risk of cancer recurrence. Cortisol binds to a cytoplasmic receptor, glucocorticoid receptor (GR) encoded by GR gene (NR3C1). We hypothesized that not only cancer cells, but even immune cells in the tumor microenvironment (TME) may contribute to GR expression in bulk tumor and influence prognosis. To test this, mRNA expression data was accessed from METABRIC and TCGA. "High" and "low" expression was based on highest and lowest quartiles of NR3C1 gene expression, respectively. Single-cell sequencing data were obtained from GSE75688 and GSE114725 cohorts. Computer algorithms CIBERSORT, Gene Set Enrichment Analysis and TIMER were used. GR-high BC has better median disease-free and disease-specific survival. Single cell sequencing data showed higher GR expression on immune cells compared to cancer and stromal cells. Positive correlation between GR-high BC and CD8+ T-cells was noted. In GR-high tumors, higher cytolytic activity (CYT) with decreased T-regulatory and T-follicular helper cells was observed. High GR expression was associated with lower proliferation index Ki67, enriched in IL-2_STAT5, apoptosis, KRAS, TGF-β signaling, and epithelial-to-mesenchymal transition. Immune cells significantly contribute to GR expression of bulk BC. GR-high BC has a favorable TME with higher CYT with favorable outcomes.

DOI： 10.3390/ijms21134635

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We generated a 4-gene score with genes upregulated in LM2-4, a metastatic variant of MDA-MB-231 (DOK 4, HCCS, PGF, and SHCBP1) that was strongly associated with disease-free survival (DFS) in TCGA cohort (hazard ratio [HR]>1.2, p < 0.02). The 4-gene score correlated with overall survival of TCGA (HR = 1 .44, p < 0 .001), which was validated with DFS and disease-specific survival of METABRIC cohort. The 4-gene score was able to predict worse survival or clinically aggressive tumors, such as high Nottingham pathological grade and advanced cancer staging. High score was associated with worse survival in the hormonal receptor (HR)-positive/Her2-negative subtype. High score enriched cell proliferation-related gene sets in GSEA. The score was high in primary tumors that originated, in and metastasized to, brain and lung, and it predicted worse progression-free survival for metastatic tumors. Good tumor response to neoadjuvant chemotherapy or hormonal therapy was accompanied by score reduction. High scores were also predictive of response to neoadjuvant chemotherapy for HR-positive/Her2-negative subtype. High score tumors had increased expression of T cell exhaustion marker genes, suggesting that the score may also be a biomarker for immunotherapy response. Our novel 4-gene score with both prognostic and predictive values may, therefore, be clinically useful particularly in HR-positive breast cancer.

DOI： 10.3390/cancers12051148

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microRNA-143(miR-143) is a well-known tumor suppressive microRNA that exhibits anti-tumoral function by targeting KRAS signaling pathways in various malignancies. We hypothesized that miR-143 suppresses breast cancer progression by targeting KRAS and its effector molecules. We further hypothesized that high expression of miR-143 is associated with a favorable tumor immune microenvironment of estrogen receptor (ER)-positive breast cancer patients which result in improved survival. Two major publicly available breast cancer cohorts; The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) were used. The miR-143 high expression group was associated with increased infiltration of anti-cancer immune cells and decreased pro-cancer immune cells, as well as enrichment of the genes relating to T helper (Th1) cells resulting in improved overall survival (OS) in ER-positive breast cancer patients. To the best of our knowledge, this is the first study to demonstrate that high expression of miR-143 in cancer cells associates with a favorable tumor immune microenvironment, upregulation of anti-cancer immune cells, and suppression of the pro-cancer immune cells, associating with better survival of the breast cancer patients.

