Eva Balslev

Retrospective Analysis of Topoisomerase IIa Amplifications and Deletions As Predictive Markers in Primary Breast Cancer Patients Randomly Assigned to Cyclophosphamide, Methotrexate, and Fluorouracil or Cyclophosphamide, Epirubicin, and Fluorouracil: Danish Breast Cancer Cooperative Group

PURPOSE The aim of the study was to evaluate the predictive value of HER2 and topoisomerase IIalpha gene (TOP2A) for the efficacy of epirubicin in the adjuvant setting of breast cancer patients. PATIENTS AND METHODS In the Danish Breast Cancer Cooperative Group trial 89D, 980 pre- and postmenopausal primary patients were randomly allocated to either CMF (cyclophosphamide, methotrexate, and fluorouracil; n = 500) or CEF (cyclophosphamide, epirubicin, and fluorouracil; n = 480) times 9, between January 1990 and November 1999. Tumor tissue was retrospectively identified from 805 patients and was analyzed for HER2-positivity and for TOP2A-amplifications and deletions. RESULTS HER2-positivity was found in 33% of the 805 investigated tumors and was not a predictive marker for epirubicin sensitivity. TOP2A changes were identified in 23% of the 773 investigated tumors: 12% had TOP2A amplifications and 11% had TOP2A deletions. We found that patients with TOP2A amplification had an increased recurrence-free (RFS; hazard ratio [HR], 0.43; 95% CI, 0.24 to 0.78) and overall survival (OS; HR, 0.57; 95% CI, 0.29 to 1.13), respectively if treated with CEF compared with CMF, and that patients with TOP2A deletions had an almost identical hazard ratio (RFS: HR, 0.63; 95% CI, 0.36 to 1.11; OS: HR, 0.56; 95% CI, 0.30 to 1.04). This is in contrast to patients with a normal TOP2A genotype for whom similar outcome was observed in both treatment arms (RFS: HR, 0.90; 95% CI, 0.70 to 1.17; OS: HR, 0.88; 95% CI, 0.66 to 1.17). CONCLUSION TOP2A amplification-and possibly deletion-seems to be predictive markers for the effect of adjuvant epirubicin containing therapy in primary breast cancer, but a final conclusion has to await a confirmative study or a meta-analysis.

Amplification of HER2 and TOP2A and deletion of TOP2A genes in breast cancer investigated by new FISH probes

New strategies for improving treatment of patients with breast carcinoma have focused on the HER2 oncoprotein with regard to response to traditional therapy regimes and the effect of a new drug specifically directed against the protein. Furthermore, the status of the topoisomerase IIα (TOP2A) gene has been suggested as a predictive marker of anthracycline treatment. In this study of 120 tumours, immunohistochemically detected HER2 overexpression with HercepTest™ has been compared to the HER2 gene amplification investigated with a new HER2 probe for fluorescence in situ hybridization (FISH). In addition, the HercepTest was evaluated as a screening tool for choosing cases for FISH investigation of TOP2A gene aberrations. The HercepTest score 3+ identified HER2 gene amplification in 27 of 30 amplified tumours (sensitivity of 0.90) with a false-negative rate of 0.10 and a false-positive rate of 0.06. TOP2A gene amplification or deletion was found in 20 cases. Sixteen (80%) of these carcinomas were in the HercepTest 3+ group, but four tumours had alterations in the TOP2A gene with normal HER2 status. Traditionally, in the FISH technique the result has been based on counting 60 cells. However, we found that a much less time-consuming method of counting 60 signals gave equally good results.

