The tumor microenvironment is highly complex, and immune escape is currently considered an important hallmark of cancer, largely contributing to tumor progression and metastasis. Named for their ...capability of killing target cells autonomously, natural killer (NK) cells serve as the main effector cells toward cancer in innate immunity and are highly heterogeneous in the microenvironment. Most current treatment options harnessing the tumor microenvironment focus on T cell-immunity, either by promoting activating signals or suppressing inhibitory ones. The limited success achieved by T cell immunotherapy highlights the importance of developing new-generation immunotherapeutics, for example utilizing previously ignored NK cells. Although tumors also evolve to resist NK cell-induced cytotoxicity, cytokine supplement, blockade of suppressive molecules and genetic engineering of NK cells may overcome such resistance with great promise in both solid and hematological malignancies. In this review, we summarized the fundamental characteristics and recent advances of NK cells within tumor immunometabolic microenvironment, and discussed potential application and limitations of emerging NK cell-based therapeutic strategies in the era of presicion medicine.
Metabolic reprogramming is a hallmark of cancer. However, systematic characterizations of metabolites in triple-negative breast cancer (TNBC) are still lacking. Our study profiled the polar ...metabolome and lipidome in 330 TNBC samples and 149 paired normal breast tissues to construct a large metabolomic atlas of TNBC. Combining with previously established transcriptomic and genomic data of the same cohort, we conducted a comprehensive analysis linking TNBC metabolome to genomics. Our study classified TNBCs into three distinct metabolomic subgroups: C1, characterized by the enrichment of ceramides and fatty acids; C2, featured with the upregulation of metabolites related to oxidation reaction and glycosyl transfer; and C3, having the lowest level of metabolic dysregulation. Based on this newly developed metabolomic dataset, we refined previous TNBC transcriptomic subtypes and identified some crucial subtype-specific metabolites as potential therapeutic targets. The transcriptomic luminal androgen receptor (LAR) subtype overlapped with metabolomic C1 subtype. Experiments on patient-derived organoid and xenograft models indicate that targeting sphingosine-1-phosphate (S1P), an intermediate of the ceramide pathway, is a promising therapy for LAR tumors. Moreover, the transcriptomic basal-like immune-suppressed (BLIS) subtype contained two prognostic metabolomic subgroups (C2 and C3), which could be distinguished through machine-learning methods. We show that N-acetyl-aspartyl-glutamate is a crucial tumor-promoting metabolite and potential therapeutic target for high-risk BLIS tumors. Together, our study reveals the clinical significance of TNBC metabolomics, which can not only optimize the transcriptomic subtyping system, but also suggest novel therapeutic targets. This metabolomic dataset can serve as a useful public resource to promote precision treatment of TNBC.
Background
Molecular subtyping of triple‐negative breast cancers (TNBCs) via gene expression profiling is essential for understanding the molecular essence of this heterogeneous disease and for ...guiding individualized treatment. We aim to devise a clinically practical method based on immunohistochemistry (IHC) for the molecular subtyping of TNBCs.
Materials and Methods
By analyzing the RNA sequencing data on TNBCs from Fudan University Shanghai Cancer Center (FUSCC) (n = 360) and The Cancer Genome Atlas data set (n = 158), we determined markers that can identify specific molecular subtypes. We performed immunohistochemical staining on tumor sections of 210 TNBCs from FUSCC, established an IHC‐based classifier, and applied it to another two cohorts (n = 183 and 214).
Results
We selected androgen receptor (AR), CD8, FOXC1, and DCLK1 as immunohistochemical markers and classified TNBCs into five subtypes based on the staining results: (a) IHC‐based luminal androgen receptor (IHC‐LAR; AR‐positive +), (b) IHC‐based immunomodulatory (IHC‐IM; AR‐negative −, CD8+), (c) IHC‐based basal‐like immune‐suppressed (IHC‐BLIS; AR−, CD8−, FOXC1+), (d) IHC‐based mesenchymal (IHC‐MES; AR−, CD8−, FOXC1−, DCLK1+), and (e) IHC‐based unclassifiable (AR−, CD8−, FOXC1−, DCLK1−). The κ statistic indicated substantial agreement between the IHC‐based classification and mRNA‐based classification. Multivariate survival analysis suggested that our IHC‐based classification was an independent prognostic factor for relapse‐free survival. Transcriptomic data and pathological observations implied potential treatment strategies for different subtypes. The IHC‐LAR subtype showed relative activation of HER2 pathway. The IHC‐IM subtype tended to exhibit an immune‐inflamed phenotype characterized by the infiltration of CD8+ T cells into tumor parenchyma. The IHC‐BLIS subtype showed high expression of a VEGF signature. The IHC‐MES subtype displayed activation of JAK/STAT3 signaling pathway.