DOI： 10.3390/ijms21093213

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Most breast cancer (BC) patients succumb to metastatic disease. MiR-34a is a well-known tumor suppressive microRNA which exerts its anti-cancer functions by playing a role in p53, apoptosis induction, and epithelial-mesenchymal transition (EMT) suppression. Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) cohorts were used to test our hypothesis that miR-34a high BCs translate to less aggressive cancer biology and better survival in large cohorts. There was no association between miR-34a expression levels and clinicopathological features of BC patients except for HER2 positivity. MiR-34a high expressing tumors were associated with lower Nottingham pathological grades and lower MKI67 expression. In agreement, high miR-34a tumors demonstrated lower GSVA scores of cell cycle and cell proliferation-related gene sets. High miR-34a tumors enriched the p53 pathway and apoptosis gene sets. Unexpectedly, high miR-34a tumors also associated with elevated EMT pathway score and ZEB1 and two expressions. MiR-34a expression did not associate with any distant metastasis. Further, high miR-34a tumors did not associate with better survival compared with miR-34a low tumors. In conclusion, the clinical relevance of miR-34a high expressing tumors was associated with suppressed cell proliferation, enhanced p53 pathway and apoptosis, but enhanced EMT and these findings did not reflect better survival outcomes in large BC patient cohorts.

DOI： 10.3390/ijms21093045

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The vast majority of breast cancer death is a result of metastasis. Thus, accurate identification of patients who are likely to have metastasis is expected to improve survival. The G2M checkpoint plays a critical role in cell cycle. We hypothesized that breast cancer tumors with high activity of G2M pathway genes are more aggressive and likely to metastasize. To test this, we used the single-sample gene set variation analysis method to calculate the score for the Hallmark G2M checkpoint pathway using gene expression data of a total of 4626 samples from 12 human breast cancer cohorts. As expected, a high G2M pathway score correlated with enriched tumor expression of other cell proliferation-related gene sets. The score was significantly associated with clinical aggressive features of tumors and patient survival in estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Interestingly, a high G2M score of metastasis tumors was also significantly associated with worse survival. In primary as well as metastasis tumors with high scores, the infiltration of both pro- and anti-cancerous immune cells increased. Tumor G2M score was also associated with treatment response to systemic chemotherapy in ER-positive/HER2-negative cancer, and was predictive of response to cyclin-dependent kinase inhibition therapy.

DOI： 10.3390/ijms21082921

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Patient-Derived Xenograft (PDX) is now accepted as a murine model that better mimics human cancer when compared to a conventional cancer cell-line inoculation model. Some claim the advantage of orthotopic site implantation of patient tumor (OS) over ectopic implantation into the subcutaneous space (SQ); however, there has been no study that describes a head-to-head comparison of oncological differences between these two models to date. We hypothesize that OS tumors re-transplant and grow better than SQ tumors and are therefore a better model to evaluate tumor aggressiveness. Breast cancer PDXs were generated using the tumors derived from 11 patients into NOD scid gamma (NSG) mice. We used six ER(+)HER2(-) tumors and five triple negative (TN) tumors for a total of 11 tumors. Five PDX lines grew for an overall engraftment rate of 45%. We present our OS implantation method in detail. The re-transplantation rate of TN tumors in each transplant site was significantly higher in OS when compared to SQ tumors (70.1% vs. 32.1%, p < 0.01). OS tumors grow significantly faster than SQ tumors. Similarly, OS tumors demonstrated significantly more mitotic figures and Ki-67 positive cells than SQ tumors. The tumor re-transplantation rate significantly increased by the second and third generations with the OS method. The time from implantation to development of a palpable tumor dramatically decreased after the first passage. PDX of ER(+) tumors demonstrated significantly lower engraftment rates and slower tumor growth than TN tumors, which remarkably improved by the first passage. Orthotopically implanted PDX tumors showed better re-transplantation rates, greater tumor size, and more significant growth compared to the subcutaneously implanted model.

DOI： 10.1007/s10911-020-09442-7

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Publishing type：Research paper (scientific journal)   Publisher：American Association for Cancer Research (AACR)  

Abstract

Background: MicroRNA-143(miR-143) is known to function as the tumor suppressor in various cancers, including breast cancer. The tumor immune microenvironment such as the balance between the T helper cell type 1 (Th1) and Th2 is reported to associate with the survival of the breast cancer patients. In this study, we hypothesized that the miR-143 has favorable effect to the tumor immune microenvironment.

