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1.  Guidance for laboratories performing molecular pathology for cancer patients 
Journal of Clinical Pathology  2014;67(11):923-931.
Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here.
PMCID: PMC4215286  PMID: 25012948
Molecular Pathology; Neoplasms; Quality Control; Laboratory Tests; Molecular Oncology
2.  An International Interpretation Study Using the ALK IHC Antibody D5F3 and a Sensitive Detection Kit Demonstrates High Concordance between ALK IHC and ALK FISH and between Evaluators 
The goal of personalized medicine is to treat patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or toxic therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK-positive non–small-cell lung cancer (NSCLC) tumors, but to date the only approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate a new automated standardized ALK IHC assay.
Archival NSCLC tumor specimens (n =103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc., Tucson, AZ). Specimens were scored binarily as positive if strong granular cytoplasmic brown staining was present in tumor cells. IHC results were compared with the FISH results and interevaluator comparisons made.
Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%), and accurate relative (93%) to the ALK FISH results. Similar results were observed using a majority score. IHC negativity was scored by seven of seven and six of seven evaluators on three and two FISH-positive cases, respectively. IHC positivity was scored on two FISH-negative cases by seven of seven readers. There was agreement among seven of seven and six of seven readers on 88% and 96% of the cases before review, respectively, and after review there was agreement among seven of seven and six of seven on 95% and 97% of the cases, respectively.
On the basis of expert evaluation the ALK IHC test is sensitive, specific, and accurate, and a majority score of multiple readers does not improve these results over an individual reader’s score. Excellent inter-reader agreement was observed. These data support the algorithmic use of ALK IHC in the evaluation of NSCLC.
PMCID: PMC4186652  PMID: 24722153
Non–small-cell lung cancer; Anaplastic lymphoma kinase; Immunohistochemistry; Fluorescence in situ hybridization; Companion diagnostics; Biomarkers; Crizotinib
3.  Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer 
Virchows Archiv  2014;465(4):409-417.
Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology.
PMCID: PMC4180906  PMID: 25218890
Breast cancer; Preanalytical; EndoPredict; Molecular pathology; Gene expression
4.  Gross cystic disease fluid protein 15 (GCDFP-15) expression in breast cancer subtypes 
BMC Cancer  2014;14(1):546.
Gross cystic disease fluid protein 15 (GCDFP-15), which is regulated by the androgen receptor (AR), is a diagnostic marker for mammary differentiation in histopathology. We determined the expression of GCDFP-15 in breast cancer subtypes, its potential prognostic and predictive value, as well as its relationship to AR expression.
602 pre-therapeutic breast cancer core biopsies from the phase III randomized neoadjuvant GeparTrio trial (NCT00544765) were investigated for GCDFP-15 expression by immunohistochemistry. Expression data were correlated with disease-free (DFS) and overall survival (OS) time as well as pathological complete response (pCR) to neoadjuvant chemotherapy.
239 tumors (39.7%) were GCDFP-15 positive. GCDFP-15 expression was positively linked to hormone receptor (HR) and HER2 positive tumor type, while most triple negative carcinomas were negative (p < 0.0001). GCDFP-15 was also strongly correlated to AR expression (p 0.001), and to the so-called molecular apocrine subtype (HR-/AR+, p < 0.0001). Higher rates of GCDFP-15 positivity were seen in tumors of lower grade (<0.0001) and negative nodal status (p = 0.008). GCDFP-15 positive tumors tended to have a more favourable prognosis than GCDFP-15 negative tumors (DFS (p = 0.052) and OS (p = 0.044)), which was not independent from other factors in multivariate analysis. GCDFP-15 expression was not linked to pCR. Histological apocrine differentiation was frequent in molecular apocrine carcinomas (60.7%), and was associated with GCDFP-15 within this group (p = 0.039).
GCDFP-15 expression is higher in tumors with favorable prognostic features. GCDFP-15 expression is further a frequent feature of AR positive tumors and the molecular apocrine subtype. It might have reduced sensitivity as a diagnostic marker for mammary differentiation in triple negative tumors as compared to HR or HER2 positive tumor types.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-546) contains supplementary material, which is available to authorized users.
