Background: Treatment resistance is a well-known problem in estrogen receptor (ER) positive breast cancer. Complementary therapies are investigated for women who do not respond or who develop resistance against standard ER targeted treatment. Insulin-like growth factor-1 receptor (IGF1R) and its signaling pathway has been suggested to cause estrogen-independent cell growth and survival. Therapy against targets within the pathway is currently investigated in clinical trials. The aim of this study was to investigate if the IGF1R/mTOR pathway was activated or deregulated in breast cancer patients and to explore if any of the markers were prognostic, with or without adjuvant tamoxifen. Material and methods: Two patient cohorts were investigated by immunohistochemistry using tissue microarrays. The first cohort (N = 264) consisted of mainly post-menopausal women with stage II breast cancer treated with tamoxifen for 2 years irrespective of ER status. The second cohort (N = 206) consisted of mainly medically untreated, pre-menopausal patients with node-negative breast cancer. The protein expression of IGF1R, p-mTOR and p-S6rp were investigated. Cytoplasmic staining was evaluated for all markers and membrane staining was additionally evaluated for IGF1R. Statistical analyses were based on the intensity (0-3) of staining. Expression of IGF1R gave similar results in the cytoplasm and membrane, and p-values for cytoplasmic staining are reported below. Distant disease free survival (DDFS) at 5 years was used as end-point. Results: IGF1R expression was positively associated with ERa (p<0.001 in Mann-Whitney ranksum test), PgR (p<0.001) and HER2 (p = 0.042) expression in cohort 1, and also with Ki67 (p = 0.006) in cohort 2. p-S6rp was positively associated with ERα in cohort 1 (p<0.001) and HER2 (p = 0.004) in cohort 2. p-mTOR was positively associated only with Ki67 (p<0.001) in cohort 1. High expression of IGF1R was associated with a significantly better prognosis in cohort 1 (HR = 0.7 per intensity step, 95% CI = 0.5-0.9, p = 0.016 using Cox regression). When stratifying for ER status the effect was found in ER negative (ER-) (N = 80, HR = 0.6, 95% CI = 0.4-1.0, p = 0.03) but not in ER positive (ER+) patients (N = 174, HR 1.2, 95% CI = 0.8-2.0, p = 0.40). Both the effect in the ER- subgroup as well as the difference between ER- and ER+ patients were confirmed in interaction analysis and remains after adjustment for age, tumor size, node status, HER2, Ki67, and menopausal status (p = 0.06 for interaction). In cohort 2, no relation to DDFS could be found for IGF1R. p-mTOR and p-S6rp showed no relationship to prognosis in either of the cohorts. Conclusion: We found that high IGF1R expression was associated with a better prognosis for tamoxifen treated women. This effect could be seen in the ER- but not in the ER+ subgroup of patients. The lack of co-activation of downstream markers (p-mTOR and p-S6rp) in the IGF1R pathway shows that the prognostic effect is not due to complete activation of this pathway. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-52.

