Kristi Krüger

In vivo MRI and histopathological assessment of tumor microenvironment in luminal-like and basal-like breast cancer xenografts

To explore tumor pathophysiology with special attention to the microenvironment in two molecular subtypes of human breast cancer using in vivo magnetic resonance imaging (MRI) and histopathology. The impact of tumor growth, size, and the influence of estradiol were also investigated.

Effect of antiangiogenic therapy on tumor growth, vasculature and kinase activity in basal- and luminal-like breast cancer xenografts

Several clinical trials have investigated the efficacy of bevacizumab in breast cancer, and even if growth inhibiting effects have been registered when antiangiogenic treatment is given in combination with chemotherapy no gain in overall survival has been observed. One reason for the lack of overall survival benefit might be that appropriate criteria for selection of patients likely to respond to antiangiogenic therapy in combination with chemotherapy, are not available.

Microvascular proliferation in luminal A and basal-like breast cancer subtypes.

Aims The aims of this study were to examine microvessel density (MVD), proliferating MVD (pMVD) and Vascular Proliferation Index (VPI) in basal-like phenotype (BP) and luminal A subtypes of breast cancer and to study their prognostic value. Methods Dual-colour immunohistochemistry for von Willebrand factor and Ki67 was done on sections from 62 luminal A and 62 BP tumours matched for grade and selected from 909 breast cancers previously reclassified into molecular subtypes. Associations between MVD, pMVD and VPI, molecular subtypes and breast cancer prognosis were estimated using linear regression and survival analyses. Results Both pMVD (difference 1.9 microvessels/mm2 (p=0.002)) and VPI (difference 1.7 percentage points (p=0.014)) were higher in BP tumours compared with luminal A. No clear difference between subtypes was found for MVD. However, only MVD was associated with prognosis. HR for breast cancer death for all cases was 1.10 (95% CI 1.02 to 1.18) per 10 vessels increase. Among luminal A tumours, HR was 1.22 per 10 vessels increase (p<0.001) and in BP it was 1.04 (p=0.37). Conclusions High MVD was associated with poor prognosis in luminal A, but not in BP cancers. Vascular proliferation was higher in BP, indicating a more active angiogenesis than in luminal A tumours. The luminal A subgroup comprised mostly histopathological grade 3 cancers in this selected series, and further studies are needed to clarify whether MVD provides additional prognostic information for luminal A tumours irrespective of grade. This may contribute to stratification of this large group of patients and may aid in identifying tumours with a particularly good prognosis.

Expression of Nestin associates with BRCA1 mutations, a basal-like phenotype and aggressive breast cancer

We here examined whether Nestin, by protein and mRNA levels, could be a predictor of BRCA1 related breast cancer, a basal-like phenotype, and aggressive tumours. Immunohistochemical staining of Nestin was done in independent breast cancer hospital cohorts (Series I-V, total 1257 cases). Also, TCGA proteomic data (n = 103), mRNA microarray data from TCGA (n = 520), METABRIC (n = 1992), and 6 open access breast cancer datasets (n = 1908) were analysed. Patients with Nestin protein expression in tumour cells more often had BRCA1 germline mutations (OR 8.7, p < 0.0005, Series III), especially among younger patients (<40 years at diagnosis) (OR 16.5, p = 0.003). Nestin protein positivity, observed in 9–28% of our hospital cases (Series I-IV), was independently associated with reduced breast cancer specific survival (HR = 2.0, p = 0.035) and was consistently related to basal-like differentiation (by Cytokeratin 5, OR 8.7–13.8, p < 0.0005; P-cadherin OR 7.0–8.9, p < 0.0005; EGFR staining, OR 3.7–8.2, p ≤ 0.05). Nestin mRNA correlated significantly with Nestin protein expression (ρ = 0.6, p < 0.0005), and high levels were seen in the basal-like intrinsic subtype. Gene expression signalling pathways linked to high Nestin were explored, and revealed associations with stem-like tumour features. In summary, Nestin was strongly associated with germline BRCA1 related breast cancer, a basal-like phenotype, reduced survival, and stemness characteristics.