Conclusion
We developed an IHC‐based approach to classify TNBCs into molecular subtypes. This IHC‐based classification can provide additional information for prognostic evaluation. It allows for subgrouping of TNBC patients in clinical trials and evaluating the efficacy of targeted therapies within certain subtypes.
Implications for Practice
An immunohistochemistry (IHC)‐based classification approach was developed for triple‐negative breast cancer (TNBC), which exhibited substantial agreement with the mRNA expression‐based classification. This IHC‐based classification (a) allows for subgrouping of TNBC patients in large clinical trials and evaluating the efficacy of targeted therapies within certain subtypes, (b) will contribute to the practical application of subtype‐specific treatment for patients with TNBC, and (c) can provide additional information beyond traditional prognostic factors in relapse prediction.
This article describes an immunohistochemistry‐based approach to classification of triple‐negative breast cancers into molecular subtypes for purposes of the translation of TNBC molecular classification into clinical practice.
Background
The standard 5 years of endocrine therapy has demonstrated additional benefits compared with short‐term (2‐3 years) treatment in patients with estrogen receptor (ER)‐positive breast ...cancer; however, data specific to ER‐low positive breast cancer (1%‐10% by immunohistochemistry) are limited, and it is unclear whether long‐term treatment is still necessary for this subgroup.
Methods
The authors used the prospectively maintained Breast Surgery Database of Fudan University Shanghai Cancer Center for this propensity‐matched analysis. The primary end point was disease‐free survival. Multivariate Cox regression analysis and propensity score‐matching methods were used to minimize bias. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated. All statistics were 2‐sided.
Results
From 2012 to 2017, 22,768 consecutive women had pathologically confirmed, early stage breast cancer, and 1013 (4.45%) were identified with ER‐low positive disease. Among these, 634 patients met the inclusion criteria and were divided into 3 groups: those who received no endocrine therapy (n = 89), those who received 2 to 3 years of endocrine therapy (n = 185), and those who received approximately 5 years of endocrine therapy (n = 360). At a median follow‐up of 65 months, there was no significant difference in disease‐free survival between patients who received 2 to 3 years and 5 years of endocrine therapy (HR, 0.82; 95% CI, 0.51‐1.33; P = .43). The findings were consistent after multivariate Cox analysis of the propensity score‐matched samples (5 vs 2‐3 years of treatment: HR, 0.74; 95% CI, 0.41‐1.31; P = .30).
Conclusions
Short‐term endocrine therapy for 2 to 3 years might be an alternative for patients who have ER‐low positive breast cancer instead of the standard 5 years of treatment.
Short‐term endocrine therapy for 2 to 3 years might be an alternative for patients who have estrogen receptor‐low positive breast cancer instead of the standard 5 years. More extensive studies and translational research on identifying endocrine‐sensitive cases within this population are still needed.
We comprehensively analyzed clinical, genomic, and transcriptomic data of a cohort of 465 primary triple-negative breast cancer (TNBC). PIK3CA mutations and copy-number gains of chromosome 22q11 were ...more frequent in our Chinese cohort than in The Cancer Genome Atlas. We classified TNBCs into four transcriptome-based subtypes: (1) luminal androgen receptor (LAR), (2) immunomodulatory, (3) basal-like immune-suppressed, and (4) mesenchymal-like. Putative therapeutic targets or biomarkers were identified among each subtype. Importantly, the LAR subtype showed more ERBB2 somatic mutations, infrequent mutational signature 3 and frequent CDKN2A loss. The comprehensive profile of TNBCs provided here will serve as a reference to further advance the understanding and precision treatment of TNBC.
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•We build the genomic and transcriptomic landscape of 465 primary TNBCs•Chinese TNBC cases demonstrate more PIK3CA mutations and LAR subtype•Transcriptomic data classify TNBCs into four subtypes•Multi-omics profiling identifies potential targets within specific TNBC subtypes
Jiang et al. characterize primary Chinese triple-negative breast cancer (TNBC) and classify it into four subtypes. They find that these TNBCs have more frequent PIK3CA mutations and chromosome 22q11 copy-number gains than non-Asian TNBCs and that the LAR subtype has more ERBB2 somatic mutations and CDKN2A loss.