PMCID: PMC4122770  PMID: 25070172
GCDFP-15; Breast cancer; Neoadjuvant chemotherapy; Apocrine; CUP
5.  The combinatorial complexity of cancer precision medicine 
Oncoscience  2014;1(7):504-509.
Precision medicine approaches have recently been developed that offer therapies targeting mainly single genetic alterations in malignant tumors. However, next generation sequencing studies have shown that tumors normally harbor multiple genetic alterations, which could explain the so far limited successes of personalized medicine, despite considerable benefits in certain cases. Combination therapies may contribute to a solution, but will pose a major challenge for clinical trials evaluating those therapies. As we discuss here, reasons include the low abundance of most of the relevant mutations and particularly the combinatorial complexity of possible combination therapies. Our report provides a systematic and quantitative account of the implications of combinatorial complexity for cancer precision medicine and clinical trial design. We also present an outlook on how systems biological approaches may be harnessed to contribute to a solution of the complexity challenge by predicting optimal combination therapies for individual patients and how clinical trial design may be adapted by combining and extending basket and umbrella design features.
PMCID: PMC4278319  PMID: 25594052
Precision Medicine; Combination Therapies; Systems Medicine; Personalized Therapy; Clinical Trial Design
6.  Prospective Validation of Immunological Infiltrate for Prediction of Response to Neoadjuvant Chemotherapy in HER2-Negative Breast Cancer – A Substudy of the Neoadjuvant GeparQuinto Trial 
PLoS ONE  2013;8(12):e79775.
We have recently described an increased lymphocytic infiltration rate in breast carcinoma tissue is a significant response predictor for anthracycline/taxane-based neoadjuvant chemotherapy (NACT). The aim of this study was to prospectively validate the tumor-associated lymphocyte infiltrate as predictive marker for response to anthracycline/taxane-based NACT.
Patients and Methods
The immunological infiltrate was prospectively evaluated in a total of 313 core biopsies from HER2 negative patients of the multicenter PREDICT study, a substudy of the neoadjuvant GeparQuinto study. Intratumoral lymphocytes (iTuLy), stromal lymphocytes (strLy) as well as lymphocyte-predominant breast cancer (LPBC) were evaluated by histopathological assessment. Pathological complete response (pCR) rates were analyzed and compared between the defined subgroups using the exact test of Fisher.
Patients with lymphocyte-predominant breast cancer (LPBC) had a significantly increased pCR rate of 36.6%, compared to non-LPBC patients (14.3%, p<0.001). LPBC and stromal lymphocytes were significantly independent predictors for pCR in multivariate analysis (LPBC: OR 2.7, p = 0.003, strLy: OR 1.2, p = 0.01). The amount of intratumoral lymphocytes was significantly predictive for pCR in univariate (OR 1.2, p = 0.01) but not in multivariate logistic regression analysis (OR 1.2, p = 0.11).
Confirming previous investigations of our group, we have prospectively validated in an independent cohort that an increased immunological infiltrate in breast tumor tissue is predictive for response to anthracycline/taxane-based NACT. Patients with LPBC and increased stromal lymphocyte infiltration have significantly increased pCR rates. The lymphocytic infiltrate is a promising additional parameter for histopathological evaluation of breast cancer core biopsies.
PMCID: PMC3846472  PMID: 24312450
7.  The EndoPredict Gene-Expression Assay in Clinical Practice - Performance and Impact on Clinical Decisions 
PLoS ONE  2013;8(6):e68252.