HER2, TOP2A, and TIMP-1 and Responsiveness to Adjuvant Anthracycline-Containing Chemotherapy in High-Risk Breast Cancer Patients

PURPOSE To evaluate whether the combination of HER2 with TIMP-1 (HT) or TOP2A with TIMP-1 (2T) more accurately identifies patients who benefit from cyclophosphamide, epirubicin, and fluorouracil (CEF) compared with cyclophosphamide, methotrexate, and fluorouracil (CMF) than these markers do when analyzed individually. PATIENTS AND METHODS The Danish Breast Cancer Cooperative Group (DBCG) 89D trial randomly assigned 980 high-risk Danish breast cancer patients to CMF or CEF. Archival tumor tissue was analyzed TIMP-1, and HER2-negative and TIMP-1 immunoreactive tumors were classified as HT nonresponsive and otherwise HT responsive. Similarly, the 2T panel was constructed by combining TOP2A and TIMP-1; tumors with normal TOP2A status and TIMP-1 immunoreactivity were classified as 2T-nonresponsive and otherwise 2T-responsive. Results In total, 623 tumors were available for analysis, of which 154 lacked TIMP-1 immunoreactivity, 188 were HER2 positive, and 139 had a TOP2A aberration. HT status was a statistically significant predictor of benefit from CEF compared with CMF (P(interaction) = .036 for invasive disease-free survival [IDFS] and .047 for overall survival [OS]). The 269 (43%) patients with a 2T-responsive profile had a significant reduction in IDFS events (adjusted hazard ratio, 0.48; 95% CI, 0.34 to 0.69; P < .001) and OS events (adjusted hazard ratio, 0.54; 95% CI, 0.38 to 0.77; P < .001). 2T status was a highly significant predictor of benefit from CEF compared with CMF (P(interaction) < .0001 for IDFS and .004 for OS). CONCLUSION The 2T profile is a more accurate predictor of incremental benefit from anthracycline-containing chemotherapy than HER2, TIMP-1, or TOP2A individually, and compared with these, 2T classifies a larger proportion of patients as sensitive to anthracyclines.

High expression of miR-21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer

Rask L, Balslev E, Jørgensen S, Eriksen J, Flyger H, Møller S, Høgdall E, Litman T, Nielsen BS. High expression of miR‐21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer. APMIS 2011; 119: 663–73.

High serum levels of YKL-40 in patients with squamous cell carcinoma of the head and neck are associated with short survival

YKL‐40 is a glycoprotein secreted by macrophages, neutrophils and malignant tumor cells. Elevated serum levels of YKL‐40 are associated with poor prognosis in several malignancies. In this study, we examined the prognostic value of serum YKL‐40 before treatment and during follow‐up in patients with squamous cell carcinoma of the head and neck (HNSCC). YKL‐40 was determined by ELISA retrospectively in serum from 173 patients with primary HNSCC before treatment and up to 2 years after treatment. Median follow‐up time was 7.9 years. YKL‐40 protein expression in tumor biopsies was assessed by immunohistochemistry in 50 patients. Pretreatment serum YKL‐40 was elevated in 53%. Patients with high serum YKL‐40 had shorter survival than patients with normal serum YKL‐40 (33 vs. 84 months; p = 0.008). Multivariate Cox analysis including pretreatment serum YKL‐40, age, sex, primary tumor site, TNM classification and treatment demonstrated that TNM classification (HR = 2.61, p = 0.02) and serum YKL‐40 (log‐transformed continuous variable: HR = 1.55, p < 0.0001) were independent prognostic variables of overall survival (OS). Multivariate Cox analysis demonstrated that TNM classification (HR = 5.77, p = 0.001) and serum YKL‐40 (dichotomous variable: HR = 2.75, p = 0.01) were independent predictors of recurrence‐free survival. During follow‐up after radiotherapy, a high serum YKL‐40 (log‐transformed continuous variable) in patients with TNM Stage III and IV disease predicted poorer OS within 6 months (HR = 1.95, p < 0.0001). Immunohistochemical analysis showed YKL‐40 expression in the malignant tumor cells. In conclusion, serum YKL‐40 was demonstrated to be an independent prognostic biomarker of recurrence‐free and overall survival in patients with HNSCC.

US-guided fine needle aspiration versus coarse needle biopsy of thyroid nodules.