Metastasis is the main death reason for triple‐negative breast cancer (TNBC). Thus, identifying the driver genes associated with metastasis of TNBC is urgently needed. CRISPR screens have ...dramatically enhanced genome editing and made it possible to identify genes associated with metastasis. In this study, we identified and explored the crucial role of ras homolog family member V (RhoV) in TNBC metastasis. Here, we performed customized in vivo CRISPR screens targeting metastasis‐related genes obtained from transcriptome analysis of TNBC. The regulatory role of RhoV in TNBC was validated using gain‐ or loss‐of‐function studies in vitro and in vivo. We further conducted immunoprecipitation and LC–MS/MS to explore the metastasis mechanism of RhoV. In vivo functional screens identified RhoV as a candidate regulator involved in tumor metastasis. RhoV was frequently upregulated in TNBC and correlated with poor survival. Knockdown of RhoV significantly suppressed cell invasion, migration, and metastasis both in vitro and in vivo. In addition, we provided evidence that p‐EGFR interacted with RhoV to activate the downstream signal pathway of RhoV, thereby promoting tumor metastasis. We further confirmed that this association was dependent on GRB2 through a specific proline‐rich motif in the N‐terminus of RhoV. This mechanism of RhoV is unique, as other Rho family proteins lack the proline‐rich motif in the N‐terminus.
In this study, we applied an in vivo CRISPR screen targeting recurrence‐related genes. Furthermore, we systematically identified RhoV as a key modulator of breast tumor migration.
Background
With the largest sample size to date, the authors’ objective was to investigate the incidence of primary‐to‐metastatic human epidermal growth factor 2 (HER2) conversion and the predictors ...for such conversion. Moreover, no previous studies have evaluated the prognosis of patients who have negative HER2 expression (HER2‐0) versus low HER2 expression (HER2‐low) when HER2 status was assessed based on all recurrent/metastatic lesions.
Methods
The authors included 1299 patients who had available HER2 status of primary breast tumors and paired recurrent/metastatic lesions at Fudan University Shanghai Cancer Center and West China Hospital.
Results
In total, 370 patients (28.5%) experienced primary‐to‐metastatic HER2 conversion. Intrapatient intermetastasis spatial heterogeneity and temporal heterogeneity of HER2 were detected. When assessing HER2 based on recurrent/metastatic tumors, patients who had HER2‐0 tumors had significantly shorter overall survival than those who had HER2‐low tumors in the overall population and in the estrogen receptor (ER)‐negative subgroup. However, when assessing HER2 based on primary tumors, there was no difference in overall survival between patients who had HER2‐0 versus HER2‐low tumors. Moreover, patients who had tumors that converted from HER2‐0 to HER2‐low had longer overall survival than those who had consistent HER2‐0 status in the ER‐negative subgroup. By combining four predictors (ER status, Ki67 index, biopsy site, and disease‐free interval), the authors established the first prediction tool to estimate the probability of HER2‐0 tumors converting to HER2‐low/positive tumors.
Conclusions
Intrapatient primary‐to‐metastatic and intermetastatic HER2 heterogeneity were observed in this large‐scale cohort study. When evaluating HER2 based on recurrent/metastatic tumors, an overall survival difference was observed between patients who had HER2‐0 versus HER2‐low, recurrent/metastatic breast tumors. The developed prediction tool might help clinicians screen out patients with primary HER2‐0 tumors that have a high probability of HER2 status conversion and recommend them for re‐biopsy, thus helping to screen out candidate patients for trastuzumab deruxtecan treatment.
Intrapatient primary‐to‐metastatic and intermetastatic HER2 heterogeneity was observed in this large‐scale cohort study. The prediction tool developed by the authors might help clinicians screen out patients with primary HER2‐negative tumors who have a high probability of HER2 status conversion and recommend them for re‐biopsy, thus helping to screen out candidate patients for trastuzumab deruxtecan treatment.
Triple-negative breast cancer (TNBC) is a highly heterogeneous disease, and molecular subtyping may result in improved diagnostic precision and targeted therapies. Our previous study classified TNBCs ...into four subtypes with putative therapeutic targets. Here, we conducted the FUTURE trial (ClinicalTrials.gov identifier: NCT03805399), a phase Ib/II subtyping-based and genomic biomarker-guided umbrella trial, to evaluate the efficacy of these targets. Patients with refractory metastatic TNBC were enrolled and stratified by TNBC subtypes and genomic biomarkers, and assigned to one of these seven arms: (A) pyrotinib with capecitabine, (B) androgen receptor inhibitor with CDK4/6 inhibitor, (C) anti PD-1 with nab-paclitaxel, (D) PARP inhibitor included, (E) and (F) anti-VEGFR included, or (G) mTOR inhibitor with nab-paclitaxel. The primary end point was the objective response rate (ORR). We enrolled 69 refractory metastatic TNBC patients with a median of three previous lines of therapy (range, 1-8). Objective response was achieved in 20 (29.0%, 95% confidence interval (CI): 18.7%-41.2%) of the 69 intention-to-treat (ITT) patients. Our results showed that immunotherapy (arm C), in particular, achieved the highest ORR (52.6%, 95% CI: 28.9%-75.6%) in the ITT population. Arm E demonstrated favorable ORR (26.1%, 95% CI: 10.2%-48.4% in the ITT population) but with more high grade (≥ 3) adverse events. Somatic mutations of TOP2A and CD8 immunohistochemical score may have the potential to predict immunotherapy response in the immunomodulatory subtype of TNBC. In conclusion, the phase Ib/II FUTURE trial suggested a new concept for TNBC treatment, demonstrating the clinical benefit of subtyping-based targeted therapy for refractory metastatic TNBC.
