Prognostic and predictive testing of molecular markers in breast cancer by real-time quantitative PCR

This chapter provides a general overview of breast cancer, including the relevance of measuring gene expression in the primary breast tumor in relation to the progression of the disease and the tumor response to treatment. To better understand the concept of breast cancer, extra emphasis will be put on breast cancer subtypes, staging and grading, and the tumor micro-environment that harbors the epithelial cancer cells. Currently available biomarkers to assess outcome of breast cancer patients in general (i.e. prognosis) as well as biomarkers currently available to assess patient outcome in response to therapy (i.e. prediction) will be discussed. After this it should become clear that there is still an urgent need for new biomarkers. How this search for additional biomarkers can be achieved by measuring mRNAgene expression in the primary tumor of patients diagnosed with breast cancer, will be explained in more detail

Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2

In previous studies we found expression of the protein colligin 2 (heat shock protein 47 (HSP47), SERPINH1) in glioma neovasculature while not in normal brain tissue. Generally, the regulation of heat shock gene expression in eukaryotes is mediated by heat shock factors (HSF). In mammals, three heat shock transcription factors, HSF-1, -2, and -4, have been isolated. Here we investigated the relation between the expression of colligin 2 and these heat shock factors at the mRNA level using real-time reverse transcriptase PCR (qRT-PCR) in different grades of astrocytic tumorigenesis, viz., low-grade glioma and glioblastoma. Endometrium samples, representing physiological angiogenesis, were included as controls. Since colligin 2 is a chaperon for collagens, the gene expression of collagen I (COL1A1) was also investigated. The blood vessel density of the samples was monitored by expression of the endothelial marker CD31 (PECAM1). Because NG2-immunopositive pericytic cells are involved in glioma neovascularization, the expression of NG2 (CSPG4) was also measured

Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients

Summary: Circulating tumor cells (CTCs) have rapidly developed as important cancer biomarkers after their enumeration proved to be prognostic in metastatic breast, colorectal and prostate cancer, and their rise or decline after the first cycle of therapy showed to predict therapy response. Besides mere counting, CTCs can be isolated and subsequently analyzed using various molecular applications, including miRNA expression analysis. Recently, miRNA expression profiling in primary tumors has yielded promising results. However, establishing miRNA expression in the circulation likely has advantages over determination in primary tumor tissue, further augmenting the potential applications of miRNA determination in oncology. Additionally to CTC-associated miRNAs, free circulating miRNAs have been identified in whole blood, plasma and serum. Since determination of miRNAs in peripheral blood, either cell-free or CTC-associated, is expected to become important in oncology, especially when linked to and interpreted together with epithelial CTCs, this review focuses on measuring miRNAs in the circulation of cancer patients

Pleiotropic actions of suramin on the proliferation of human breast-cancer cells in vitro

Suramin, a non‐specific growth factor antagonist, is currently under investigation for treatment of cancer patients. We studied its action on 6 different human breast‐cancer cell lines in vitro. In complete growth medium, pleiotropic effects were observed with respect to cell proliferation, i.e. suramin is stimulatory at low concentrations and inhibitory at higher concentrations, for 4 of the 6 cell lines studied. The various cell lines showed marked differences with respect to the antiproliferative action of suramin, the Evsa‐T cells being by far the most sensitive ones. A suramin concentration of 100 μg/ml brought about a 100% stimulation of the proliferation of ZR/HERc cells, ZR 75.1 cells ectopically expressing a human epidermal growth factor receptor (EGF‐R) cDNA. Although less pronounced (10 to 60% stimulation), a similar response was observed for the parent ZR 75.1 cells, as well as for T‐47D and MDA‐MB‐231 cells. The non‐specificity of the action of suramin was established by the observation that suramin‐induced inhibition of cell proliferation could be abolished by insulin‐like growth factor‐1 (IGF‐I) or basic fibroblast growth factor (bFGF), and even by estradiol, both in complete growth medium and under defined serum‐free conditions. Our data indicate that suramin exerts pleiotropic effects on the proliferation of human breast cancer cells in vitro, and confirm the non‐specific nature of its action. The stimulatory effect of low concentrations of suramin on the proliferation of breast cancer cells may have important consequences for breast cancer patients treated with suramin. Copyrigh

