Andreas M. Stark

Breast Cancer Metastasis to the Central Nervous System

Clinically symptomatic metastases to the central nervous system (CNS) occur in approximately 10 to 15% of patients with metastatic beast cancer. CNS metastases are traditionally viewed as a late complication of systemic disease, for which few effective treatment options exist. Recently, patients with Her-2-positive breast tumors who were treated with trastuzumab have been reported to develop CNS metastases at higher rates, often while responding favorably to treatment. The blood:brain barrier and the unique brain microenvironment are hypothesized to promote distinct molecular features in CNS metastases that may require tailored therapeutic approaches. New research approaches using cell lines that reliably and preferentially metastasize in vivo to the brain have been reported. Using such model systems, as well as in vitro analogs of blood-brain barrier penetration and tissue-based studies, new molecular leads into this disease are unfolding.

Her-2 Overexpression Increases the Metastatic Outgrowth of Breast Cancer Cells in the Brain

Retrospective studies of breast cancer patients suggest that primary tumor Her-2 overexpression or trastuzumab therapy is associated with a devastating complication: the development of central nervous system (brain) metastases. Herein, we present Her-2 expression trends from resected human brain metastases and data from an experimental brain metastasis assay, both indicative of a functional contribution of Her-2 to brain metastatic colonization. Of 124 archival resected brain metastases from breast cancer patients, 36.2% overexpressed Her-2, indicating an enrichment in the frequency of tumor Her-2 overexpression at this metastatic site. Using quantitative real-time PCR of laser capture microdissected epithelial cells, Her-2 and epidermal growth factor receptor (EGFR) mRNA levels in a cohort of 12 frozen brain metastases were increased up to 5- and 9-fold, respectively, over those of Her-2-amplified primary tumors. Co-overexpression of Her-2 and EGFR was also observed in a subset of brain metastases. We then tested the hypothesis that overexpression of Her-2 increases the colonization of breast cancer cells in the brain in vivo. A subclone of MDA-MB-231 human breast carcinoma cells that selectively metastasizes to brain (231-BR) overexpressed EGFR; 231-BR cells were transfected with low (4- to 8-fold) or high (22- to 28-fold) levels of Her-2. In vivo, in a model of brain metastasis, low or high Her-2-overexpressing 231-BR clones produced comparable numbers of micrometastases in the brain as control transfectants; however, the Her-2 transfectants yielded 3-fold greater large metastases (>50 microm(2); P < 0.001). Our data indicate that Her-2 overexpression increases the outgrowth of metastatic tumor cells in the brain in this model system.

Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases

Purpose Brain metastases are an increasingly common complication in breast cancer patients. The Metastasis Suppressor Genes (MSG) Nm23, KISS1, KAI1, BRMS1, and Mkk4 have been associated with the metastatic potential of breast cancer in vitro and in vivo.Methods The mRNA expression of Nm23, KISS1, KAI1, BRMS1, and Mkk4 in fresh frozen tissue samples of brain metastases from ductal invasive breast cancer specimens was examined in relation to primary tumors. In a first step, mRNA expression screening was carried out using a semi-quantitative RT-PCR approach, in a second step quantitative real-time RT-PCR was performed on selected specimens. By immunohistochemical staining, gene products were visualized on the protein level.Results Semi-quantitative RT-PCR revealed reduced mRNA expression of Nm23, KISS1, KAI1, BRMS, and Mkk4 in brain metastases. Results for KISS1, KAI1, BRMS, and Mkk4 were confirmed by real-time RT-PCR. In detail, mRNA expression reduction in breast cancer brain metastases was tenfold. Expression of MSG could be confirmed by immunohistochemical staining on protein level.Conclusions Our investigations revealed significantly reduced mRNA expression of metastases suppressor genes KISS1, KAI1, BRMS1, and Mkk4 in breast cancer brain metastasis. Particularly, in the case of KISS1 and Mkk4, an important role for future treatment of patients with breast cancer brain metastatic lesions can be assumed.

Analyses of Resected Human Brain Metastases of Breast Cancer Reveal the Association between Up-Regulation of Hexokinase 2 and Poor Prognosis

Brain metastases of breast cancer seem to be increasingin incidence as systemic therapy improves. Metastatic disease in the brain is associated with high morbidity and mortality. We present the first gene expression analysis of laser-captured epithelial cells from resected human brain metastases of breast cancer compared with unlinked primary breast tumors. The tumors were matched for histology, tumor-node-metastasis stage, and hormone receptor status. Most differentially expressed genes were down-regulated in the brain metastases, which included, surprisingly, many genes associated with metastasis. Quantitative real-time PCR analysis confirmed statistically significant differences or strong trends in the expression of six genes: BMP1, PEDF, LAMγ3, SIAH, STHMN3, and TSPD2. Hexokinase 2 (HK2) was also of interest because of its increased expression in brain metastases. HK2 is important in glucose metabolism and apoptosis. In agreement with our microarray results, HK2 levels (both mRNA and protein) were elevated in a brain metastatic derivative (231-BR) of the human breast carcinoma cell line MDA-MB-231 relative to the parental cell line (231-P) in vitro. Knockdown of HK2 expression in 231-BR cells using short hairpin RNA reduced cell proliferation when cultures were maintained in glucose-limiting conditions. Finally, HK2 expression was analyzed in a cohort of 123 resected brain metastases of breast cancer. High HK2 expression was significantly associated with poor patient survival after craniotomy (P = 0.028). The data suggest that HK2 overexpression is associated with metastasis to the brain in breast cancer and it may be a therapeutic target. (Mol Cancer Res 2009;7(9):1438–45)