The validated EndoPredict assay is a novel tool to predict the risk of metastases of patients with estrogen receptor positive, HER2 negative breast cancer treated with endocrine therapy alone. It has been designed to integrate genomic and clinical information and includes clinico-pathological factors such as tumor size and nodal status. The test is feasible in a decentral setting in molecular pathology laboratories. In this project, we investigated the performance of this test in clinical practice, and performed a retrospective evaluation of its impact on treatment decisions in breast cancer. During one year, EndoPredict assays from 167 patients could be successfully performed. For retrospective evaluation of treatment decisions, a questionnaire was sent to the clinical partner. Regarding the molecular EP class, samples from 56 patients (33.5%) had a low-risk, whereas 111 patients (66.5%) showed a high-risk gene profile. After integration of the clinicopathological factors the combined clinical and molecular score (EPclin) resulted in a low-risk group of 77 patients (46.4%), while 89 (53.6%) had a high risk EPclin score. The EPclin-based estimated median 10-year-risk for metastases with endocrine therapy alone was 11% for the whole cohort. The median handling time averaged three days (range: 0 to 11 days), 59.3% of the tests could be performed in three or less than three days. Comparison of pre- and post-test therapy decisions showed a change of therapy in 37.7% of patients. 16 patients (12.3%) had a change to an additional chemotherapy while 25.4% of patients (n = 33) changed to an endocrine therapy alone. In 73 patients (56.2%) no change of therapy resulted. In 6.1% of patients (n = 8), the patients did not agree to the recommendation of the tumor board. Our results show that the EndoPredict assay could be routinely performed in decentral molecular pathology laboratories and the results markedly change treatment decisions.
PMCID: PMC3694878  PMID: 23826382
8.  Differential expression of histone deacetylases HDAC1, 2 and 3 in human breast cancer - overexpression of HDAC2 and HDAC3 is associated with clinicopathological indicators of disease progression 
BMC Cancer  2013;13:215.
In breast cancer, the role of epigenetic alterations including modifications of the acetylation status of histones in carcinogenesis has been an important research focus during the last years. An increased deacetylation of histones leads to increased cell proliferation, cell migration, angiogenesis and invasion. Class 1 histone deacetylases (HDAC) seem to be most important during carcinogenesis.
The immunhistochemical expression of HDAC1, 2 and 3 was analyzed on tissue microarrays (TMAs) from 238 patients with primary breast cancer. We analyzed the nuclear staining intensity (negative, weak, moderate, strong) as well as the percentage of positive tumor cells and calculated the immunoreactivity score (0–12). Expression was correlated with clinicopathological parameters and patient survival.
In this cohort, we found a differential positive expression of HDAC1, HDAC2 and HDAC3. HDAC2 and HDAC3 expression was significantly higher in less differentiated tumors: HDAC2 (n=207), p<0.001 and HDAC3 (n=220), p<0.001 and correlated with negative hormone receptor status: HDAC2 (n=206), p=0.02 and HDAC3 (n=219), p=0.04. Additionally, a high HDAC2 expression was significantly associated with an overexpression of HER2 (n=203, p=0.005) and the presence of nodal metastasis (n=200, p=0.04).
HDAC1 was highly expressed in hormone receptor positive tumors (n=203; p<0.001).
As a conclusion, our results show that the class-1 HDAC isoenzymes 1, 2 and 3 are differentially expressed in breast cancer. HDAC2 and HDAC3 are strongly expressed in subgroups of tumor with features of a more aggressive tumor type.
PMCID: PMC3646665  PMID: 23627572
HDAC; Breast cancer; Immunohistochemistry
9.  CognitionMaster: an object-based image analysis framework 
Diagnostic Pathology  2013;8:34.
Automated image analysis methods are becoming more and more important to extract and quantify image features in microscopy-based biomedical studies and several commercial or open-source tools are available. However, most of the approaches rely on pixel-wise operations, a concept that has limitations when high-level object features and relationships between objects are studied and if user-interactivity on the object-level is desired.
In this paper we present an open-source software that facilitates the analysis of content features and object relationships by using objects as basic processing unit instead of individual pixels. Our approach enables also users without programming knowledge to compose “analysis pipelines“ that exploit the object-level approach. We demonstrate the design and use of example pipelines for the immunohistochemistry-based cell proliferation quantification in breast cancer and two-photon fluorescence microscopy data about bone-osteoclast interaction, which underline the advantages of the object-based concept.