Association of microRNA-7 and its binding partner CDR1-AS with the prognosis and prediction of 1st-line tamoxifen therapy in breast cancer

The large number of non-coding RNAs (ncRNAs) and their breadth of functionalities has fuelled many studies on their roles in cancer. We previously linked four microRNAs to breast cancer prognosis. One of these microRNAs, hsa-miR-7, was found to be regulated by another type of ncRNA, the circular non-coding RNA (circRNA) CDR1-AS, which contains multiple hsa-miR-7 binding sites. Based on this finding, we studied the potential clinical value of this circRNA on breast cancer prognosis in a cohort based on a cohort that was previously analysed for hsa-miR-7 and in an adjuvant hormone-naïve cohort for 1st-line tamoxifen treatment outcomes, in which we also analysed hsa-miR-7. A negative correlation was observed between hsa-miR-7 and CDR1-AS in both cohorts. Despite associations with various clinical metrics (e.g., tumour grade, tumour size, and relapse location), CDR1-AS was neither prognostic nor predictive of relevant outcomes in our cohorts. However, we did observe stromal CDR1-AS expression, suggesting a possible cell-type specific interaction. Next to the known association of hsa-miR-7 expression with poor prognosis in primary breast cancer, we found that high hsa-miR-7 expression was predictive of an adverse response to tamoxifen therapy and poor progression-free and post-relapse overall survival in patients with recurrent disease

ER and PI3K pathway activity in primary ER positive breast cancer is associated with progression-free survival of metastatic patients under first-line tamoxifen

Estrogen receptor positive (ER+) breast cancer patients are eligible for hormonal treatment, but only around half respond. A test with higher specificity for prediction of endocrine therapy response is needed to avoid hormonal overtreatment and to enable selection of alternative treatments. A novel testing method was reported before that enables measurement of functional signal transduction pathway activity in individual cancer tissue samples, using mRNA levels of target genes of the respective pathway-specific transcription factor. Using this method, 130 primary breast cancer samples were analyzed from non-metastatic ER+ patients, treated with surgery without adjuvant hormonal therapy, who subsequently developed metastatic disease that was treated with first-line tamoxifen. Quantitative activity levels were measured of androgen and estrogen receptor (AR and ER), PI3K-FOXO, Hedgehog (HH), NFκB, TGFβ, and Wnt pathways. Based on samples with known pathway activity, thresholds were set to distinguish low from high activity. Subsequently, pathway activity levels were correlated with the tamoxifen treatment response and progression-free survival. High ER pathway activity was measured in 41% of the primary tumors and was associated with longer time to progression (PFS) of metastases during first-line tamoxifen treatment. In contrast, high PI3K, HH, and androgen receptor pathway activity was associated with shorter PFS, and high PI3K and TGFβ pathway activity with worse treatment response. Potential clinical utility of assessment of ER pathway activity lies in predicting response to hormonal therapy, while activity of PI3K, HH, TGFβ, and AR pathways may indicate failure to respond, but also opens new avenues for alternative or complementary targeted treatments

MRNA expression profiles of colorectal liver metastases as a novel biomarker for early recurrence after partial hepatectomy

Background: Identification of specific risk groups for recurrence after surgery for isolated colorectal liver metastases (CRLM) remains challenging due to the heterogeneity of the disease. Classical clinicopathologic parameters have limited prognostic value. The aim of this study was to identify a gene expression signature measured in CRLM discriminating early from late recurrence after partial hepatectomy. Methods: CRLM from two patient groups were collected: I) with recurrent disease ≤12 months after surgery (N = 33), and II) without recurrences and disease free for ≥36 months (N = 30). The patients were clinically homogeneous; all had a low clinical risk score (0-2) and did not receive (neo-) adjuvant chemotherapy. Total RNA was hybridised to Illumina arrays, and processed for analysis. A leave-one-out cross validation (LOOCV) analysis was performed to identify a prognostic gene expression signature. Results: LOOCV yielded an 11-gene profile with prognostic value in relation to recurrent disease ≤12 months after partial hepatectomy. This signature had a sensitivity of 81.8%, with a specificity of 66.7% for predicting recurrences (≤12 months) versus no recurrences for at least 36 months after surgery (X2 P < 0.0001). Conclusion: The current study yielded an 11-gene signature at mRNA level in CRLM discriminating early from late or no relapse after partial hepatectomy

