Quantification of multiple steroid hormones in serum and human breast cancer tissue by liquid chromatography-tandem mass spectrometry analysis.

Introduction: Systemic and local steroid hormone levels may function as novel prognostic and predictive biomarkers in breast cancer patients. We aimed at developing a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of multiple, biologically pivotal steroid hormones in human serum and breast cancer tissue. Methods: The quantitative method consisted of liquid-liquid extraction, Sephadex LH-20 chromatography for tissue extracts, and analysis of steroid hormones by liquid-chromatography-tandem mass spectrometry. We analyzed serum and tissue steroid hormone levels in 16 and 40 breast cancer patients, respectively, and assessed their correlations with clinical parameters. Results: The method included quantification of nine steroid hormones in serum [including cortisol, cortisone, corticosterone, estrone (E1), 17ß-estradiol (E2), 17α-hydroxyprogesterone, androstenedione (A4), testosterone and progesterone) and six (including cortisone, corticosterone, E1, E2, A4, and testosterone) in cancer tissue. The lower limits of quantification were between 0.003-10 ng/ml for serum (250 µl) and 0.038-125 pg/mg for tissue (20 mg), respectively. Accuracy was between 98%-126%, intra-assay coefficient of variations (CV) was below 15%, and inter-assay CV were below 11%. The analytical recoveries for tissue were between 76%-110%. Tissue levels of E1 were positively correlated with tissue E2 levels (p<0.001), and with serum levels of E1, E2 and A4 (p<0.01). Tissue E2 levels were positively associated with serum E1 levels (p=0.02), but not with serum E2 levels (p=0.12). The levels of tissue E2 and ratios of E1 to A4 levels (an index for aromatase activity) were significantly higher in patients with larger tumors (p=0.03 and p=0.02, respectively). Conclusions: The method was convenient and suitable for a specific and accurate profiling of clinically important steroid hormones in serum. However, the sensitivity of the profile method in steroid analysis in tissue samples is limited, but it can be used for the analysis of steroids in breast cancer tissues if the size of the sample or its steroid content is sufficient.

Association of serum cortisol and cortisone levels and risk of recurrence after endocrine treatment in breast cancer.

Metabolic reprogramming in breast cancer involves changes in steroid hormone synthesis and metabolism. Alterations in estrogen levels in both breast tissue and blood may influence carcinogenesis, breast cancer growth, and response to therapy. Our aim was to examine whether serum steroid hormone concentrations could predict the risk of recurrence and treatment-related fatigue in patients with breast cancer. This study included 66 postmenopausal patients with estrogen receptor-positive breast cancer who underwent surgery, radiotherapy, and adjuvant endocrine treatment. Serum samples were collected at six different time points [before the start of radiotherapy (as baseline), immediately after radiotherapy, and then 3, 6, 12 months, and 7-12 years after radiotherapy]. Serum concentrations of eight steroid hormones (cortisol, cortisone, 17α-hydroxyprogesterone, 17ß-estradiol, estrone, androstenedione, testosterone, and progesterone) were measured using a liquid chromatography-tandem mass spectrometry-based method. Breast cancer recurrence was defined as clinically proven relapse/metastatic breast cancer or breast cancer-related death. Fatigue was assessed with the QLQ-C30 questionnaire. Serum steroid hormone concentrations measured before and immediately after radiotherapy differed between relapse and relapse-free patients [(accuracy 68.1%, p = 0.02, and 63.2%, p = 0.03, respectively, partial least squares discriminant analysis (PLS-DA)]. Baseline cortisol levels were lower in patients who relapsed than in those who did not (p < 0.05). The Kaplan-Meier analysis showed that patients with high baseline concentrations of cortisol (≥ median) had a significantly lower risk of breast cancer recurrence than patients with low cortisol levels (

The association of women's birth size with risk of molecular breast cancer subtypes: a cohort study.

BACKGROUND: Because birth size appears to be positively associated with breast cancer risk, we have studied whether this risk may differ according to molecular breast cancer subtypes. METHODS: A cohort of 22,931 women born 1920-1966 were followed up for breast cancer occurrence from 1961 to 2012, and 870 were diagnosed during follow-up. Archival diagnostic material from 537 patients was available to determine molecular breast cancer subtype, specified as Luminal A, Luminal B (human epidermal growth factor receptor 2 (HER2)-), Luminal B (HER2+), HER2 type, and Triple negative (TN) breast cancer. Information on the women's birth weight, birth length and head circumference at birth was used to estimate hazard ratios (HR) with 95% confidence intervals (CI) for each molecular subtype, applying Cox regression, and stratified by maternal height. RESULTS: Birth length (per 2 cm increments) was positively associated with Luminal A (HR = 1.2, 95% CI, 1.0-1.3), Luminal B (HER2+) (HR = 1.3, 95% CI, 1.0-1.7), and TN breast cancer (HR = 1.4, 95% CI, 1.0-1.9). No clear association was found for birth weight and head circumference. The positive associations of birth length were restricted to women whose mothers were relatively tall (above population median). CONCLUSION: We found a positive association of birth length with risk of Luminal A, Luminal B (HER2+) and TN breast cancer that appears to be restricted to women whose mothers were relatively tall. This may support the hypothesis that breast cancer risk is influenced by determinants of longitudinal growth and that this finding deserves further scrutiny.