Background: Detection and enumeration of circulating tumor cells (CTCs) allows real time monitoring of disease evolvement. In women with metastatic breast cancer (MBC), a CTC count of ≥5 CTCs is associated with decreased progression-free survival (PFS) and overall survival (OS). Serial sampling after therapy initiation has indicated that longitudinal CTC enumeration adds prognostic information, but data from long time sampling is sparse. The aim of this study was to evaluate if prospective longitudinal detection of CTC count and CTC clusters in women with newly diagnosed MBC can improve prognostication and monitoring of patients in the clinical setting. Methods: Longitudinal blood samples were collected at baseline (BL) and after 1, 3 and 6 months in 156 women with MBC scheduled for 1st line systemic therapy. CTC enumeration and cluster detection were performed by the CellSearch® system in a prospective monitoring trial (NCT01322893). 115 patients had evaluable samples at all time-points. Primary endpoint was PFS and secondary endpoint was OS at BL in relation to CTC count and as landmark analyses during treatment. In addition, change in CTC count during therapy was compared to progressive disease (PD) versus non-PD. Structured clinical and radiological evaluation for PD was performed every 3rd month. Results: Seventy-nine (52%) of 152 evaluable patients had ≥5 CTC and 14/79 patients had CTC-clusters (33 clustered CTC) at BL. Median follow-up time was 25 (7-69) months. Patients with ≥5 CTCs had inferior PFS and OS in uni-(data not shown) and multivariable analysis (HRPFS 1.91 (1.26-2.91), P=0.003) (HROS 3.57 (2.02-6.31), P<0.001) at BL. Presence of clusters at BL was prognostic for OS (HROS 2.37 (1.25-4.51), P=0.008). Longitudinal landmark analysis of number of CTCs and presence of CTC clusters showed a time-dependent increase in HR during treatment for CTCs and CTC-clusters and predicted worse PFS and OS at all time-points. Stratifying patients based on CTC count and presence of clusters revealed four risk groups (0, 1-4, ≥5 CTC, ≥5 CTC + clusters) where patients with clusters had inferior PFS and OS at all time points. Change in CTC count from BL to 1 and 3 months, and from 3 to 6 months was significantly related to evaluation at 3 and 6 months (PD vs non-PD, P=0.013 (3 months), P=0.016 (6 months)) and change in CTC count from BL to 1, 3 and 6 months was also significantly predictive of both PFS and OS. Notably, survival was significantly inferior for patients with persistent CTC ≥5 during treatment. Discussion: CTC is an independent prognostic factor for MBC patients scheduled for 1st line systemic therapy. By longitudinal monitoring during treatment, the prognostic information by presence of ≥5 CTC and clusters increases over time and supports long time monitoring of patients. Importantly, detection of CTC-clusters identifies a subgroup of patients with dismal prognosis at all time-points indicating that CTC-clusters renders important clinical information. Change in CTC count during systemic therapy is related to outcome of evaluation and prognosis at all time-points. Citation Format: Larsson A-M, Jansson S, Bendahl P-O, Baker S, Graffman C, Lundgren C, Loman N, Aaltonen KE, Rydén L. Improved prognostic information by serial monitoring of CTC enumeration and CTC-clusters from baseline to six months in patients with metastatic breast cancer scheduled for 1st line systemic therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-03.

Introduction: Triple-negative breast cancer (TNBC) accounts for approximately 15% of all female breast cancer and is associated with aggressive clinical behaviour. No targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and c-KIT, have however shown some promising results for patients with TNBC. The aim of the present study was to investigate whether gains and/or amplifications of VEGFR2 and c-KIT occur in TNBC. These genes may constitute novel candidate biomarkers for selecting TNBC patients for treatment with tyrosine kinase inhibitors. Material & Methods: Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and c-KIT in 83 primary human breast cancers, of which 31 were classified as TNBC. Gains were defined as ≥4 gene copies in more than 40% of the cancer cells, while amplification was defined as gene copy/CEP ratio >2 in more than 10% of the cancer cells. A tumour was considered FISH positive for c-KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Immunohistochemical (IHC) staining was performed for assessment of c-KIT protein expression. Results: Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were c-KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and c-KIT in nine (18%) cases for both genes. No significant difference in frequency between TNBCs and non-TNBCs was found. There was a correlation between FISH positivity for VEGFR2 and c-KIT (c2 test, P<0.001), and VEGFR2 and c-KIT FISH positivity correlated to ER/PgR negativity and high Nottingham histological grade (NHG). A significantly worse breast cancer specific survival (BCSS) was seen for FISH positive cases in the whole cohort as well as among untreated patients and non-TNBCs, but not among TNBC cases. Discussion: The high correlation between VEGFR2 and c-KIT FISH positivity suggests that the genes are co-amplified. Increased copy number of both genes was related to aggressive disease and a worse prognosis, and thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-11.