We introduce an open source software system that offers object-based image analysis. The object-based concept allows for a straight-forward development of object-related interactive or fully automated image analysis solutions. The presented software may therefore serve as a basis for various applications in the field of digital image analysis.
PMCID: PMC3626931  PMID: 23445542
Software; Open source; Image analysis; Object-based image analysis
10.  Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test 
BMC Cancer  2012;12:456.
EndoPredict (EP) is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE) tissue by reverse transcription-quantitative real-time PCR (RT-qPCR). Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory.
Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability.
PCR assays were linear up to Cq values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/μl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots) resulted in a total noise (standard deviation) of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14) was caused by the replicate-to-replicate noise of the PCR assays (repeatability) and was not associated with different operating conditions (reproducibility). Performance characteristics established in the manufacturer’s laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units.
The EP test showed reproducible performance characteristics with good precision and negligible laboratory-to-laboratory variation. This study provides further evidence that the EP test is suitable for decentralized testing in specialized molecular pathological laboratories instead of a reference laboratory. This is a unique feature and a technical advance in comparison with existing RNA-based prognostic multigene expression tests.
PMCID: PMC3534340  PMID: 23039280
Breast cancer; Prognostic multigene expression test; Analytical validation; PCR; Pathology
11.  EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations 
Virchows Archiv  2012;461(3):245-257.
In non-small cell lung cancer, epidermal growth factor receptor gene mutations and anaplastic lymphoma kinase (ALK) gene rearrangements have a major impact upon the level of response to treatment with specific tyrosine kinase inhibitors. This review describes the molecular basis of ALK inhibition, summarizes current data on the effectiveness and safety of ALK inhibition therapy, describes the different testing methodologies with their advantages and disadvantages, provides a suggested testing algorithm and puts forward a proposal for an external quality assessment program in ALK testing.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-012-1281-4) contains supplementary material, which is available to authorized users.
PMCID: PMC3432214  PMID: 22825000
Anaplastic lymphoma kinase; Rearrangement; Crizotinib; Algorithm; Guidelines; Non-small cell lung cancer
12.  Remodeling of central metabolism in invasive breast cancer compared to normal breast tissue – a GC-TOFMS based metabolomics study 
BMC Genomics  2012;13:334.
Changes in energy metabolism of the cells are common to many kinds of tumors and are considered a hallmark of cancer. Gas chromatography followed by time-of-flight mass spectrometry (GC-TOFMS) is a well-suited technique to investigate the small molecules in the central metabolic pathways. However, the metabolic changes between invasive carcinoma and normal breast tissues were not investigated in a large cohort of breast cancer samples so far.
A cohort of 271 breast cancer and 98 normal tissue samples was investigated using GC-TOFMS-based metabolomics. A total number of 468 metabolite peaks could be detected; out of these 368 (79%) were significantly changed between cancer and normal tissues (p<0.05 in training and validation set). Furthermore, 13 tumor and 7 normal tissue markers were identified that separated cancer from normal tissues with a sensitivity and a specificity of >80%. Two-metabolite classifiers, constructed as ratios of the tumor and normal tissues markers, separated cancer from normal tissues with high sensitivity and specificity. Specifically, the cytidine-5-monophosphate / pentadecanoic acid metabolic ratio was the most significant discriminator between cancer and normal tissues and allowed detection of cancer with a sensitivity of 94.8% and a specificity of 93.9%.
For the first time, a comprehensive metabolic map of breast cancer was constructed by GC-TOF analysis of a large cohort of breast cancer and normal tissues. Furthermore, our results demonstrate that spectrometry-based approaches have the potential to contribute to the analysis of biopsies or clinical tissue samples complementary to histopathology.
PMCID: PMC3430598  PMID: 22823888
Breast cancer; Metabolomics; Gas chromatography; Mass spectrometry; Cancer detection
13.  Detection and Segmentation of Cell Nuclei in Virtual Microscopy Images: A Minimum-Model Approach 
Scientific Reports  2012;2:503.