Characterization of FGD5 Expression in Primary Breast Cancers and Lymph Node Metastases.

Faciogenital dysplasia 5 ( FGD5) amplification drives tumor cell proliferation, and is present in 9.5% of breast cancers. We describe FGD5 expression, assess associations between FGD5 amplification and FGD5 expression, and assess FGD5 expression in relation to proliferation and prognosis. FGD5 immunohistochemistry was done on primary tumors ( n=829) and lymph node metastases ( n=231) from a cohort of Norwegian patients. We explored associations between FGD5 amplification, FGD5 expression, and proliferation, and analyzed the prognostic value of FGD5 expression by estimating cumulative risks of death and hazard ratios (HRs). We identified nuclear and cytoplasmic expression in 64% and 73% of primary tumors, respectively, and found an association between gene amplification and nuclear expression ( p=0.02). The proportion of cases with FGD5 expression was higher in lymph node metastases, compared with primary tumors ( p=0.004 for nuclear and p=0.001 for cytoplasmic staining). Neither proliferation nor prognosis was associated with FGD5 expression (age-adjusted HR 1.12 [95% confidence interval = 0.89-1.41] for nuclear expression; and 0.88 [95% CI = 0.70-1.12] for cytoplasmic expression). FGD5 is expressed in a high proportion of breast cancers and lymph node metastases. There was a correlation between FGD5 amplification and nuclear expression, but no association between FGD5 expression and proliferation or prognosis.

Basal markers and prognosis in luminal breast cancer.

PURPOSE: Basal marker expression in triple-negative breast cancers identifies basal-like tumours, and thus separates the TN group into two prognostic groups. However, the expression and prognostic significance of basal markers in luminal breast cancers are poorly described. The aim of this study was to investigate the expression and prognostic value of basal markers (CK5, CK14 and EGFR) in luminal breast cancer. METHODS: A total of 1423 formalin-fixed, paraffin-embedded breast cancer tumours from a well-characterized cohort of Norwegian women, previously reclassified into molecular subtypes using IHC and ISH, were included in the study. For the present study, tumours expressing at least one of the basal markers CK5, CK14 or EGFR were defined as basal marker positive. Cumulative incidence of death from breast cancer and hazard ratio analyses were used to assess prognosis according to basal marker expression. RESULTS AND CONCLUSION: In total, 470 cases (33.0%) were basal marker positive. A higher proportion of the basal marker-positive tumours were of histopathological grade 3 compared to basal marker negative. For hormone receptor-positive, HER2-negative cases, we found better prognosis for basal marker-positive breast cancer compared to basal marker negative. For all subtypes combined, poorer prognosis for basal marker-negative cases was found in histopathological grade 2 tumours but not among grade 1 and 3.

HER2 gene copy number and breast cancer-specific survival.

AIMS: HER2 amplification occurs in 10-15% of breast cancers. It is associated with poor breast cancer-specific survival (BCSS) and is an important prognostic and predictive marker. While it has been accepted that the HER2:chromosome 17 centromere (CEP17) ratio determines HER2 status, recent guidelines acknowledge the significance of HER2 copy number alone. The aims of this study were to assess BCSS according to mean HER2 copy number and HER2 status expressed as a HER2:CEP17 ratio with and without increased CEP17 copy number. METHODS AND RESULTS: The study population comprised breast cancer patients treated with surgery only and with long-term follow-up. In situ hybridization for HER2:CEP17 was performed on tissue microarrays and was successful in 679 cases. These were included in the study. Kaplan-Meier methods were used to estimate BCSS. A total of 47 cases had ≥4 < 6 HER2 copies; 16 were HER2+ and 31 were HER2- by ratio. Eighty-five cases had ≥6 copies of HER2 and only two of these were HER2- by ratio. The risk of death from breast cancer was increased among those with ≥6 HER2 compared to cases with 0-3.9 HER2 signals [hazard ratio (HR): 2.05; confidence interval (CI): 1.49-2.82 (unadjusted)]. After adjusting for stage, there was increased risk of death from breast cancer during the first 5 years after diagnosis in cases that were HER2- by ratio but with ≥4 < 6 HER2 (HR: 2.38; CI: 1.23-4.60). CONCLUSIONS: Increased copy number of HER2 may confer an increased risk of death from breast cancer despite negative HER2 status by ratio.

Invasive lobular breast cancer: the prognostic impact of histopathological grade, E-cadherin and molecular subtypes.

