Pathogenesis and vascular integrity of breast cancer brain metastasis

Dogma dictates that brain metastasis originate from the proliferation of extravasated tumor cells and that the blood-brain barrier (BBB) prevents the delivery of chemotherapeutic drugs to the tumors. The purpose of this study was to clarify the relationship between tumor localization and progression and the involvement of BBB function in a murine model of breast cancer brain metastasis. Green fluorescent protein expressing MDA-MB435 breast cancer cells were injected into the left ventricle of nude mice. At various time points, the entire vasculature was labeled with rhodamine-conjugated albumin. The tumors and vasculature were then imaged by laser-scanning confocal and stereo fluorescence microscopy. About 75% of the cells that reached the brain extravasated and grew perivascularly. Twenty five percent of the cells, however, proliferated within the vasculature and ultimately led to thrombosis-like infarction of the brain parenchyma. The tumorigenic "embolus" served as a sustained release source of tumor cells to downstream sites. Continuing intravascular tumor expansion led to disruption of the BBB and to overflow of cells that progressed along the vessels perivascularly to distant sites that regained protection of the BBB. Breast cancer brain metastases involve both extravascular and intravascular growth of tumor cells. These distinct pathways contribute to different pathological phenotypes that generate a heterogeneous BBB that facilitates or inhibits the delivery of chemotherapeutic drugs to the tumor.     

Treatment and outcome of brain metastasis as first site of distant metastasis from breast cancer

Twenty-eight consecutive patients with breast cancer were analyzedwho presented with a single brain metastasis asfirst site of distant metastasis. The response tosurgery with postoperative radiation therapy (RT) (9 patients)and to non-surgical therapy as first-line treatment was100% and 89% respectively with a significant differencein median recurrence-free intervals of 23 months andof 5 months respectively (p=0.033). Retreatmentof a local relapse by surgery (± RT,± chemotherapy) or by non-surgical treatment resulted ina response in 6 of the 7 operatedpatients and in 5 of the 6 non-operatedpatients with a median duration of response of7 months (range 2–20 months) and of 3months (range 2–4 months) respectively. The overall mediansurvival of the 28 patients with a singlebrain metastasis was 16 months (range 2–39 months).The median survival in the primarily operated patientswas 23 months, in the primarily not-operated group10 months, and in the never-operated group 9months. In comparison, the response to non-surgical treatmentin 20 consecutive patients who presented with multiplebrain metastases as first site of distant metastasiswas 55% with a median recurrence free intervalof 4 months. The median survival in thisgroup was 4 months, which was significantly shorterthan survival of patients with single brain metastasis(p=0.0036). These results suggest that breastcancer patients with a single brain metastasis asfirst presentation of relapse constitute a specific subgroupwith a favorable response to treatment and along survival especially if they can be treatedby surgery with postoperative RT.     

Mutational profiling of brain metastasis from breast cancer: matched pair analysis of targeted sequencing between brain metastasis and primary breast cancer

Although breast cancer is the second most common cause of brain metastasis with a notable increase of incidence, genes that mediate breast cancer brain metastasis (BCBM) are not fully understood. To study the molecular nature of brain metastasis, we performed gene expression profiling of brain metastasis and matched primary breast cancer (BC). We used the Ion AmpliSeq Cancer Panel v2 covering 2,855 mutations from 50 cancer genes to analyze 18 primary BC and 42 BCBM including 15 matched pairs. The most common BCBM subtypes were triple-negative (42.9%) and basal-like (36.6%). In a total of 42 BCBM samples, 32 (76.2%) harbored at least one mutation (median 1, range 0–7 mutations). Frequently detected somatic mutations included TP53 (59.5%), MLH1 (14.3%), PIK3CA (14.3%), and KIT (7.1%). We compared BCBM with patient-matched primary BC specimens. There were no significant differences in mutation profiles between the two groups. Notably, gene expression in BCBM such as TP53, PIK3CA, KIT, MLH1, and RB1 also seemed to be present in primary breast cancers. The TP53 mutation frequency was higher in BCBM than in primary BC (59.5% vs 38.9%, respectively). In conclusion, we found actionable gene alterations in BCBM that were maintained in primary BC. Further studies with functional testing and a delineation of the role of these genes in specific steps of the metastatic process should lead to a better understanding of the biology of metastasis and its susceptibility to treatment.     

