Discovery and Validation of Biomarkers in Breast Cancer

Worldwide, breast cancer is the most common malignancy among women, and although treatment and prognosis have improved substantially over the last decades, for some patients the risk of recurrence remains for several years following diagnosis. Meanwhile, many breast cancer patients receive systemic adjuvant treatment unnecessarily, since their tumours will never recur. Implicitly, these patients are being overtreated while others are being undertreated. The challenge is to identify patients with a higher risk of developing recurrences and metastasis, from those who do not need additional treatment. These women may be spared potential treatment-induced side effects. Breast cancer is a highly complex and very heterogeneous disease, displaying both inter- and intratumoural biological variation. To ensure correct diagnosis and treatment, we need more precise and improved biomarkers. Equally important as discovering new and better biomarkers is the validation of existing ones. The work described in this thesis focuses on the discovery of novel candidate biomarkers for breast cancer, but also emphasize the equally important value of validating existing ones. The first study examined the expression of the protein MARCKSL1 by immunohistochemistry. Increased expression of MARCKSL1 was previously associated with risk for metastasis and worse prognosis in breast cancer patients, especially in those with highly proliferating tumours. In this study, we set out to validate these findings. However, in contrast to previous findings, MARCKSL1 protein expression was not prognostic in this independent patient cohort. In the search for novel prognostic and predictive biomarkers in breast cancer, microRNAs are now emerging as potential candidates. In previous studies, gene expression of miR-18a and miR-18b correlated with high proliferation and basal-like features of breast cancer. In the second study, we applied chromogenic in situ hybridization to investigate the in situ expression of these microRNAs in both ER+ and ER- tumours. Our findings revealed that miR-18a and miR-18b are specifically expressed in the stroma surrounding the tumour, especially in ER- breast tumours that present with a high degree of tumour infiltrating lymphocytes. Additional investigations suggested that the expression of these miRNAs might be associated with macrophages. Cell proliferation is a fundamental feature of cancer cells, and high proliferation correlates with a higher risk of recurrence and reduced survival in breast cancer. Ki-67 is a well-known marker for proliferation, but its use is controversial because of the lack of consensus regarding pre-analytical processing, optimal clinical cut-off value and a high degree of variability across laboratories. Digital pathology is becoming increasingly important in routine diagnostics and is soon to be implemented in Norway. In the third study, we employed digital image analysis to evaluate the expression of Ki-67 in tissue microarrays, in a case-control study of tamoxifen-treated patients with and without recurrence. However, our findings do not support an increased risk of recurrence associated with Ki-67 expression. The resulting discrepancies with previous studies discussed in this thesis, highlights the importance of performing replication and validation studies, and to critically re-evaluate previous biomarkers

Nucleus pulposus application onto rat spinal dorsal nerve roots leads to a persistent increase in spinal C-fibre responses, possibly due to upregulation of IL1α, IL1β and TNF

Sensitization of sensory neurons after noxious conditioning may be involved in many chronic pain states, including radiating low back pain and sciatica following disc herniation. Here, we examine such sensitization induced by two types of noxious conditioning: I) electrical sciatic high frequency stimulation (HFS), and II) nucleus pulposus (NP), harvested from intervertebral discs, applied onto the spinal dorsal nerve roots. In addition, we investigate the gene expression of the proinflammatory cytokines IL1α, IL1β, TNF and the protease MMP1 in NP tissue. Electrophysiological extracellular potentials were recorded from the spinal dorsal horn in anaesthetized Sprague-Dawley- or Lewis rats. A single test stimulus was applied to the sciatic nerve every 4th minute and the A- and C-fibre responses were separated according to latencies. For the gene expression analysis, total RNA was isolated from NP tissue and mRNA expression quantified by RT-qPCR. First, the spinal neuronal responses were studied by field potential recordings following HFS conditioning of the sciatic nerve. The HFS conditioning produced a clear long-term potentiation (LTP), which outlasted the experimental time period of 180 minutes. Next, the spinal neuronal responses were studied by single unit recordings following NP application onto the dorsal nerve roots. The NP conditioning produced a persistent increase in the spinal C-fibre responses, also outlasting the experimental time period of 180 minutes. In addition, the present study demonstrated a significant upregulation in the gene expression of IL1α, IL1β and TNF 180 minutes after application of NP onto the spinal dorsal nerve roots. No changes, however, were seen in the expression of MMP1. In summary, the HFS caused a robust LTP in the spinal cord. Furthermore, application of NP onto the spinal dorsal nerve roots induced an LTP-like phenomenon which was also associated with an increase in gene expression of IL1α, IL1β and TNF in NP tissue 180 minutes after application. The present data suggests that herniated NP in contact with the dorsal nerve roots may cause a persistent spinal hyperexcitability of nociceptive neurons, possibly due to biochemical mediators intrinsic to the NP tissue

