Clonal evolution in breast cancer revealed by single nucleus genome sequencing.

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

[Comparison of the effectiveness of platinum-based chemotherapy versus non-platinum-based chemotherapy for triple-negative breast cancer with metastases confined to the lungs].

OBJECTIVE: To compare the effect of first-line treatment with platinum-based chemotherapy and non-platinum-based chemotherapy in patients with lung metastases from triple negative breast cancer (TNBC). METHODS: Sixty-five eligible patients were divided into platinum-treated group and non-platinum-treated group according to the first-line therapy. Factors predicting the chemotherapeutic efficacy included overall survival (OS), progression-free survival (PFS) and objective response (OR). RESULTS: In the platinum-treated group of 32 patients, 2 cases (6.3%) achieved CR, 16 cases (50.0%) achieved PR, 11 (34.4%) cases achieved SD, and 3 patients (9.4%) achieved PD. In the non-platinum-treated group of 33 patients, 2 cases (6.1%) achieved CR, 6 cases (18.2%) achieved PR, 16 cases (48.5%) achieved SD, and 9 cases (27.3%) achieved PD. Median PFS was significantly longer in the platinum-treated group than in the non-platinum-treated group (10 months vs. 6.0 months, P = 0.012), and OS was also improved (32 months vs. 22 months, P = 0.006). Multivariate analysis of several factors including local-regional lymph node involvement, lung metastasis-related symptoms, first-line platinum-based chemotherapy, disease-free interval, size and number of lung lesions, showed that first-line platinum-based chemotherapy was an independent prognostic factor for TNBC patients with lung metastases. CONCLUSIONS: Compared with non-platinum-based chemotherapy, the first-line platinum-based chemotherapy can improve PFS and OS in TNBC patients with metastases confined to the lungs.

Preventive effect of aspirin on peripherally inserted central catheter-related vein thrombosis in patients with malignant tumors.

The majority of patients receiving chemotherapy undergo PICC catheterization. However, PICCs are significantly associated with catheter related complications, including deep vein thrombosis, blood infection, fibrin sheath, catheter prolapse, catheter displacement and blockage. Of all the risks, PICC-related VT was the most prevailing clinic symptom and resulted in a high risk of death. AIM: The study aimed to investigate the preventive efficacy and safety of aspirin for patients with malignant tumors receiving venous thrombosis (VT) related with peripherally inserted central catheters (PICC) treatment. PATIENTS AND METHODS: A randomized controlled trial was conducted. Participants with malignant tumors receiving chemotherapy who accepted PICC insertion operation were randomly allocated to the aspirin treatment group (n = 235) or the control group (n = 246). The patients in the aspirin group were administrated aspirin (100mg) for 30 days, whereas the patients in control group were administrated a placebo drug. The incidence of PICC-related VT in both groups and the aspirin related adverse effects were evaluated. RESULTS: The incidence of PICC-related VT was 0.4% in the aspirin group, compared with 3.3% in the control group (P = 0.038). In addition, aspirin related bleeding was not observed. CONCLUSION: PICC-related VT could be effectively prevented by aspirin in patients with malignant tumors.

Tumor-Derived Exosomal miR-29b Reduces Angiogenesis in Pancreatic Cancer by Silencing ROBO1 and SRGAP2.

Background: Exosomal miR-29b reportedly plays a role during cancer metastasis. However, its exact function and underlying mechanism during pancreatic cancer (PC) have not been investigated. Methods: Exosomes from PC cells were prepared and identified. Transmission electron microscopy (TEM) and confocal microscopy were used to examine structural characteristics of the exosomes and verify their internalization by human umbilical vein endothelial cells (HUVECs). The tube formation and migration abilities of HUVECs were detected. VEGF content was assessed by ELISA. GW4869 was used to suppress exosome release. Luciferase reporter assays were performed to verify the predicted interaction of miR-29b with ROBO1 and SRGAP2 mRNA. Results: Exosomal miRNA-29b was differentially expressed in the conditioned medium of PC cells. Exosomes from PC cells were verified by TEM and western blotting. Treatment with the exosomal inhibitor (GW4869) prevented an increase in miR-29b expression and recused the reduced VEGF expression and tube formation and migration abilities of HUVECs cocultured with BxPC3 and AsPC-1 cells that overexpressed miR-29b. Furthermore, the downregulation of ROBO1 and SRGAP2 in cocultured HUVECs was also reduced after additional treatment with GW4869. After incubation with miR-29b exosomes, HUVECs had lower VEGF concentrations and reduced migration and tube formation rates; however, those effects were eliminated by subsequent transfection with the miR-29b inhibitor. Luciferase reporter assays verified the interaction of miR-29b with ROBO1 and SRGAP2. That interaction was also supported by rescue assays showing that overexpression of ROBO1 and SRGAP2 also reduced the antiangiogenic effect of exosomal miR-29b in HUVECs. Conclusion: Exosomal miR-29b originating from PC cells protected HUVECs from PC cell-induced angiogenesis by attenuating ROBO1 and SRGAP2 expression. Our findings suggest a strategy for treating PC.

Long non-coding RNA EGFR-AS1 sponges micorRNA-381 to upregulate ROCK2 in bladder cancer.