CAVEOLIN-1 expression in brain metastasis from lung cancer predicts worse outcome and radioresistance,irrespective of tumor histotype

Brain metastases develop in one-third of patients with non-small-cell lung cancer and are associated with a dismal prognosis, irrespective of surgery or chemo-radiotherapy. Pathological markers for predicting outcomes after surgical resection and radiotherapy responsiveness are still lacking. Caveolin 1 has been associated with chemo- and radioresistance in various tumors, including non-small-cell lung cancer. Here, caveolin 1 expression was assessed in a series of 69 brain metastases from non-small-cell lung cancer and matched primary tumors to determine its role in predicting survival and radiotherapy responsiveness. Only caveolin 1 expression in brain metastasis was associated with poor prognosis and an increased risk of death (log rank test, p = 0.015). Moreover, in the younger patients (median age of <54 years), caveolin 1 expression neutralized the favorable effect of young age on survival compared with the older patients. Among the radiotherapy-treated patients, an increased risk of death was detected in the group with caveolin 1-positive brain metastasis (14 out of 22 patients, HR=6.839, 95% CI 1.849 to 25.301, Wald test p = 0.004). Overall, caveolin 1 expression in brain metastasis from non-small-cell lung cancer is independently predictive of worse outcome and radioresistance and could become an additional tool for personalized therapy in the critical subset of brain-metastatic non-small-cell lung cancer patients.     

Prediction of outcomes for patients with brain parenchymal metastases from breast cancer (BC): a new BC-specific prognostic model and a nomogram

The purpose of this study is to validate the recently published Breast-Graded Prognostic Assessment (GPA) and propose a new prognostic model and nomogram for patients with brain parenchymal metastases (BM) from breast cancer (BC). We retrospectively investigated 171 consecutive patients who received a diagnosis of BM from BC during 2000-2008. We appraised the recently proposed Sperduto's BC-specific GPA in training cohort through Kaplan-Meier survival curve using log-rank test and area under the curve for the BC-GPA predicting overall survival at 1 year and developed a new nomogram to predict outcomes using multivariate Cox-regression analysis. By putting the Sperduto's Breast-GPA together with our nomogram, we developed a new prognostic model. We validated our new prognostic model with an independent external patient cohort from 2 institutes for the same period. On the basis of our Cox-regression analysis, therapeutic effect of trastuzumab and status of extracranial systemic disease control were incorporated into our new prognostic model in addition to Karnofsky performance status, age, and hormonal status. Our new prognostic model showed significant discrimination in median survival time, with 3.7 months for class I (n = 15), 7.8 months for class II (n = 82), 10.7 months for class III (n = 42), and 19.2 months for class IV (n = 32; P < .0001). The new prognostic model accurately predicted survival among patients with BC from BM in an external validation cohort (P < .0001). We propose a new prognostic model and a nomogram reflecting the different biological features of BC, including treatment effect and status of extracranial disease control, which was excellently validated in an independent external cohort.     

Loss of CADM1 expression is associated with poor prognosis and brain metastasis in breast cancer patients

Breast cancer brain metastases (BCBM) are detected with increasing incidence. In order to detect potential genes involved in BCBM, we first screened for genes down-regulated by methylation in cell lines with site-specific metastatic ability. The expression of five genes, CADM1, SPARC, RECK, TNFAIP3 and CXCL14, which were also found down-regulated in gene expression profiling analyses of BCBM tissue samples, was verified by qRT-PCR in a larger patient cohort. CADM1 was chosen for further down-stream analyses. A higher incidence of CADM1 methylation, correlating with lower expression levels, was found in BCBM as compared to primary BC. Loss of CADM1 protein expression was detected most commonly among BCBM samples as well as among primary tumors with subsequent brain relapse. The prognostic role of CADM1 expression was finally verified in four large independent breast cancer cohorts (n=2136). Loss of CADM1 protein expression was associated with disease stage, lymph node status, and tumor size in primary BC. Furthermore, all analyses revealed a significant association between loss of CADM1 and shorter survival. In multivariate analyses, survival was significantly shorter among patients with CADM1-negative tumors. Loss of CADM1 expression is an independent prognostic factor especially associated with the development of brain metastases in breast cancer patients.     