Automated image analysis of cells and tissues has been an active research field in medical informatics for decades but has recently attracted increased attention due to developments in computer and microscopy hardware and the awareness that scientific and diagnostic pathology require novel approaches to perform objective quantitative analyses of cellular and tissue specimens. Model-based approaches use a priori information on cell shape features to obtain the segmentation, which may introduce a bias favouring the detection of cell nuclei only with certain properties. In this study we present a novel contour-based “minimum-model” cell detection and segmentation approach that uses minimal a priori information and detects contours independent of their shape. This approach avoids a segmentation bias with respect to shape features and allows for an accurate segmentation (precision = 0.908; recall = 0.859; validation based on ∼8000 manually-labeled cells) of a broad spectrum of normal and disease-related morphological features without the requirement of prior training.
PMCID: PMC3394088  PMID: 22787560
14.  Who Is at Risk for Diagnostic Discrepancies? Comparison of Pre- and Postmortal Diagnoses in 1800 Patients of 3 Medical Decades in East and West Berlin 
PLoS ONE  2012;7(5):e37460.
Autopsy rates in Western countries consistently decline to an average of <5%, although clinical autopsies represent a reasonable tool for quality control in hospitals, medically and economically. Comparing pre- and postmortal diagnoses, diagnostic discrepancies as uncovered by clinical autopsies supply crucial information on how to improve clinical treatment. The study aimed at analyzing current diagnostic discrepancy rates, investigating their influencing factors and identifying risk profiles of patients that could be affected by a diagnostic discrepancy.
Methods and Findings
Of all adult autopsy cases of the Charité Institute of Pathology from the years 1988, 1993, 1998, 2003 and 2008, the pre- and postmortal diagnoses and all demographic data were analyzed retrospectively. Based on power analysis, 1,800 cases were randomly selected to perform discrepancy classification (class I-VI) according to modified Goldman criteria. The rate of discrepancies in major diagnoses (class I) was 10.7% (95% CI: 7.7%–14.7%) in 2008 representing a reduction by 15.1%. Subgroup analysis revealed several influencing factors to significantly correlate with the discrepancy rate. Cardiovascular diseases had the highest frequency among class-I-discrepancies. Comparing the 1988-data of East- and West-Berlin, no significant differences were found in diagnostic discrepancies despite an autopsy rate differing by nearly 50%. A risk profile analysis visualized by intuitive heatmaps revealed a significantly high discrepancy rate in patients treated in low or intermediate care units at community hospitals. In this collective, patients with genitourinary/renal or infectious diseases were at particularly high risk.
This is the current largest and most comprehensive study on diagnostic discrepancies worldwide. Our well-powered analysis revealed a significant rate of class-I-discrepancies indicating that autopsies are still of value. The identified risk profiles may aid both pathologists and clinicians to identify patients at increased risk for a discrepant diagnosis and possibly suboptimal treatment intra vitam.
PMCID: PMC3358345  PMID: 22629399
15.  Comparison of the RNA-based EndoPredict multigene test between core biopsies and corresponding surgical breast cancer sections 
Journal of Clinical Pathology  2012;65(7):660-662.
This study compared the perfomance of the RNA-based EndoPredict multigene test on core biopsies and surgical breast cancer specimens and analysed the influence of biopsy-induced tissue injuries on the test result.
80 formalin-fixed paraffin-embedded samples comprising paired biopsies and surgical specimens from 40 ER-positive, HER2-negative patients were evaluated. Total RNA was extracted and the EndoPredict score was determined.
RNA yield was considerably lower in core biopsies, but sufficient to measure the assay in all samples. The EndoPredict score was highly correlated between paired samples (Pearson r=0.92), with an excellent concordance of classification into a low or high risk of metastasis (overall agreement 95%).
The measurements are comparable between core biopsies and surgical sections, which suggest that the EndoPredict assay can be performed on core biopsy tissue. Inflammatory changes induced by presurgical biopsies had no significant effect on the RNA-based risk assessment in surgical specimens.
PMCID: PMC3426896  PMID: 22447922
Breast; breast cancer; breast pathology; cancer; cancer genetics; cancer research; EGFR; endocrine pathology; gynaecological pathology; molecular oncology; molecular pathology; oncology; ovary; statistics; tumour markers
16.  Decentral gene expression analysis for ER+/Her2− breast cancer: results of a proficiency testing program for the EndoPredict assay 
Virchows Archiv  2012;460(3):251-259.
Gene expression profiles provide important information about the biology of breast tumors and can be used to develop prognostic tests. However, the implementation of quantitative RNA-based testing in routine molecular pathology has not been accomplished, so far. The EndoPredict assay has recently been described as a quantitative RT-PCR-based multigene expression test to identify a subgroup of hormone–receptor-positive tumors that have an excellent prognosis with endocrine therapy only. To transfer this test from bench to bedside, it is essential to evaluate the test–performance in a multicenter setting in different molecular pathology laboratories. In this study, we have evaluated the EndoPredict (EP) assay in seven different molecular pathology laboratories in Germany, Austria, and Switzerland. A set of ten formalin-fixed paraffin-embedded tumors was tested in the different labs, and the variance and accuracy of the EndoPredict assays were determined using predefined reference values. Extraction of a sufficient amount of RNA and generation of a valid EP score was possible for all 70 study samples (100%). The EP scores measured by the individual participants showed an excellent correlation with the reference values, respectively, as reflected by Pearson correlation coefficients ranging from 0.987 to 0.999. The Pearson correlation coefficient of all values compared to the reference value was 0.994. All laboratories determined EP scores for all samples differing not more than 1.0 score units from the pre-defined references. All samples were assigned to the correct EP risk group, resulting in a sensitivity and specificity of 100%, a concordance of 100%, and a kappa of 1.0. Taken together, the EndoPredict test could be successfully implemented in all seven participating laboratories and is feasible for reliable decentralized assessment of gene expression in luminal breast cancer.
PMCID: PMC3306560  PMID: 22371223
Breast cancer; Prognosis; mRNA; Quality control
17.  An intracellular targeted antibody detects EGFR as an independent prognostic factor in ovarian carcinomas 
BMC Cancer  2011;11:294.
In ovarian cancer, the reported rate of EGFR expression varies between 4-70% depending on assessment method and data on patient outcome are conflicting. Methods: In this study we investigated EGFR expression and its prognostic value in a cohort of 121 invasive ovarian carcinomas, using a novel antibody against the intracellular domain of the receptor. We further evaluated an association between EGFR, the nuclear transporter CRM1 as well as COX-2. Furthermore, we evaluated EGFR expression in ten ovarian cancer cell lines and incubated cancer cells with Leptomycin B, a CRM1 specific inhibitor.
We observed a membranous and cytoplasmic EGFR expression in 36.4% and 64% of ovarian carcinomas, respectively. Membranous EGFR was an independent prognostic factor for poor overall survival in ovarian cancer patients (HR 2.7, CI 1.1-6.4, p = 0.02) which was also found in the serous subtype (HR 4.6, CI 1.6-13.4, p = 0.004). We further observed a significant association of EGFR with COX-2 and nuclear CRM1 expression (chi-square test for trends, p = 0.006 and p = 0.013, respectively). In addition, combined membranous EGFR/COX-2 expression was significantly related to unfavorable overall survival (HR 7.2, CI 2.3-22.1, p = 0.001).
In cell culture, we observed a suppression of EGFR protein levels after exposure to Leptomycin B in OVCAR-3 and SKOV-3 cells.
Our results suggest that the EGFR/COX-2/CRM1 interaction might be involved in progression of ovarian cancer and patient prognosis. Hence, it is an interesting anti-cancer target for a combination therapy. Further studies will also be needed to investigate whether EGFR is also predictive for benefit from EGFR targeted therapies.
PMCID: PMC3149030  PMID: 21756326
EGFR; CRM1; COX-2; ovarian cancer; prognosis
18.  Estrogen Receptor Alpha Expression in Ovarian Cancer Predicts Longer Overall Survival 
Pathology Oncology Research  2011;17(3):511-518.
Estrogen as a potential factor of ovarian carcinogenesis, acts via two nuclear receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), but the cellular signal pathways involved are not completely clear so far. In this study we have described the expression of ERα, detected by immunocytochemistry in 11 ovarian carcinoma cell lines and by immunohistochemistry in 43 Federation Internationale des Gyneacologistes et Obstetristes stage III ovarian carcinoma specimens prepared before and after treatment with cisplatin-based schemes. For cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma, analysis of cisplatin sensitivity in 11 ovarian carcinoma cell line was also performed. The strong nuclear ERα expression was only shown in the single A2780P cell line. Expression of ERα in tissue specimens did not reveal any correlations between histopathological parameters (histologic type and grading). We demonstrated a significant association with ERα expression in specimens from primary laparotomies (PL) and cause–specific survival. In the cases terminated by death of the patient, overall immunoreactivity score of ERα expression at PL was significantly lower than in surviving patients. In addition, Kaplan-Meier analysis revealed significantly shorter overall survival time and progression-free time in cases with lower immunoreactivity score of ERα expression at PL. Our findings support the hypothesis that aberrant hormone activity, by way of altered receptor expression, might be an important factor in the malignant transformation of ovarian cancer.
PMCID: PMC3158974  PMID: 21207255
Estrogen receptor alpha; Ovarian cancer; Immunohistochemistry; Cisplatin
19.  Prevalence of TMPRSS2-ERG and SLC45A3-ERG gene fusions in a large prostatectomy cohort 
The majority of prostate cancers harbor recurrent gene fusions between the hormone-regulated TMPRSS2 and members of the ETS family of transcription factors, most commonly ERG. Prostate cancer with ERG rearrangements represent a distinct subclass of tumor based on studies reporting associations with histomorphologic features, characteristic somatic copy number alterations, and gene expression signatures. The current study describes the frequency of ERG rearrangement prostate cancer and three 5 prime (5') gene fusion partners (i.e., TMPRSS2, SLC45A3 and NDRG1) in a large prostatectomy cohort.
ERG gene rearrangements and mechanism of rearrangement, as well as rearrangements of TMPRSS2, SLC45A3, and NDRG1 were assessed using fluorescence in-situ hybridization (FISH) on prostate cancer samples from 614 patients treated by radical prostatectomy. ERG rearrangement occurred in 53% of the 540 assessable cases. TMPRSS2 and SLC45A3 were the only 5' partner in 78% and 6% of these ERG rearranged cases, respectively. Interestingly, 11% of the ERG rearranged cases demonstrated concurrent TMPRSS2 and SLC45A3 rearrangements. TMPRSS2 or SLC45A3 rearrangements could not be identified for 5% of the ERG rearranged cases. From these remaining cases we identified one case with NDRG1 rearrangement. We did not observe any associations with pathologic parameters or clinical outcome.
This is the first study to describe the frequency of SLC45A3-ERG fusions in a large clinical cohort. Most studies have assumed that all ERG rearrangement prostate cancers harbor TMPRSS2-ERG fusions. This is also the first study to report concurrent TMPRSS2 and SLC45A3 rearrangements in the same tumor focus suggesting additional complexity that had not been previously appreciated. This study has important clinical implications for the development of diagnostic assays to detect ETS rearrangement prostate cancer. Incorporation of these less common ERG rearrangement prostate cancer fusion assays could further increase the sensitivity of these PCR-based approaches.
PMCID: PMC2848699  PMID: 20118910
Prostate cancer; ETS rearrangements; prevalence
20.  Multicentre validation study of nucleic acids extraction from FFPE tissues 
Virchows Archiv  2010;457(3):309-317.
In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (, isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp–250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-010-0917-5) contains supplementary material, which is available to authorized users.
PMCID: PMC2933807  PMID: 20665046
FFPE; Multicentre study; Molecular analyses standardisation; PCR; DNA; RNA; Isolation
21.  HER2 diagnostics in gastric cancer—guideline validation and development of standardized immunohistochemical testing 
Virchows Archiv  2010;457(3):299-307.