MiR-18a and miR-18b are expressed in the stroma of oestrogen receptor alpha negative breast cancers

Background Previously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER−), cytokeratin 5/6 positivity and basal-like features of breast cancer. Methods We investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed. Results CISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b. Conclusions Our study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER− tumourspublishedVersio

Digital Image Analysis of Ki-67 Stained Tissue Microarrays and Recurrence in Tamoxifen-Treated Breast Cancer Patients

Purpose: The proliferation marker Ki-67 has been used as a prognostic marker to separate low- and high-risk breast cancer subtypes and guide treatment decisions for adjuvant chemotherapy. The association of Ki-67 with response to tamoxifen therapy is unclear. High-throughput automated scoring of Ki-67 might enable standardization of quantification and definition of clinical cut-off values. We hypothesized that digital image analysis (DIA) of Ki-67 can be used to evaluate proliferation in breast cancer tumors, and that Ki-67 may be associated with tamoxifen resistance in early-stage breast cancer. Patients and Methods: Here, we apply DIA technology from Visiopharm using a custom designed algorithm for quantifying the expression of Ki-67, in a case–control study nested in the Danish Breast Cancer Group clinical database, consisting of stages I, II, or III breast cancer patients of 35– 69 years of age, diagnosed during 1985– 2001, in the Jutland peninsula, Denmark. We assessed DIA-Ki-67 score on tissue microarrays (TMAs) from breast cancer patients in a case–control study including 541 ER-positive and 300 ER-negative recurrent cases and their non-recurrent controls, matched on ER-status, cancer stage, menopausal status, year of diagnosis, and county of residence. We used logistic regression to estimate odds ratios and associated 95% confidence intervals to determine the association of Ki-67 expression with recurrence risk, adjusting for matching factors, chemotherapy, type of surgery, receipt of radiation therapy, age category, and comorbidity. Results: Ki-67 was not associated with increased risk of recurrence in tamoxifen-treated patients (ORadj =0.72, 95% CI 0.54, 0.96) or ER-negative patients (ORadj =0.85, 95% CI 0.54, 1.34). Conclusion: Our findings suggest that Ki-67 digital image analysis in TMAs is not associated with increased risk of recurrence among tamoxifen-treated ER-positive breast cancer or ER-negative breast cancer patients. Overall, our findings do not support an increased risk of recurrence associated with Ki-67 expression.publishedVersio

Metabolic consequences of perioperative oral carbohydrates in breast cancer patients - an explorative study

Background The metabolic consequences of preoperative carbohydrate load in breast cancer patients are not known. The present explorative study investigated the systemic and tumor metabolic changes after preoperative per-oral carbohydrate load and their influence on tumor characteristics and survival. Methods The study setting was on university hospital level with primary and secondary care functions in south-west Norway. Serum and tumor tissue were sampled from a population-based cohort of 60 patients with operable breast cancer who were randomized to either per-oral carbohydrate load (preOp™; n = 25) or standard pre-operative fasting (n = 35) before surgery. Magnetic resonance (MR) metabolomics was performed on serum samples from all patients and high-resolution magic angle spinning (HR-MAS) MR analysis on 13 tumor samples available from the fasting group and 16 tumor samples from the carbohydrate group. Results Fourteen of 28 metabolites were differently expressed between fasting and carbohydrate groups. Partial least squares discriminant analysis showed a significant difference in the metabolic profile between the fasting and carbohydrate groups, compatible with the endocrine effects of insulin (i.e., increased serum-lactate and pyruvate and decreased ketone bodies and amino acids in the carbohydrate group). Among ER-positive tumors (n = 18), glutathione was significantly elevated in the carbohydrate group compared to the fasting group (p = 0.002), with a positive correlation between preoperative S-insulin levels and the glutathione content in tumors (r = 0.680; p = 0.002). In all tumors (n = 29), glutamate was increased in tumors with high proliferation (t-test; p = 0.009), independent of intervention group. Moreover, there was a positive correlation between tumor size and proliferation markers in the carbohydrate group only. Patients with ER-positive / T2 tumors and high tumor glutathione (≥1.09), high S-lactate (≥56.9), and high S-pyruvate (≥12.5) had inferior clinical outcomes regarding relapse-free survival, breast cancer-specific survival, and overall survival. Moreover, Integrated Pathway Analysis (IPA) in serum revealed activation of five major anabolic metabolic networks contributing to proliferation and growth. Conclusions Preoperative carbohydrate load increases systemic levels of lactate and pyruvate and tumor levels of glutathione and glutamate in ER-positive patients. These biological changes may contribute to the inferior clinical outcomes observed in luminal T2 breast cancer patients.publishedVersio