KIF14 mRNA expression is a predictor of grade and outcome in breast cancer

Gain of chromosome 1q is a hallmark of breast cancer, and likely reflects oncogene amplification. We previously identified mitotic kinesin KIF14 (kinesin family member 14) as an overexpressed candidate oncogene in the 1q31.3-1q32.1 minimal region of genomic gain in breast cancer cell lines. KIF14 also showed high expression in other cancers, notably an association with survival in lung tumors. We now report KIF14 expression in 99 primary breast tumors and 10 normal breast controls. Measured by real-time RT-PCR, KIF14 was overexpressed 10-fold on average in tumors relative to normals (t test p = 0.000054); expression increased with grade (ANOVA p = 0.000006). Infiltrating ductal carcinomas had higher KIF14 levels than lobular (p = 0.017), and estrogen receptor (ER) negative tumors had higher KIF14 levels than ER positive tumors (t test p = 0.030). KIF14 expression correlated positively with Ki-67 mRNA level (Spearman r = 0.692, p = 0.000001), fraction of positive nodes (r = 0.227, p = 0.024) and percent invasive cells (r = 0.360, p = 0.0002), and negatively with percent fatty stroma (r = -0.258, p = 0.010) and percent normal epithelium (r = -0.291, p = 0.003). KIF14 expression is thus tumor-specific and increased in more aggressive tumors. Indeed, KIF14 expression predicted overall survival (univariate Cox p = 0.010), with an odds ratio of 3.60 (1.37-9.48), in 50 tumors with available outcome data. KIF14 overexpression also predicted decreased disease-free survival (log-rank p = 0.049). These findings are the first evidence of association between expression of a mitotic kinesin and prognostic variables in breast cancer.     

Disruption of the blood brain barrier by brain metastases of triple‐negative and basal‐type breast cancer but not HER2/neu‐positive breast cancer

BACKGROUND:

Generally, the blood‐brain barrier (BBB) of brain metastasis was thought to be disrupted.

METHODS:

We retrospectively performed immunohistochemical staining for glucose transporter 1 (GLUT1) and breast cancer resistance protein (BCRP) to evaluate the status of the BBB in resected brain metastases. Associations between expression of GLUT1 and/or BCRP and the immunohistochemical profiles of breast cancers, such as the statuses of hormone receptors, human epidermal growth factor receptor 2 (HER2/neu), and a basal‐type marker (cytokeratin 5/6, HER1), were also analyzed.

RESULTS:

The study included 29 breast cancer patients with brain metastasis who had undergone brain tumor resections. Among the 29 patients, there was no expression of GLUT1 and BCRP in the intratumor microvessels of 9 (32%) and 11 (38%) patients, respectively. There was no expression of both GLUT1 and BCRP in 8 patients (28%). The expression of GLUT1 was significantly associated with that of BCRP (P < .001). A positive correlation was observed between the expression of GLUT1 and/or BCRP and brain metastases of HER2/neu‐positive breast cancer (P = .012), while a negative correlation was observed between the expression of GLUT1 and/or BCRP and brain metastases of triple negative or basal‐type breast cancer (P = .014 and P = .003 for triple negative and basal‐type, respectively).

CONCLUSIONS:

Brain metastases of triple negative or basal‐type breast cancers may often disrupt the BBB, whereas brain metastases of HER2/neu‐positive breast cancer tend to preserve the BBB. Cancer 2010. © 2010 American Cancer Society.     