Material and Methods: We obtained the clinicopathological data and survival information of 755 breast cancer patients from The Cancer Genome Atlas (TCGA) database. Survival analysis, Overall survival (OS) and Disease free survival (DFS) was conducted comparing the high and low expression groups. CYT score, CIBERSORT, and other immunological factors were used to evaluate intratumoral immune cell composition within estrogen receptor (ER) positive breast cancer patients. Also, gene set enrichment analysis (GSEA) was performed between miR-143 high and low expression groups within the ER positive group.

Results: High expression of miR-143 was significantly associated with prolonged OS only in ER positive breast cancer patients (p=0.014). Expression of miR-143 was not associated with DFS, stage, TNM factors of breast cancer patients. Within ER positive patients, the result of CIBERSORT revealed that high expression of miR-143 was associated with higher percentage of macrophage M1 (p&amp;lt;0.03) and lower percentage of macrophage M2 (p&amp;lt;0.001). There was more Th1 cells in miR-143 high group (p&amp;lt;0.001) whereas Th2 cells were less (p&amp;lt;0.001). This result was strikingly echoed by GSEA, where miR-143 high significantly enriched the genes related to Th1 cells compared with that of Th2 cells. Regulatory T cell, Lymphocyte infiltration signature score, and T cell receptor diversity were higher in the patients with high miR-143 expression (p&amp;lt;0.03, p&amp;lt;0.001, and p&amp;lt;0.001 respectively). CYT score that reflect cytolytic activity was higher in the high miR-143 expression group (p&amp;lt;0.04).

Conclusion: High expression of miR-143 was associated with improved OS in ER positive breast cancer patients. Also, miR-143 was found to associate with high Th1 and low Th2 cells as well as enriching the genes relating to Th1 cells, which may explain the favorable role of miR-143 in ER positive breast cancer.

Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Mariko Asaoka, Takashi Takeshita, Eriko Katsuta, Manabu Futamura, Kazuhiro Yoshida, Kazuaki Takabe. High expression microRNA-143 is associated with favorable tumor immune microenvironment and better survival of ER positive breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-59.

DOI： 10.1158/1538-7445.sabcs19-p3-08-59

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The vast majority of mortality in breast cancer results from distant metastasis. Brain metastases occur in as many as 30% of patients with advanced breast cancer, and the 1-year survival rate of these patients is around 20%. Pre-clinical animal models that reliably reflect the biology of breast cancer brain metastasis are needed to develop and test new treatments for this deadly condition. The patient-derived xenograft (PDX) model maintains many features of a donor tumor, such as intra-tumor heterogeneity, and permits the testing of individualized treatments. However, the establishment of orthotopic PDXs of brain metastasis is procedurally difficult. We have developed a method for generating such PDXs with high tumor engraftment and growth rates. Here, we describe this method and identify variables that affect its outcomes. We also compare the brain-orthotopic PDXs with ectopic PDXs grown in mammary pads of mice, and show that the responsiveness of PDXs to chemotherapeutic reagents can be dramatically affected by the site that they are in.

DOI： 10.3390/cancers12020444

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While cancer cells gain aggressiveness by mutations, abundant mutations release neoantigens, attracting anti-cancer immune cells. We hypothesized that in breast cancer (BC), where mutation is less common, tumors with high mutation rates demonstrate aggressive phenotypes and attract immune cells simultaneously. High mutation rates were defined as the top 10% of the mutation rate, utilizing TCGA and METABRIC transcriptomic data. Mutation rate did not impact survival although high mutation BCs were associated with aggressive clinical features, such as more frequent in ER-negative tumors (p < 0.01), in triple-negative subtype (p = 0.03), and increased MKI-67 mRNA expression (p < 0.01) in both cohorts. Tumors with high mutation rates were associated with APOBEC3B and homologous recombination deficiency, increasing neoantigen loads (all p < 0.01). Cell proliferation and immune activity pathways were enriched in BCs with high mutation rates. Furthermore, there were higher lymphocytes and M1 macrophage infiltration in high mutation BCs. Additionally, T-cell receptor diversity, cytolytic activity score (CYT), and T-cell exhaustion marker expression were significantly elevated in BCs with high mutation rates (all p < 0.01), indicating strong immunogenicity. In conclusion, enhanced immunity due to neoantigens can be one of possible forces to counterbalance aggressiveness of a high mutation rate, resulting in similar survival rates to low mutation BCs.

DOI： 10.1038/s41598-020-58995-4

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Inflammation has been linked with cancer, but whether it is part of the problem or part of the solution remains to be a matter of debate in breast cancer. Our group and others have demonstrated that inflammation aggravates cancer progression; however, some claim that inflammation may support immune cell infiltration and suppress cancer. We defined the gene set variation analysis of the Molecular Signatures Database Hallmark inflammatory response gene set as the inflammatory pathway score and analyzed 3632 tumors in total from 4 breast cancer cohorts (METABRIC, TCGA, GSE25066, and GSE21094). In the whole breast cancer cohort, high-score tumors were associated with aggressive clinical characteristics, such as worse disease specific survival, higher Nottingham histological grade, and younger age. Inflammatory score was significantly higher in triple-negative (TNBC) as well as basal and normal subtypes compared with the other subtypes, which suggest that the detrimental effect of high level of inflammation may be because it includes a more aggressive subtype. On the contrary, high score within TNBC was significantly associated with better survival. TNBC with high score enriched not only IFN-α, IFN-γ response, IL-2/STAT5 signaling, Allograft rejection, Complement, p53 pathway, Reactive Oxygen, and Apoptosis but also TNF-α signaling, IL6-JAK-STAT signaling, TGF-β signaling, Coagulation, Angiogenesis, EMT, KRAS signaling, and PI3K-AKT-MTOR signaling gene sets. High score was associated with mainly favorable anticancerous immune cell infiltration as well as Leukocyte fraction, TIL regional fraction, Lymphocyte infiltration, IFN-γ response, TGF-β response, and cytolytic activity scores. Although the inflammatory pathway score was not associated with neoadjuvant treatment response, it associated with expressions of immune checkpoint molecules. In conclusion, inflammation was associated with worse outcome in the whole breast cancer cohort, but with better outcome in TNBC, which was associated with favorable anticancerous immune response and immune cell infiltrations.

DOI： 10.1155/2020/5618786

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KRAS signaling is associated with cancer progression in several cancers. Upregulation of KRAS signaling is often seen in cancers that harbor high KRAS mutation rate, such as pancreatic cancer and non-small cell lung cancer (NSCLC). Less than 2% of breast cancers have KRAS mutation, however, the alteration of the effector signaling such as PI3K/AKT and MAPK pathways are well known. Mutated KRAS is known to function as immune suppressor in other cancers, but the role of KRAS signaling on tumor immune microenvironment (TIME) in breast cancer is not known. We hypothesize that the enrichment of KRAS signaling is associated with reduced patient survival as well as TIME in triple negative breast cancer (TNBC). Patient cohorts from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n = 1903) and The Cancer Genome Atlas (TCGA; n = 982) were used. Higher expression of KRAS in breast cancer cell-lines (MCF7, BT474, and MDA-MB231) compared to MCF10A, which is a model of benign mammary cells was found. Both MEK and PI3K inhibitors suppressed MB231 cell proliferation in dose dependent manner. Gene Set Variant Analysis (GSVA) of the patient cohorts demonstrated two peaks by KRAS_SIGNALING_UP gene sets which were divided into KRAS-high and -low groups using median cutoff. There was no difference in KRAS mutation between KRAS-high and low. Despite its cell proliferation promoting role, KRAS-high patients demonstrated significantly better Disease-Free Survival and Overall Survival in triple negative breast cancer (TNBC). KRAS-high TNBC was associated with favorable tumor immune microenvironment with elevated B cells and CD8 T cells, monocytes, or M1 macrophage. It was associated with decreased CD4 central memory T-cells, but not Regulatory T-cells, or M2 macrophage detected by xCell. To elucidate the mechanism of this association, Gene Set Enrichment Analysis was performed. Inflammatory response, IL6/JAK-STAT3 signaling, and Interferon gamma response gene sets were enriched in KRAS-high TNBC patients in both METABRIC and TCGA cohorts. In agreement, cytolytic activity score, interferon gamma response score, and lymphocyte infiltrating signature score, were all significantly elevated in KRAS-high TNBC. In conclusion, we found that patients with enrichment of KRAS signaling gene sets were associated with inflammation and favorable tumor immune microenvironment as well as improved survival in TNBC.

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Annexin A1 (ANXA1) is a phospholipid-linked protein involved in inflammation, immune response, and mast cell reactivity. Recently, we reported that ANXA1 is associated with aggressive features of triple-negative breast cancer (TNBC); however, its clinical relevance remains controversial. We hypothesized that human TNBC with high expression of ANXA1 mRNA is associated with pro-cancerous immune cell infiltration, including mast cells, and with an aggressive phenotype. Clinical and RNA-seq data were obtained from The Cancer Genome Atlas (TCGA, n = 1079) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (n = 1904). TNBC patients had significantly higher levels of ANXA1 expression compared to the other subtypes in both TCGA and METABRIC cohorts (p < 0.001). ANXA1 protein expression was assessed by immunohistochemistry in Japanese TNBC patient cohort (n = 48), where 17 cases (35.4%) had positive ANXA1 staining, and their overall survival was significantly shorter compared with negative staining group (p = 0.008). The CIBERSORT algorithm was used to calculate immune cell infiltrations. ANXA1 high tumors were associated with activated mast cells and M2 macrophages (p > 0.01), but did not show any association with tumor heterogeneity nor cytolytic activity. High expression of ANXA1 group enriched inflammation, epithelial-to-mesenchymal transition (EMT), and angiogenesis-related genes in a gene set enrichment assay in both cohorts. To our knowledge, this is the first study to demonstrate that ANXA1 is associated with infiltration of mast cells and inflammation that is associated with the aggressive phenotype of TNBC, such as EMT and angiogenesis.

DOI： 10.3390/ijms20174197

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Estrogen receptor (ER) positive breast cancer (BC), the most abundant BC subtype, is notorious for poor response to neoadjuvant chemotherapy (NAC). The androgen receptor (AR) was reported to support estradiol-mediated ER activity in an in vitro system. Recently, ER-positive BC with fewer tumor infiltrating lymphocytes (TILs) was shown to have a better prognosis, opposite to the trend seen with ER-negative BC. We hypothesized that ER-positive BC with high expression of AR will have fewer TILs and an inferior response to NAC, but with a better prognosis. In both TCGA and METABRIC cohorts, AR expression was significantly higher in ER-positive BCs compared to ER-negatives (p < 0.001, p < 0.001, respectively) and it correlated with ER expression (R = 0.630, R = 0.509, respectively). In ER-positive tumors, AR high tumors enriched UV response down (NES = 2.01, p < 0.001), and AR low tumors enriched DNA repair (NES = -2.02, p < 0.001). AR high tumors were significantly associated with procancer regulatory T-cells, and AR low tumors were associated with anticancer immune cells, such as CD4, CD8, and Gamma-Delta T-cells and memory B-cells in ER-positive BC (p < 0.01). Further, cytolytic activity was significantly lower in AR high BC in both cohorts. Finally, AR high tumors had a significantly lower rate of attaining pathological complete response to NAC (GSE22358), but better survival. In conclusion, our results demonstrated that high AR has fewer tumor infiltrating lymphocytes as well as cytolytic activity and an inferior response to NAC, but better survival in ER-positive BC.

DOI： 10.3390/ijms20112655

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In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

DOI： 10.3791/57849

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