Abstract 2429: Nestin expression in tumor cells is associated with BRCA1 positive genotype and a basal-like phenotype in breast cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: The basal-like subtype of breast cancer is one of at least five molecular categories. These poor prognosis tumors are most often triple negative, i.e. negative for hormone receptors and HER2, and therefore lack targeted treatment options. There is at present no consensus on how to best define this subtype, and different surrogate immunohistochemistry based markers have been suggested. We here present data supporting nestin as a marker of the basal-like subtype. Originally reported in neuronal progenitor cells, nestin was later observed in the stem cell/progenitor compartment in a variety of tissues as well as in different cancer types, and suggested to be a cancer stem cell marker. In breast cancer, nestin has been reported in the basal-like and triple negative phenotypes. Here, we examined nestin expression in different breast cancer cohorts, including BRCA familial cancer, and breast cancer metastasis. Materials and methods: Immunohistochemical staining of nestin was done in four series of breast cancer, and a semi-quantitative measure of nestin positivity (staining index) was used for evaluation of expression. Series I include 190 breast cancer patients diagnosed during 1996-2001 in Hordaland, Norway. Series II comprise 192 breast cancer cases identified in Kampala, Uganda in 1990-2002. Series III includes 151 patients with breast cancer, 53 BRCA1 and 45 BRCA2 mutation carriers, identified during 1980-1995 in Montreal, Canada, and 51 BRCA negative patients from Norway. Series IV consists of 35 patients with tissue from the primary breast cancer and matched metastasis collected in 1996-2007. Finally, level 3 mRNA microarray data from TCGA (n = 520) and four open access breast cancer gene expression data sets (n = 1148) with information on molecular subtype were included. Results: The proportion of nestin positive breast tumors was 11%, 24% and 28% in Series I-III. Nestin expression was significantly associated with basal cell markers Cytokeratin 5/6 and P-cadherin (p-values <0.0005), five immunohistochemical profiles for the basal-like phenotype (Odds ratio, OR, range 10.9-27.5) and the triple negative phenotype (OR range 11.8-28.2). Further, patients with BRCA1 mutation more often had nestin positive tumors (OR 8.7, Series III). Higher nestin mRNA levels were seen in basal-like tumors, compared with the luminal-like and HER2-enriched subtypes. In Series IV, nestin expression in the primary tumor correlated with expression in the corresponding metastasis, with no statistically significant difference between the two groups (McNemar test, p = 1.0). Conclusion: We found nestin positivity in breast cancer cells to be significantly and strongly associated with the BRCA1 genotype, individual basal cell markers, and the basal-like and triple negative phenotypes. Citation Format: Kristi Kruger, Elisabeth Wik, Hawa Nalwoga, Monica Mannelqvist, Ingunn M. Stefansson, Jarle B. Arnes, Goril Knutsvik, Turid Aas, William D. Foulkes, Lars A. Akslen. Nestin expression in tumor cells is associated with BRCA1 positive genotype and a basal-like phenotype in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2429. doi:10.1158/1538-7445.AM2015-2429

Abstract 2651: Nestin expression in tumor cells is associated with the basal-like phenotype and aggressive features in breast cancer.

Background: Nestin, neuroepithelial stem cell protein, is an intermediate filament protein originally described as a neuronal stem cell/progenitor marker during central nervous system development. Nestin is also expressed in immature or progenitor cells in non-neural tissues, including the basal/myoepithelial layer of the mammary gland and immature blood vessels. Recently, nestin positivity has been reported in different cancer types, and on this background we wanted to evaluate nestin positivity in breast cancer. Materials and methods: Immunohistochemical staining of nestin was done on tissue microarray slides from two independent series of breast cancer. Series 1 was a nested case-control study of interval and screening detected breast cancer based on the Norwegian Mammography Screening Program in Hordaland County during 1996-2001, giving a series of 190 patients with invasive breast cancer. Series 2 included 192 cases of invasive breast carcinoma from 1990-2002 identified in the Kampala Cancer Registry, and at the Department of Pathology, Makerere University College of Health Sciences (MakCHS), Kampala, Uganda. Evaluation of staining was done using staining index, where the staining index (values 0-9) was obtained as a product of staining intensity (0-3) and proportion of immunopositive cells ( 50%, 3). A staining index above 0 was regarded as positivity for nestin. Results: In both series, nestin positivity in tumor cells was significantly associated with higher histologic grade; grade 1-2 vs. grade 3, (p Concerning basal cell markers, nestin positivity in tumor cells was significantly associated with cytokeratin 5/6 and p-cadherin, with p-values Conclusion: In these two series of breast cancer, nestin expression in tumor cells was significantly associated with features of aggressive breast cancer disease such as higher histologic grade, negativity for estrogen and progesterone receptors and high tumor cell proliferation by Ki-67. Further, nestin positivity was significantly associated with basal cell markers, the triple negative phenotype and the basal-like phenotype. Citation Format: Kristi Kruger, Hawa Nalwoga, Karin Collett, Jarle B. Arnes, Turid Aas, Lars A. Akslen. Nestin expression in tumor cells is associated with the basal-like phenotype and aggressive features in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2651. doi:10.1158/1538-7445.AM2013-2651