OBJECTIVE The aim of this study was retrospectively to evaluate ultrasound (US) guided fine-needle aspiration (FNA), in combination with US-guided coarse-needle biopsies, (CNB) from solitary or dominant thyroid nodules routinely performed during a 2 year period. METHODS Seventy seven patients were biopsied using US-guided FNA and CNB. FNA was performed using a 21-Gauge needle and CNB using a 18-Gauge single action spring-activated needle biopsy system. The biopsies were performed with local anaesthesia. The Department of Pathology routinely examined the biopsy specimens. The retrieval rate in obtaining material for diagnostic evaluation was FNA (97%), CNB (88%), FNA and CNB (100%). RESULTS In all, 41 of the 77 patients underwent neck-surgery. The surgical specimens were used to determine the results of diagnosing neoplasia. The accuracy, sensitivity and specificity for FNA were 80, 83, and 77%. For CNB 86, 78, and 94%. For both FNA and CNB 80, 89 and 73%. The diagnostic value of the two methods showed no significant difference (P < 0.05). CNB revealed contrary to FNA, however, one additional cancer. Also a higher number of false positive findings was noticed using FNA. No serious complications were registered. Adequate biopsies were obtained in all the patients using the combination of US-guided FNA and CNB. No patient underwent rebiopsy. CONCLUSIONS The study demonstrated that neither US-guided CNB nor the combination of US-guided FNA and CNB were superior to US-guided FNA. US-guided CNB is only recommended in few selected patients.

Identification and analysis of miRNAs in human breast cancer and teratoma samples using deep sequencing

BackgroundMiRNAs play important roles in cellular control and in various disease states such as cancers, where they may serve as markers or possibly even therapeutics. Identifying the whole repertoire of miRNAs and understanding their expression patterns is therefore an important goal.MethodsHere we describe the analysis of 454 pyrosequencing of small RNA from four different tissues: Breast cancer, normal adjacent breast, and two teratoma cell lines. We developed a pipeline for identifying new miRNAs, emphasizing extracting and retaining as much data as possible from even noisy sequencing data. We investigated differential expression of miRNAs in the breast cancer and normal adjacent breast samples, and systematically examined the mature sequence end variability of miRNA compared to non-miRNA loci.ResultsWe identified five novel miRNAs, as well as two putative alternative precursors for known miRNAs. Several miRNAs were differentially expressed between the breast cancer and normal breast samples. The end variability was shown to be significantly different between miRNA and non-miRNA loci.ConclusionPyrosequencing of small RNAs, together with a computational pipeline, can be used to identify miRNAs in tumor and other tissues. Measures of miRNA end variability may in the future be incorporated into the discovery pipeline as a discriminatory feature. Breast cancer samples show a distinct miRNA expression profile compared to normal adjacent breast.

Sentinel node biopsy in breast cancer: Five years experience from Denmark

Introduction. Danish experience from the first five years with sentinel lymph node biopsy (SLNB) as a routine staging procedure in early breast cancer is reported. Methods. During the period January 1, 2002 to December 31, 2006, 14 923 patients were diagnosed at Danish breast surgical centers certified for the sentinel node method. SLNB was performed in 8 338 patients (55.9%). The fraction increased steadily from 43% in 2002 to 67% in 2006. The median follow-up was 1.7 year (range 0–5.2 years). Results. Patients staged with SLNB were younger, had more often BCS, had smaller tumor size, were more often hormone receptor positive, and had lower grade, than patients staged with lymph node dissection (ALND). Blue dye and radio colloid were used in combination in 82%. Lymphoscintigraphy was performed in 61%, and frozen section was performed in 87%. Originally, peritumoral injection of tracer was most often used, but the recommendations have changed, and in 2006 90% of cases had sub-or periareolar injection of radioactive tracer. In the sentinel nodes 25% had macrometastases, 17% micrometastases only, and 3.2% isolated tumor cells only (ITC). ALND was performed in 2 714 patients, whose lymph node classification by SN was known. In the group of 1 563 patients with macrometastases in SN, 45% had non-sentinel node metastases, and in the group of 942 patients with micrometastases only, 23% had more positive nodes. Regional lymph node metastases were found in 15% with ITC in sentinel nodes. Lymph node recurrence among node negative patients was observed more often after staging by SLNB (0.5%) than after ALND (0.2%, p =0.04). Conclusion. Two thirds of breast cancer patients can be safely staged with the sentinel node technique, half of these will need no further axillary surgery. The loco-regional control in node negative patients classified by SLNB is high, but seems not quite comparable to what is seen after ALND.