三阴性乳腺癌细胞系MDA-MB-231与其脑转移细胞系中microRNA差异表达分析

目的：分析三阴性乳腺癌（TNBC）细胞系MDA-MB-231（MB-231）与其脑转移细胞系MDA-MB-231Brm （MB-231Brm）中microRNA （miRNA）的表达差异,寻找有价值的三阴性乳腺癌脑转移相关的miRNA。方法：Trizol法提取MB-231和MB-231Brm细胞总RNA,用Illumina高通量测序技术分离并检测miRNA表达情况,将其与已知的miRNA数据库比对,采用miRDeep2软件进行新的miRNA预测;使用差异基因检测法筛选差异表达的miRNA,构建差异表达谱。应用生物信息学分析进行miRNA靶基因的功能分析,并用实时荧光定量PCR （qPCR）进行验证。结果：MB-231和MB-231Brm细胞中miRNA的种类和表达存在差异,两者共有的miRNA为961种,前者自有164种,后者自有196种;与MB-231相比,MB-231Brm细胞的miRNA中,有145个表达显著上调,54个表达显著下调,1 122个表达未见明显差异;差异表达分析显示,与MB-231相比,MB-231Brm中的miR-199a-3p和miR-103a-3p等表达显著增加,而miR-1246和miR-4787-5p等表达显著下降;基因本体分析（GO）发现差异表达的miRNA所调控的靶基因与细胞组分、分子功能及生物过程均相关;qPCR验证发现,与MB-231相比,miR-1246在MB-231Brm中表达显著降低且差异有统计学意义（P < 0.05）。结论：MB-231和MB-231Brm中存在差异表达的miRNA,且在TNBC脑转移过程中其调控的靶基因通过参与细胞组分形成、调节分子功能及调控生物过程等发挥作用;并发现miR-1246在脑转移细胞系中的表达下降,其可能通过某种途径参与TNBC脑转移的发生、发展过程。     

Enhanced brain delivery and therapeutic activity of trastuzumab after blood-brain barrier opening by NEO100 in mouse models of brain-metastatic breast cancer

BackgroundThe antitumor efficacy of human epidermal growth factor receptor 2 (HER2)-targeted therapies, such as humanized monoclonal antibody trastuzumab (Herceptin^®, Roche), in patients with breast-to-brain cancer metastasis is hindered by the low permeability of the blood-brain barrier (BBB). NEO100 is a high-purity version of the natural monoterpene perillyl alcohol, produced under current good manufacturing practice (cGMP) regulations, that was shown previously to reversibly open the BBB in rodent models. Here we investigated whether NEO100 could enable brain entry of trastuzumab to achieve greater therapeutic activity.MethodsAn in vitro BBB, consisting of human astrocytes and brain endothelial cells, was used to determine trastuzumab penetration in the presence or absence of NEO100. For in vivo studies, we administered intravenous (IV) trastuzumab or the trastuzumab-drug conjugate ado-trastuzumab emtansine (T-DM1; Kadcyla^®, Roche), to mouse models harboring intracranial HER2+ breast cancer, with or without BBB opening via IA NEO100. Brain and tumor tissues were examined for the presence of trastuzumab and infiltration of immune cells. Therapeutic impact was evaluated based on overall survival.ResultsNEO100 greatly increased trastuzumab penetration across an in vitro BBB. In vivo, IA NEO100-mediated BBB opening resulted in brain tumor-selective accumulation of trastuzumab, without detectable presence in normal brain tissue, along with increased presence of immune cell populations. IV delivery of trastuzumab or T-DM1 achieved significantly greater overall survival of tumor-bearing mice when combined with IA NEO100.ConclusionIA NEO100 facilitates brain tumor entry of trastuzumab and T-DM1 and significantly enhances their therapeutic efficacy, along with increased antibody-dependent immune cell recruitment.     

Brain metastasis of breast tumors and blood brain barrier

Brain metastases are prevalent in solid tumours and lymphomas. They are associated with poor survival. The brain is regarded as a sanctuary site for metastatic tumour cells where they exist partially protected from drugs by the blood brain barrier. Amongst the different molecular sub-types of breast cancer, HER2 positive tumours and triple negative tumours exhibit the highest incidence of brain metastasis. Specific strategies are needed to fight brain metastatic disease. Preclinical models for brain metastasis have been developed, yielding mechanistic molecular knowledge and new therapeutic approaches.