Abstract 1384: Axl and VE-cadherin expression as markers of angiogenesis in human breast cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The aim of our study was to investigate vascular expression of Axl and VE-cadherin as potential markers of angiogenesis in human breast carcinoma. Materials and methods: Immunohistochemical staining was performed using antibodies against Axl and VE-cadherin. Expression was examined in a population-based series of breast cancers collected in Hordaland County (Norway) during 1996-2001. The series is a nested case-control study as part of the Norwegian Breast Cancer Screening Program, and consists of 95 invasive interval cancers and 95 invasive screen detected cancers matched by tumor diameter. Positive microvessels were counted in 10 consecutive high power fields within hot-spot areas according to previously established criteria and expressed as microvessel density (MVD) by mean number of microvessels per mm2. Results: The MVD for Axl was associated with progesterone receptor status (p=0.026), HER-2 status (p=0.030) and basal-like phenotypes: CK5/6+ P-cadherin+ (p=0.009), ER- HER2-CK5/6+ (p=0.014) and ER- HER2- CK5/6+ and/or EGFR+ (p=0.027). In contrast, MVD by VE-cadherin expression (n=127) indicated negative associations with basal-like phenotypes: CK5/6+ or P-cadherin+ or EGFR+ (p=0.034), and ER- HER2- P-cadherin+ (p=0.003). VE-cadherin expression also tended to be associated with better patient survival (p=0.067). Conclusion: High MVD by vascular expression of Axl was associated with aggressive features in our breast cancer series, especially the basal-like phenotype, whereas MVD by VE-cadherin expression appeared to be related to less aggressive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1384. doi:1538-7445.AM2012-1384

Abstract LB-299: Effect of antiangiogenic therapy on tumor growth, vasculature and kinase activity in luminal and basal-like breast cancer xenografts

Angiogenesis is important for the growth and dissemination of solid tumors, and VEGF-A is one of the main stimulators of this process. Bevacizumab binds VEGF and increases the progression-free, but not overall, survival in breast cancer patients treated with chemotherapy. For determining factors of importance for treatment response and resistance, two different human breast cancer xenografts (luminal -and basal-like) were used, which respond differently to antiangiogenic mono -or combination therapy. Both doxorubicin and bevacizumab inhibited tumor growth significantly in basal-like xenografts, but with a superior effect when given in combination. In contrast, luminal-like tumors demonstrated no additional effect of combination therapy. Vascular characterization demonstrated a total inhibition of endothelial cell proliferation (pMVD) and reduction in mean vascular density (MVD) in the basal-like tumors three days after bevacizumab treatment, not seen in the luminal-like tumors. At day 10, combination treatment reduced pMVD and MVD in both tumors, however only MVD lasted to the end of the experiment and in the basal-like tumors. Analysis of kinase activity in the basal-like tumors after monotherapy demonstrated an early (day 3) increase in the phosphorylation of several targets, followed by a reduction at day 10. Furthermore, the most effective treatment in vivo was accompanied with increased phosphorylation of growth control -and proangiogenic kinase substrates, like PLCγ1, EGFR, PDGFRβ, VEGFR2, and MAP kinases. Early inhibition of vascular proliferation and upregulation of specific signaling pathways, as described above, may therefore indicate important markers for response. Such markers may also be of value in patient stratification, for the prediction of treatment effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-299. doi:10.1158/1538-7445.AM2011-LB-299