Differential expression of miR-139, miR-486 and miR-21 in breast cancer patients sub-classified according to lymph node status

PurposeTherapeutic decisions in breast cancer are increasingly guided by prognostic and predictive biomarkers. Non-protein-coding microRNAs (miRNAs) have recently been found to be deregulated in breast cancers and, in addition, to be correlated with several clinico-pathological features. One of the most consistently up-regulated miRNAs is miR-21. Here, we specifically searched for differentially expressed miRNAs in high-risk breast cancer patients as compared to low-risk breast cancer patients. In the same patients, we also compared miR-21 expression with the expression of its presumed target PTEN.MethodsBoth microarray and RT-qPCR techniques were used to assess miRNA expression levels in lymph node-positive and -negative human invasive ductal carcinoma tissues. Simultaneously, PTEN protein expression levels were assessed using immunohistochemistry.ResultsmiR-486-5p and miR-139-5p were found to be down-regulated in patients with lymph node metastases, whereas miR-21 was found to be up-regulated in patients with a positive lymph node status. miR-21 expression levels were found to significantly correlate with tumour size (r = 0.403, p = 0.009; Spearman’s rank), whereas no relation was found between miR-21 and PTEN expression levels (Kruskal-Wallis test).ConclusionDown-regulation of miR-486-5p and miR-139-5p, in conjunction with up-regulation of miR-21, may represent a useful signature for the identification of high-risk breast cancer patients.

Lack of TIMP-1 tumour cell immunoreactivity predicts effect of adjuvant anthracycline-based chemotherapy in patients (n = 647) with primary breast cancer. A Danish Breast Cancer Cooperative Group Study

PURPOSE A number of prospective studies have shown that adjuvant CEF significantly improves disease-free and overall survival as compared to CMF in breast cancer patients. Our aim was to determine whether the benefit of epirubicin versus methotrexate differs according to TIMP-1 tumour cell immunoreactivity. EXPERIMENTAL DESIGN Tissue micro arrays from 647 patients randomly assigned to CMF or CEF in DBCG trial 89D were included. The primary end-point was invasive disease-free survival (IDFS). A central assessment of tissue inhibitor of metalloproteinases 1 (TIMP-1) status was performed using immunohistochemistry (IHC). Tumours were regarded as TIMP-1 positive if epithelial breast cancer cells were stained using the anti-TIMP-1 monoclonal antibody VT7. RESULTS By central assessment 75% of tumours were classified as tumour cell TIMP-1 positive. Among CEF-treated patients, individuals with TIMP-1 negative tumours had a significant longer IDFS than patients with TIMP-1 positive tumours (p=0.047). The multivariate Cox regression analysis of IDFS showed that CEF was superior to CMF among patients with TIMP-1 negative tumours (hazard ratio (HR)=0.51; 95% confidence interval (CI): 0.31-0.84, p=0.0085), while no significant difference could be demonstrated among patients with TIMP-1 positive tumours (HR=0.88; 95% CI: 0.68-1.13, p=0.32). A non-significant TIMP-1 status (positive or negative) versus treatment (CMF or CEF) interaction was detected for IDFS (p=0.06) and OS (p=0.21). CONCLUSION Lack of TIMP-1 tumour cell immunoreactivity seems to predict a favourable effect of epirubicin-containing adjuvant therapy in primary breast cancer. However, an independent study is awaited to validate the potential predictive value of TIMP-1 immunoreactivity.