Progressive APOBEC3B mRNA expression in distant breast cancer metastases

__Background:__ APOBEC3B was recently identified as a gain-of-function enzymatic source of mutagenesis, which may offer novel therapeutic options with molecules that specifically target this enzyme. In primary breast cancer, APOBEC3B mRNA is deregulated in a substantial proportion of cases and its expression is associated with poor prognosis. However, its expression in breast cancer metastases, which are the main causes of breast cancer-related death, remained to be elucidated. __Patients and methods:__ RNA was isolated from 55 primary breast cancers and paired metastases, including regional lymph node (N = 20) and distant metastases (N = 35). APOBEC3B mRNA levels were measured by RT-qPCR. Expression levels of the primary tumors and corresponding metastases were compared, including subgroup analysis by estrogen receptor (ER/ESR1) status. __Results:__ Overall, APOBEC3B mRNA levels of distant metastases were significantly higher as compared to the corresponding primary breast tumor (P = 0.0015), an effect that was not seen for loco-regional lymph node metastases (P = 0.23). Subgroup analysis by ER-status showed that increased APOBEC3B levels in distant metastases were restricted to metastases arising from ER-positive primary breast cancers (P = 0.002). However, regarding ERnegative primary tumors, only loco-regional lymph node metastases showed increased APOBEC3B expression when compared to the corresponding primary tumor (P = 0.028). __Conclusion:__ APOBEC3B mRNA levels are significantly higher in breast cancer metastases as compared to the corresponding ER-positive primary tumors. This suggests a potential role for APOBEC3B in luminal breast cancer progression, and conse

Generation of in situ sequencing based OncoMaps to spatially resolve gene expression profiles of diagnostic and prognostic markers in breast cancer

Background: Gene expression analysis of breast cancer largely relies on homogenized tissue samples. Due to the high degree of cellular and molecular heterogeneity of tumor tissues, bulk tissue-based analytical approaches can only provide very limited system-level information about different signaling mechanisms and cellular interactions within the complex tissue context. Methods: We describe an analytical approach using in situ sequencing (ISS), enabling highly multiplexed, spatially and morphologically resolved gene expression profiling. Ninety-one genes including prognostic and predictive marker profiles, as well as genes involved in specific cellular pathways were mapped within whole breast cancer tissue sections, covering luminal A/B-like, HER2-positive and triple negative tumors. Finally, all these features were combined and assembled into a molecular-morphological OncoMap for each tumor tissue. Findings: Our in situ approach spatially revealed intratumoral heterogeneity with regard to tumor subtype as well as to the OncotypeDX recurrence score and even uncovered areas of minor cellular subpopulations. Since ISS-resolved molecular profiles are linked to their histological context, a deeper analysis of the core and periphery of tumor foci enabled identification of specific gene expression patterns associated with these morphologically relevant regions. Interpretation: ISS generated OncoMaps represent useful tools to extend our general understanding of the biological processes behind tumor progression and can further support the identification of novel therapeutical targets as well as refine tumor diagnostics. Fund: Swedish Cancerfonden, UCAN, Vetenskapsrådet, Cancer Genomics Netherlands, Iris, Stig och Gerry Castenbäcks Stiftelse, BRECT, PCM Program, King Gustaf V Jubilee Fund, BRO, KI and Stockholm County Council, Alice Wallenberg Foundation

BCAR4 induces antioestrogen resistance but sensitises breast cancer to lapatinib

Background: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression. Methods :Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease.Results:BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group. Conclusion :This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy