Karen Ege Olsen

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.

The value of mediastinal staging with endobronchial ultrasound-guided transbronchial needle aspiration in patients with lung cancer

OBJECTIVE To evaluate the diagnostic yield, the learning curve and the safety of endobronchial ultrasound-guided transbronchial needle biopsy (EBUS-TBNA) in mediastinal staging of patients with lung cancer. METHODS Mediastinal staging was performed with EBUS-TBNA according to the Danish national guidelines in patients fulfilling one or more of the following criteria: (1) central tumour; (2) enlarged (>10 mm) mediastinal lymph nodes on computed tomography; or (3) positron emission tomography (PET)-positive mediastinal lymph nodes. The study period began in January 2006 when EBUS-TBNA was introduced in the department and ended in December 2007. All records were reviewed retrospectively. None of the four examiners had any previous experience with EBUS-TBNA or ultrasound when the study began. All examinations were performed under general anaesthesia. Patients without useful cytological material from the EBUS-TBNA were subjected to a supplementary standard cervical mediastinoscopy if the mediastinal lymph nodes were found to be enlarged (>10 mm), PET positive or if the examiner was insecure of the result of the EBUS-TBNA. Patients with mediastinal lymph node involvement, detected by EBUS-TBNA or standard cervical mediastinoscopy, were referred to oncological treatment, while those without mediastinal lymph node involvement underwent--if they were otherwise eligible for surgery--resection and systematic lymph node sampling either by thoracotomy or by video-assisted thoracoscopy. Final mediastinal staging was defined as positive if mediastinal lymph node involvement was detected by EBUS-TBNA, standard cervical mediastinoscopy or surgery, or defined as negative otherwise. RESULTS A total of 157 patients were included in the study. N2/N3 disease was found in 67 patients (42.6%). EBUS-TBNA missed the mediastinal spread in 10 patients. Five of the ten patients had lymph node metastases in station 5, 6 or 8--out of reach of EBUS-TBNA or standard cervical mediastinoscopy. EBUS-TBNA had a sensitivity of 0.85 (0.74-0.93) and a negative predictive value of 0.90 (0.82-0.95). No complications occurred from EBUS-TBNA. The number of supplementary standard cervical mediastinoscopies decreased significantly in the study period. CONCLUSION The results of this study suggest that staging of the mediastinum with EBUS-TBNA is safe and easy to learn--even without previous experience with ultrasound. The diagnostic yield of EBUS-TBNA is in accordance with the yield of standard cervical mediastinoscopy reported in the literature. We do not find any indications in the present study of the recommended necessity for mediastinoscopy in all EBUS-TBNA-negative patients.

Handling of radical prostatectomy specimens: total or partial embedding?

Vainer B, Toft B G, Olsen K E, Jacobsen G K & Marcussen N
(2011) Histopathology58, 211–216
Handling of radical prostatectomy specimens: total or partial embedding?

Occupational Asbestos Exposure and Lung Cancer—A Systematic Review of the Literature

ABSTRACT The objective of this study was to evaluate the scientific literature concerning asbestos and lung cancer, emphasizing low-level exposure. A literature search in PubMed and Embase resulted in 5,864 citations. Information from included studies was extracted using SIGN. Twenty-one statements were evidence graded. The results show that histology and location are not helpful in differentiating asbestos-related lung cancer. Pleural plaques, asbestos bodies, or asbestos fibers are useful as markers of asbestos exposure. The interaction between asbestos and smoking regarding lung cancer risk is between additive and multiplicative. The findings indicate that the association between asbestos exposure and lung cancer risk is basically linear, but may level off at very high exposures. The relative risk for lung cancer increases between 1% and 4% per fiber-year (f-y)/mL, corresponding to a doubling of risk at 25–100 f-y/mL. However, one high-quality case-control study showed a doubling at 4 f-y/mL.

Quantitative PCR – new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real‐time PCR in comparison with immunohistochemistry and fluorescence in situ hybridization

Previously, polymerase chain reaction (PCR) technology has been hampered by its inability to generate quantitative results, a drawback inherent to the high degree of amplification taking place in the reaction. Recently, PCR techniques have been described with the potential of quantifying the amount of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2, and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real‐time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Tumor biopsies were collected from 112 patients diagnosed with early breast cancer from January 1990 to March 1994. The samples were analyzed for HER2 DNA amplification by real‐time PCR on LightCycler and by FISH and for HER2 protein expression by IHC. Inter‐assay variation for HER2 measured by LightCycler was 10% (×=3.1; n=17). Amplification ≥2 was observed in 19% of the patients. Concordance rates between real‐time PCR and the other methods were 91% (IHC) and 92% (FISH). The correlation between real‐time PCR and FISH was highly significant (p<0.001). The “LightCycler‐HER2/neu DNA quantification kit” produces results with a high level of reproducibility and its ease of use allows rapid screening for amplification of HER2. In this paper useful information is given on how real‐time PCR compares with FISH and IHC. The data show that results obtained for amplification of HER2 by real‐time PCR on the LightCycler instrument are comparable to results obtained by IHC and FISH.

Pregnancy associated plasma protein-A (PAPP-A) is not a marker of the vulnerable atherosclerotic plaque

OBJECTIVE To investigate if pregnancy associated plasma protein-A (PAPP-A) was present in the vulnerable plaque, and if not, to find alternative hypothesis for the release of PAPP-A. DESIGN AND METHODS Vulnerable plaques and control tissues were examined by immunohistochemistry. Volunteers and patients with non-atherosclerotic disease were examined for release of PAPP-A during ischemia and medical treatment. Non-atherosclerotic tissue samples were examined after incubation with heparins. RESULTS We were not able to detect PAPP-A in vulnerable plaques. Patients and volunteers experiencing ischemic events without atherosclerotic lesions only had elevated PAPP-A when treated with heparin. When tissue from normal artery wall was incubated with heparin, PAPP-A was eluted. This was not the case for non-arterial tissue samples. CONCLUSION Elevation of PAPP-A in patients with acute coronary syndromes seems to be caused by heparin induced release of PAPP-A from the arterial wall and not due to excretion from vulnerable plaques.

A comparison among HER2, TP53, PAI-1, angiogenesis, and proliferation activity as prognostic variables in tumours from 408 patients diagnosed with early breast cancer.

Background. The prognostic potential of HER2, TP53 mutations, PAI-1 protein levels, angiogenesis and proliferation were investigated in tumours from 408 patients with early breast cancer followed >10 years. One hundred and sixty seven patients (41%) died from breast cancer. Materials and methods. Tumour sections were stained for HER2, CD34, and MIB-1. HER2 scores were based on staining intensity, 3+ being considered HER2+. Angiogenesis was scored by the Chalkley method. MIB-1 was evaluated using systematic random sampling. PAI-1 was measured by ELISA. TP53 mutations were evaluated by DGGE analysis and DNA sequencing. Results. Ninety one patients (22%) were HER2 positive. TP53 was mutated in 101 cases (25%). Median PAI-1, Chalkley and MIB-1 was 0.72 ng/mg protein (range, 0–90 ng/mg protein), 5.00 (range, 2.67–12.00) and 15% (range, 1–83%). MIB-1 was correlated with HER2+, Chalkley counts, TP53 mutations (all p <0.0001), and PAI-1 (p =0.002). In univariate analyses with DSS as endpoint, HER2+ (p <0.0001), mutated TP53 (p <0.0001), high Chalkley (p =0.008), MIB-1 (p =0.002), tumour size (p =0.008), grade (p <0.0001), negative estrogen receptor (p =0.0001), and lymph node status (p <0.0001) were prognostic markers. Among node-negative patients, HER2+ (p =0.0002), mutated TP53 (p =0.001), high PAI-1 levels (p =0.02), and grade (p =0.03) indicated poor DSS. In node-positive patients, HER2+ (p =0.0002), mutated TP53 (p <0.0001), MIB-1 (p =0.01), Chalkley scores (p =0.007), negative estrogen receptor (p <0.0001) and grade (p =0.001) indicated poor prognosis. In multivariate analysis, metastatic nodes (1–3 positive: RR 1.56 95% CI 1.02–2.38; >3 positive: RR 3.70 95% CI 2.54–5.38), HER2+ (RR 1.91, 95% CI 1.35–2.70), mutated TP53 (RR 1.70, 95% CI 1.21–2.38), PAI-1 (RR 1.04, 95% CI 1.01–1.07) and grade 3 (RR 1.96, 95% CI 1.83–3.22) were independent markers of poor outcome. Conclusion. Compared to PAI-1 protein levels, Chalkley counts and MIB-1, HER2+ and mutations of TP53 were the strongest independent markers of poor prognosis irrespective of nodal status.

Sentinel Node Localization in Breast Cancer Patients Using Intradermal Dye Injection

In a series of 161 consecutive breast cancer operations, intradermal injection of Patent Blue was used to localize the sentinel node (SN). The surgical localization rate was 60%. Including the blue lymph nodes found by the pathologist, localization rate was 70%. After the first 103 operations, the surgical procedure was changed, resulting in a localization rate of 83%. Ten surgeons participated, but only one had previous experience with SN dissection. The others experienced a steep learning curve. Metastasis was found in 42 of 97 SNs (43%). In 15 cases (36%) metastasis was recognized only after step-sectioning and immunohistochemical staining for cytokeratin. In one case a benign epithelial inclusion was found. The sentinel node was false negative in 9.1% of cases. The consensus from the literature is that the best results are achieved using a combination of dye and isotopic techniques.

Endobronchial ultrasound-guided transbronchial needle aspiration of undiagnosed intrathoracic lesions

Endobronchial ultrasound-guided transbronchial fine-needle aspiration (EBUS-FNA) is a minimally invasive method used routinely for mediastinal staging of patients with lung cancer. We have used it in 135 consecutive patients with a radiologically suspicious intrathoracic lesion that remained undiagnosed despite bronchoscopy and CT-guided fine-needle aspiration (CT-FNA). There was no operative mortality or surgical complications. In 98 patients with a suspicious lesion in the lung parenchyma, adequate tissue was obtained in 83 patients (85%) and in 37 patients with enlarged lymph nodes or a mediastinal tumor adequate tissue was obtained in 35 cases (95%). However, a final diagnosis was only reached in 45% of the patients and further investigations led to malignancy in 13. We believe that EBUS-FNA represents a good alternative to more invasive diagnostic procedures when conventional methods fail, even though the diagnostic yield is lower compared with mediastinal staging in patients with known lung cancer. In almost half of the cases, EBUS-FNA provides the final diagnosis without exposing the patient to the risk of complications from more invasive procedures.