Matrix-degrading proteases ADAMTS4 and ADAMTS5 (disintegrins and metalloproteinases with thrombospondin motifs 4 and 5) are expressed in human glioblastomas

Brain tumors, in particular glioblastomas, have a high morbidity and mortality, mainly due to their invasive nature. A prerequisite for this invasiveness is cell migration based on increased expression of proteases digesting the extracellular matrix. Brevican, an important extracellular proteoglycan that is upregulated in glioblastomas, can be degraded by certain proteases. We demonstrate that in human glioblastomas secretory proteases like ADAMTS4 and ADAMTS5 (aggrecanases 1 and 2; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs) are expressed on the mRNA and protein levels in considerable amounts. Real‐time RT‐PCR shows a higher levels of ADAMTS4 and 5 expressions in glioblastomas in situ, compared to cultured human glioblastoma cells. The upregulation of these proteases in vivo by cytokines may explain this difference. In vitro, transforming growth factor‐β induces ADAMTS4, but less ADAMTS5, and interleukin‐1β ADAMTS5, but not ADAMTS4. As demonstrated by immunohistochemistry and confocal microscopy in situ, ADAMTS5 expression is confined to proliferating glioblastoma cells of surgical tumor sections and with lower intensity to astroglial cells in normal brain sections, as opposed to brevican. In vitro, glioblastoma‐derived ADAMTS5 degrades recombinant human brevican to several smaller fragments. Our results show that ADAMTS4 and 5 are upregulated on proliferating glioblastoma cells and these proteases may contribute to their invasive potential.

Malignant peripheral nerve sheath tumours--report of 8 cases and review of the literature.

Summary Background. Though Malignant peripheral nerve sheath tumours (MPNST) are a rare entity accounting for 5–10% of soft-tissue sarcomas they are an important differential diagnosis to benign tumours of the peripheral nervous system regarding treatment and prognosis. Method. We present our experience with eight patients who underwent surgery for MPNST at the Department of Neurosurgery between 10/1990 and 9/1999. The median age was 37 years [range: 13–64], the male/female ratio was 1:1. Two patients suffered from Neurofibromatosis type 1. Paraffin embedded tumour specimens were immunohistochemically stained for S-100, p53 and Ki67/MIB-1. Findings. The most frequent initial symptoms were local swelling and pain followed by irritation of cranial nerves and spinal ataxia. Four tumours were localised at the head & neck region, three were found in the extremities and one tumour was located on the trunk. All patients underwent surgery with curative intent, but total resection, defined by negative surgical margins, was achieved in only 3 cases. All of these developed local recurrence with a mean disease free survival time of 10,6 months. In five cases, adjuvant radiation was given. During follow up, three patients developed distant metastases located in the lung, liver and subcutaneous tissue. Five out of eight patients died during follow-up with a mean survival time of 11,6 months after diagnosis. Results of immunohistochemical staining were as followed: S-100 (7/8), p53 (7/8). The Ki67/MIB-1 proliferation index was detectable in all tumour samples, it differed from 10–30%. Interpretation. MPNST is a rare and fatal diagnosis in neurosurgery with high risk of local recurrence and occurence of distant metastases. Though mulitimodal therapy including surgical resection and adjuvant radiotherapy including brachytherapy is available, the prognosis remains dismal. Modern clinical studies and the development of effective chemotherapy is needed in order to gain control of the disease.

Glioblastoma: Clinical characteristics, prognostic factors and survival in 492 patients