AIMS: The aim of this study was to compare breast cancer specific survival (BCSS) for invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) and, further, to evaluate critically the prognostic value of histopathological grading of ILC and examine E-cadherin as a prognostic marker in ILC. METHODS AND RESULTS: The study comprised 116 lobular and 611 ductal breast carcinomas occurring between 1961 and 2008. All cases had been classified previously according to histopathological type and grade, stained for oestrogen receptor (ER), progesterone receptor (PR), antigen Ki67 (Ki67), epithelial growth factor receptor (EGFR), cytokeratin 5 (CK5) and human epidermal growth factor receptor 2 (HER2) and classified into molecular subtypes. For the present study, immunohistochemical staining for E-cadherin was performed. The Kaplan-Meier method and Cox proportional hazards models were used in the analyses. Grade 2 tumours comprised 85.3% of the lobular tumours and 51.9% of the ductal tumours. BCSS in ILC grade 2 was comparable to that of IDC grade 3. E-cadherin-negative ILC had a poorer prognosis compared to E-cadherin positive ILC and to IDC regardless of E-cadherin status. CONCLUSIONS: The implication of histopathological grading may differ in ILC compared to IDC. E-cadherin may be useful in prognostication in ILC and thereby influence the determination of treatment strategies for this group of women.

Alterations in collagen fibre patterns in breast cancer. A premise for tumour invasiveness?

Stromal tissue in the breast plays a key role in cancer invasiveness due to molecular and cellular changes. Collagen is the main component of the stroma. The purposes of this study were to investigate differences in collagen fibre patterns between tumour-induced stromal tissue and normal stroma, and between high-grade and low-grade breast cancer stroma, using second harmonic generation microscopy. Thirty-seven ductal carcinomas were examined: Twenty-one Luminal A phenotype and sixteen HER2 or Basal-like phenotype. Three regions were examined in each case: intratumoral, juxtatumoral and extratumoral. Two images were captured in each region. Two characteristics of collagen fibres were examined: the degree of straightness, and the degree of alignment. Collagen fibres were visually classified as curly, intermediate or straight, and as parallel or not parallel. The results of angle measurement and visual analysis showed that collagen fibres were straightest in the intratumoral region and curliest in the extratumoral region. Collagen fibres were more parallel in the juxtatumoral region compared to the two other regions. There were no significant differences between high-grade and low-grade tumours. As a breast tumour progresses, collagen fibres appear to straighten and align at the tumour boundary. This could facilitate invasion of the tumour into the surrounding stroma.

Reproductive history and the risk of molecular breast cancer subtypes in a prospective study of Norwegian women.

PURPOSE: Breast cancer can be classified into molecular subtypes that differ in clinical characteristics and prognosis. There is some but conflicting evidence that reproductive risk factors may differ between distinct breast cancer subtypes. METHODS: We investigated associations of reproductive factors with the risk for six molecular breast cancer subtypes in a cohort of 21,532 Norwegian women who were born between 1886 and 1928 and followed up for breast cancer incidence between 1961 and 2008. We obtained stored tumor tissue from incident breast cancers and used immunohistochemistry and in situ hybridization to classify 825 invasive tumors into three luminal subtypes [Luminal A, Luminal B (HER2-) and Luminal B (HER2+)] and three non-luminal subtypes [human epidermal growth factor receptor 2 (HER2) subtype, basal-like phenotype (BP) and five negative phenotype (5NP)]. We used Cox regression to assess reproductive factors and risk for each subtype. RESULTS: We found that young age at menarche, old age at first birth and low parity were associated with increased risk for luminal breast cancer subtypes. For the HER2 subtype, we either found no association or associations in the opposite direction compared to the luminal subtypes. The BP subtype appeared to have a similar reproductive risk profile as the luminal subtypes. Breastfeeding was associated with a reduced risk for HER2 and 5NP subtypes, but was not associated with any other subtype. CONCLUSIONS: The results suggest that molecular breast cancer subtypes differ in their reproductive risk factors, but associations with non-luminal subtypes are still poorly understood and warrant further study.

Anthropometric factors and risk of molecular breast cancer subtypes among postmenopausal Norwegian women.

Adult height and body weight are positively associated with breast cancer risk after menopause, but few studies have investigated these factors according to molecular breast cancer subtype. A total of 18,562 postmenopausal Norwegian women who were born between 1886 and 1928 were followed up for breast cancer incidence from the time (between 1963 and 1975) height and weight were measured until 2008. Immunohistochemical and in situ hybridization techniques were used to subtype 734 incident breast cancer cases into Luminal A, Luminal B [human epidermal growth factor receptor 2 (HER2-)], Luminal B (HER2+), HER2 subtype, basal-like phenotype (BP) and five-negative phenotype (5NP). We used Cox regression analysis to assess adult height and body mass index (BMI) in relation to risk of these subtypes. We found a positive association of height with risk of Luminal A breast cancer (ptrend , 0.004), but there was no clear association of height with any other subtype. BMI was positively associated with risk of all luminal breast cancer subtypes, including Luminal A (ptrend , 0.002), Luminal B (HER2-) (ptrend , 0.02), Luminal B (HER2+) (ptrend , 0.06), and also for the HER2 subtype (ptrend , 0.04), but BMI was not associated with risk of the BP or 5NP subtypes. Nonetheless, statistical tests for heterogeneity did not provide evidence that associations of height and BMI differed across breast cancer subtypes. This study of breast cancer risk among postmenopausal women suggests that height is positively associated with risk of Luminal A breast cancer. BMI is positively associated with risk of all luminal subtypes and for the HER2 subtype.