To characterize the prevalence of BRCA mutations and characteristics of BRCA carriers in China and to update the clinical recommendations for BRCA testing, we conducted a wide screen for BRCA ...mutations using next‐generation sequencing (NGS). A total of 4,034 Chinese subjects were screened for germline BRCA1/2 mutations, including 2,991 breast cancer patients and 1,043 healthy individuals from the community enrolled as controls. We developed an NGS‐based approach to perform BRCA1/2 screening. BRCA mutations were identified in 9.1% (232/2,560) of cases with at least one risk factor, in 3.5% (15/431) of sporadic patients and in 0.38% (4/1,043) of healthy controls. The mutation frequency ranged from 8.9 to 15.2% in cohorts with a single risk factor to 16.6–100% in groups with multiple risk factors. We identified 70 novel BRCA mutations. A high frequency of BRCA1 c.5470_5477del was detected, accounting for 13.9% (16/115) of the BRCA1 mutations detected in our study. Clinical characteristics such as family history, invasive carcinoma, negative human epidermal growth factor receptor 2 (HER2), high Ki67 index, lymph node status, and high tumour grade were closely related to BRCA mutations. BRCA2 carriers had poorer disease‐free survival among HER2‐ or hormone receptor‐positive patients (hazard ratio = 1.892; 95% confidence interval: 1.132–3.161; p = 0.013). This study shows that BRCA mutation carriers could be frequently identified among breast cancer patients with multiple risk factors. Importantly, we established an NGS‐based pipeline for BRCA1/2 testing in clinical practice and strongly suggest that breast cancer patients of premier‐ and moderate‐grade risks receive BRCA1/2 mutations testing in China.
What's new?
Chinese women carrying BRCA mutations are at lower risk of breast cancer compared with female carriers in other countries, a phenomenon that may explained by associations between ethnic background and specific BRCA variants. Little is known, however, about BRCA1/2 mutation prevalence and related clinical characteristics among Chinese women. Here, overall BRCA1/2 mutation frequency was found to be 9.1% in Chinese breast cancer patients, compared with 0.38% in healthy Chinese controls, based on next‐generation sequencing (NGS) analyses. BRCA mutations were closely associated with clinical characteristics, including family history and tumour grade. BRCA mutation carriers could be readily identified by NGS testing.
The E3 ubiquitin ligase ring finger protein 115 (RNF115) is overexpressed in more than half of human breast tumors and is implicated in the pathogenesis and progression of breast cancer. However, the ...mechanism behind RNF115 overexpression in breast tumors remains largely unknown. Here we report that ubiquitin‐specific protease 9X (USP9X), a substrate‐specific deubiquitinating enzyme, stabilizes RNF115 and thereby regulates its biological functions in breast cancer cells. Immunoprecipitation and GST pull‐down assays showed that USP9X interacted with RNF115. Depletion of RNF115 by siRNAs or overexpression of RNF115 did not significantly affect USP9X expression. In contrast, knockdown of USP9X in breast cancer cells by siRNAs reduced RNF115 protein abundance, which was partially restored following treatment with proteasome inhibitor MG‐132. Moreover, depletion of USP9X reduced the half‐life of RNF115 and increased its ubiquitination. Conversely, overexpression of USP9X resulted in an accumulation of RNF115 protein, accompanied by a decrease in its ubiquitination. RNF115 mRNA levels were unaffected by overexpression or knockdown of USP9X. Furthermore, USP9X protein expression levels correlated positively with RNF115 in breast cancer cell lines and breast tumor samples. Importantly, reintroduction of RNF115 in USP9X‐depleted cells partially rescued the reduced proliferation, migration, and invasion of breast cancer cells by USP9X knockdown. Collectively, these findings indicate that USP9X is a stabilizer of RNF115 protein and that the USP9X‐RNF115 signaling axis is implicated in the breast cancer malignant phenotype.
Findings presented here establish the ubiquitin‐specific protease 9X‐ring finger protein 115 signaling axis as an important regulatory mechanism of breast cancer and provide a rationale for potential therapeutic interventions in the treatment of breast cancer.
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