OBJECTIVE Glioblastoma is the most common and most malignant primary brain tumor in adults. The only overall accepted independent prognostic factors are patient age and performance. We present a large single institution patient series examined for prognostic factors using uni- and multivariate survival analysis. METHODS 492 patients were included who underwent craniotomy for newly diagnosed glioblastoma WHO grade IV between 1990 and 2007 at our department. The association to patient survival was estimated using log-rank test for univariate analysis and cox regression method for multivariate analysis. RESULTS Median patient age was 62 years (mean: 60.4 years, range: 22-93 years), the male: female ratio was 1.26:1. Primary genesis was found in 91.0% of cases. A multifocal tumor was present in 110 cases (22.4%). The median pre- and post-operative Karnofsky Performance Score was 70. Total tumor resection was performed in 288 cases (58.5%), subtotal removal in 134 cases (27.2%). The following parameters were significantly associated with survival in univariate analysis: age, performance, primary genesis, multifocal tumor, neurological deficit, neuropsychological findings, seizures, incidental finding, total or subtotal resection, radiotherapy, chemotherapy, combined radio-/chemotherapy with temozolomide, re-craniotomy, second tumor in patient history. The following parameters were significantly associated with survival in multivariate analysis: age, performance, multifocal tumor, total or subtotal resection, radiotherapy, chemotherapy, combined radio-/chemotherapy with temozolomide. CONCLUSION In addition to patient age and performance, we identified multiple lesions and resection status as independent prognostic factors. Radiotherapy, chemotherapy and combined radio-/chemotherapy with temozolomide were significantly associated with prolonged survival.

Surgical treatment for brain metastases: prognostic factors and survival in 177 patients

Brain metastases are an increasingly frequent complication of cancer. Advances in diagnosis and treatment have led to wider indications for surgery. We present a single-institution series of 177 patients and discuss our results with regard to the literature. Special focus is on patients with advanced age, multiple brain metastases, extracranial metastases, and brain metastasis recurrence. All patients underwent craniotomy for the resection of solid tumor brain metastases between 1994 and 2001 in our department. Perioperative morbidity and mortality as well as survival were evaluated. The median patient age was 59 years (range 32–86 years). In 177 patients, 348 brain metastases were detected, of which 68.0% were supratentorial and 32.0% were infratentorial. According to univariate analysis, the following parameters were significantly associated with prolonged patient survival: (1) age <70 years, (2) one to three intracranial metastases, (3) favorable postoperative performance, (4) resection of all intracranial lesions, and (5) recraniotomy for brain metastasis recurrence. In contrast, the presence of extracranial metastases, metachronous diagnosis, and solitary brain metastases had no influence on survival. As expected, younger age and limited number of brain metastases (up to three) are favorable prognostic factors. Remarkably, the presence of extracranial metastases had no influence on patient survival.

Endoglin expression in metastatic breast cancer cells enhances their invasive phenotype

Endoglin is a cell-surface adhesion protein as well as a coreceptor for transforming growth factor-β (TGF-β). It is located on endothelial and few other cells, but also found on certain tumor cells. Brain metastatic breast tumor cells derived from the MDA-MB-231 cell line heavily express endoglin in contrast to the corresponding parental ones. To clarify whether this determines their invasive phenotype, we compared their biological properties with endoglin-silenced brain-metastatic cells, low-expressing parental cells and these transfected with L- and S-endoglins, isoforms transducing or lacking TGF-β signals. All L-endoglin-overexpressing cells were characterized by numerous invadopodia where endoglin was preferentially localized. Endoglin-expression resulted in elevated levels of the matrix metalloproteinases (MMP-1 and MMP-19) and downregulation of the plasminogen activator inhibitor-1. In Boyden-chamber and wound-healing assays, endoglin-overexpressing cells showed a considerably higher migration and chemotaxis to TGF-β. In 3D spheroid confrontation assays between breast tumor cells and TGF-β-secreting glioma cells, high L-endoglin-expressing cells invaded into the glioma-spheroids whereas low-endoglin-expressing cells dissociated in the culture; invasion was blocked by TGF-β antibodies. In contrast to parental cells, endoglin-overexpressing cells invaded deeply into mouse brain slices. Thus, endoglin expression on tumor cells enhances their invasive character by formation of invadopodia, extracellular proteolysis, chemotaxis and migration.

Skull metastases: clinical features, differential diagnosis, and review of the literature.

BACKGROUND Most metastatic skull lesions are asymptomatic, although they can cause severe disability due to compression of dural sinuses and cranial nerves. The authors present current cases of calvarial and skull base metastases. Clinical features are compared to those of primary skull tumors and tumor-like lesions. METHODS We retrospectively reviewed the charts and radiographic images of 38 patients who underwent surgery for a skull lesion at our department between 1991 and 2001. The literature on skull metastases was reviewed. RESULTS In 12 cases, histologic examination revealed skull metastases. Eleven patients were known to suffer from cancer at the time of presentation. However, in 5 cases metastatic lesions were the first evidence of disseminated disease. Radical resection was possible in 9 cases. Removal and reconstruction of the infiltrated dura mater was necessary in 5 patients, whereas reconstruction of the bone was required in 8 patients. In comparison to 18 cases with primary skull tumors, patients with skull metastases presented less frequently with a neurologic deficit (3/12 vs. 9/18), reported a shorter history of symptoms (median 2 months vs. 24 months), and were older (median 70 years vs. 51 years). CONCLUSION Patients presenting with skull metastases are often in an advanced stage of disease, although surgery can relieve symptoms quickly and effectively with low morbidity. In particular, patients with signs of dura infiltration and related neurologic deficit should be offered neurosurgical therapy.