MiR-18a and miR-18b are expressed in the stroma of oestrogen receptor alpha negative breast cancers

Background Previously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER−), cytokeratin 5/6 positivity and basal-like features of breast cancer. Methods We investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed. Results CISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b. Conclusions Our study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER− tumour

Validation study of MARCKSL1 as a prognostic factor in lymph node-negative breast cancer patients.

Protein expression of Myristoylated alanine-rich C kinase substrate like-1 (MARCKSL1) has been identified as a prognostic factor in lymph-node negative (LN-) breast cancer patients. We aim to validate MARCKSL1 protein expression as a prognostic marker for distant metastasis-free survival (DMFS) in a new cohort of LN- breast cancer patients. MARCKSL1 expression was evaluated in 151 operable T1,2N0M0 LN- breast cancer patients by immunohistochemistry. Median follow-up time was 152 months, range 11-189 months. Results were compared with classical prognosticators (age, tumor diameter, grade, estrogen receptor, and proliferation) using single (Kaplan-Meier) and multivariate (Cox model) survival analysis. Thirteen patients (9%) developed distant metastases. With both single and multiple analysis of all features, MARCKSL1 did not show a significant prognostic value for DMFS (p = 0.498). Of the assessed classical prognosticators, only tumor diameter showed prognostic value (hazard ratio 9.3, 95% confidence interval 2.8-31.0, p <0.001). MARCKSL1 expression could not be confirmed as a prognostic factor in this cohort. Possible reasons include changes in diagnostic and treatment guidelines between the discovery and validation cohorts. Further studies are needed to reveal the potential biological role of this protein in breast cancer

Digital Image Analysis of Ki-67 Stained Tissue Microarrays and Recurrence in Tamoxifen-Treated Breast Cancer Patients

Purpose: The proliferation marker Ki-67 has been used as a prognostic marker to separate low- and high-risk breast cancer subtypes and guide treatment decisions for adjuvant chemotherapy. The association of Ki-67 with response to tamoxifen therapy is unclear. High-throughput automated scoring of Ki-67 might enable standardization of quantification and definition of clinical cut-off values. We hypothesized that digital image analysis (DIA) of Ki-67 can be used to evaluate proliferation in breast cancer tumors, and that Ki-67 may be associated with tamoxifen resistance in early-stage breast cancer. Patients and Methods: Here, we apply DIA technology from Visiopharm using a custom designed algorithm for quantifying the expression of Ki-67, in a case–control study nested in the Danish Breast Cancer Group clinical database, consisting of stages I, II, or III breast cancer patients of 35– 69 years of age, diagnosed during 1985– 2001, in the Jutland peninsula, Denmark. We assessed DIA-Ki-67 score on tissue microarrays (TMAs) from breast cancer patients in a case–control study including 541 ER-positive and 300 ER-negative recurrent cases and their non-recurrent controls, matched on ER-status, cancer stage, menopausal status, year of diagnosis, and county of residence. We used logistic regression to estimate odds ratios and associated 95% confidence intervals to determine the association of Ki-67 expression with recurrence risk, adjusting for matching factors, chemotherapy, type of surgery, receipt of radiation therapy, age category, and comorbidity. Results: Ki-67 was not associated with increased risk of recurrence in tamoxifen-treated patients (ORadj =0.72, 95% CI 0.54, 0.96) or ER-negative patients (ORadj =0.85, 95% CI 0.54, 1.34). Conclusion: Our findings suggest that Ki-67 digital image analysis in TMAs is not associated with increased risk of recurrence among tamoxifen-treated ER-positive breast cancer or ER-negative breast cancer patients. Overall, our findings do not support an increased risk of recurrence associated with Ki-67 expression