The present study aimed to investigate the role of the long non-coding RNA EGFR-AS1 in bladder cancer (BC). In this study gene expression of both BC and non-tumor tissues from BC patients were measured by quantitative PCR. Cell transfections were performed to analyze gene interactions in HT-1197 cells. Transwell assays were performed to analyze cell invasion and migration of HT-1197 cells. It was revealed that epidermal growth factor receptor-antisense RNA 1 (EGFR-AS1) was upregulated in BC and positively associated with rho associated coiled-coil containing protein kinase 2 (ROCK2). Analysis of data collected in follow-ups indicated that EGFR-AS1 expression was significantly associated with poorer overall survival of patients with BC. Moreover, in bladder cancer cells, EGFR-AS1 overexpression mediated the upregulation of ROCK2, while microRNA (miR)-381 mediated the downregulation of ROCK2. However, EGFR-AS1 and ROCK2 failed to affect each other. Bioinformatics analysis indicated that miR-381 binds EGFR-AS1. In addition, EGFR-AS1 and ROCK2 overexpression resulted in the promotion of cell invasiveness and migration of HT-1197 BC cells. Conversely, miR-381 was revealed to partially reverse the effect of EGFR-AS1 overexpression. Therefore, EGFR-AS1 may sponge miR-381 to upregulate ROCK2 in BC, thereby promoting cell invasion and migration.

Phase I safety and pharmacokinetic study of cipatinib, an original dual tyrosine kinase inhibitor.

BACKGROUND: Cipatinib is a novel tyrosine kinase inhibitor against both EGFR and HER2/neu. This phase I trial was conducted to assess the safety, dose-limiting toxicities (DLTs), and maximum-tolerated dose of cipatinib in HER2-positive patients with advanced breast cancer. METHODS: Eligible adults with advanced breast cancer were administered cipatinib 200 mg/day (n = 3) as an initial dose, with escalating dosages of 400 mg (n = 4), 800 mg (n = 2), 1200 mg (n = 3), 1400 mg (n = 3), 1600 mg (n = 3), and 1800 mg (n = 2) in 21 day cycles. DLTs were monitored until the end of cycle 2. Physical examinations, vital signs, blood sampling for hematology, clinical chemistry, and pharmacokinetics were performed throughout the trial. RESULTS: Of the 26 subjects enrolled, 23 completed the trial. A total of 143 adverse events (AEs) were reported, of which 87 were associated with cipatinib treatment and comprised: neutropenia (38%), hypertriglyceridemia (15%), fatigue (15%), nausea (12%), fever (19%), and myocardial ischemia (19%). Six AEs were graded 3-4 (neutropenia, increases in aspartate aminotransferase, and total bilirubin, fatigue, dizziness and nodal tachycardia), but none of the AEs observed were considered to be DLTs. CONCLUSION: This tolerability study revealed that despite a mild toxicity profile, cipatinib was well tolerated up to the anticipated maximum dosage of 1800 mg/m2 . Further clinical trials are warranted.

SPARC overexpression in primary tumors correlates with disease recurrence and overall survival in patients with triple negative breast cancer.

SPARC/osteonectin expression is reportedly altered in various malignancies. However, little is known regarding to the prognostic value of SPARC in triple-negative breast cancer (TNBC) patients. In this study, immunohistochemistry and immunoreactive scores (IRSs) were used to evaluate SPARC protein expression in primary tumors from 211 TNBC patients with up to 10 years of clinical follow-up data. High SPARC expression (IRS ≥3) was detected in 52.1% of primary tumors. Patients expressing high SPARC levels had worse disease-free survival (DFS) (HR=1.58, 95% CI: 1.01-2.47, P=0.044) and overall survival (OS) (HR=1.74, 95% CI: 1.06-2.85, P=0.029) than patients with lower SPARC levels. Furthermore, high SPARC expression was an independent prognostic factor for both DFS (HR=1.73, 95% CI: 1.10-2.73, P=0.018) and OS (HR=1.90, 95% CI: 1.14-3.16, P=0.014) in TNBC patients. These results suggest that increased SPARC expression may be an indicator of greater aggressiveness, and may serve as a prognostic factor for triple-negative breast cancer.

Punctuated copy number evolution and clonal stasis in triple-negative breast cancer.

Aneuploidy is a hallmark of breast cancer; however, knowledge of how these complex genomic rearrangements evolve during tumorigenesis is limited. In this study, we developed a highly multiplexed single-nucleus sequencing method to investigate copy number evolution in patients with triple-negative breast cancer. We sequenced 1,000 single cells from tumors in 12 patients and identified 1-3 major clonal subpopulations in each tumor that shared a common evolutionary lineage. For each tumor, we also identified a minor subpopulation of non-clonal cells that were classified as metastable, pseudodiploid or chromazemic. Phylogenetic analysis and mathematical modeling suggest that these data are unlikely to be explained by the gradual accumulation of copy number events over time. In contrast, our data challenge the paradigm of gradual evolution, showing that the majority of copy number aberrations are acquired at the earliest stages of tumor evolution, in short punctuated bursts, followed by stable clonal expansions that form the tumor mass.

Enriched CD44(+)/CD24(-) population drives the aggressive phenotypes presented in triple-negative breast cancer (TNBC).

The mechanism underlying the aggressive behaviors of triple negative breast cancer (TNBC) is not well characterized yet. The association between cancer stem cell (CSC) population and the aggressive behaviors of TNBC has not been established. We found the CD44(+)/CD24(-) cell population was enriched in TNBC tissues and cell lines, with a higher capacity of proliferation, migration, invasion and tumorigenicity as well as lower adhesion ability. The CD44(+)/CD24(-) cell population with cancer stem cell-like properties may play an important role in the aggressive behaviors of TNBC. This discovery may lead to new therapeutic strategies targeting CD44(+)/CD24(-) cell population in TNBC.