Trastuzumab-based therapy has been shown to confer overall survival benefit in HER2-positive patients with advanced gastric cancer in a large multicentric trial (ToGA study). Subgroup analysis identified adenocarcinomas of the stomach and gastroesophageal (GE) junction with overexpression of HER2 according to immunohistochemistry (IHC) as potential responders. Due to recent approval of trastuzumab for HER2 positive metastatic gastric and GE-junction cancer in Europe (EMEA) HER2 diagnostics is now mandatory with IHC being the primary test followed by fluorescence in situ hybridization (FISH) in IHC2+ cases. However, in order to not miss patients potentially responding to targeted therapy determination of a HER2-positive status for gastric cancer required modification of scoring as had been proposed in a pre-ToGA study. To validate this new HER2 status testing procedure in terms of inter-laboratory and inter-observer consensus for IHC scoring a series of 547 gastric cancer tissue samples on a tissue microarray (TMA) was used. In the first step, 30 representative cores were used to identify specific IHC HER2 scoring issues among eight French and German laboratories, while in the second step the full set of 547 cores was used to determine IHC HER2 intensity and area score concordance between six German pathologists. Specific issues relating to discordance were identified and recommendations formulated which proved to be effective to reliably determine HER2 status in a prospective test series of 447 diagnostic gastric cancer specimens.
PMCID: PMC2933810  PMID: 20665045
HER2; Immunohistochemistry (IHC); Gastric carcinoma; Diagnostic test
22.  GOLPH2 expression may serve as diagnostic marker in seminomas 
BMC Urology  2010;10:4.
GOLPH2 (Golgi phosphoprotein 2) is a novel Golgi membrane protein. Despite its unknown physiologic function, however, it has been proposed as a biomarker for hepatocellular and prostate carcinoma due to its upregulation in those cancer entities. Whether the overexpression of GOLPH2 is tumour specific or a generic parameter of malignancy and whether this finding is true for additional carcinomas has not been determined. In this study, we aimed to evaluate the expression pattern of GOLPH2 in testicular seminomas, the most common histologic subtype of testicular neoplasm.
GOLPH2 protein expression was assessed by immunohistochemistry in 69 testicular seminomas and compared to the expression rates in matching normal testicular tissue and intratubular germ cell neoplasia of unclassified type (IGCNU). In addition, a subset of Leydig cell tumours was analyzed accordingly.
GOLPH2 was consistently overexpressed (89.9%) in seminomas. Matching non-neoplastic tissue showed weak or negative staining. The observed differences between non-neoplastic and neoplastic tissue were statistically highly significant (p < 0.001). There were no significant associations with tumour status. Interestingly, GOLPH2 was also highly expressed in the intertubular Leydig cells as well as in Leydig cell tumours.
GOLPH2 protein is highly expressed in seminomas and in Leydig cell tumours. This study fosters the association of GOLPH2 with malignant neoplastic processes. The staining pattern is easily assessable and consistent which is a favourable property especially in clinical settings. GOLPH2 could be a novel immunohistochemical marker for the assessment of testicular neoplasms, especially against the background that in analogy to hepatocellular carcinomas complementary GOLPH2 serum levels might be helpful in detecting metastases or recurrent tumour. Therefore serum studies and analyses of GOLPH2 expression in non-seminomatous germ cell tumours are strongly warranted.
PMCID: PMC2843682  PMID: 20184749
23.  High class I HDAC activity and expression are associated with RelA/p65 activation in pancreatic cancer in vitro and in vivo 
BMC Cancer  2009;9:395.
The strong association between aberrant HDAC activity and the occurrence of cancer has led to the development of a variety of HDAC inhibitors (HDIs), which emerge as promising new targeted anticancer therapeutics.
Due to the pivotal role of RelA/p65 in the tumorigenesis of pancreatic neoplasia we examined the expression of class I HDACs 1, 2 and 3 in a large cohort of human pancreatic carcinomas and correlated our findings with RelA/p65 expression status. Furthermore, we investigated the impact of the HDIs SAHA and VPA on RelA/p65 activity in pancreatic cancer cell culture models.
Class I HDACs were strongly expressed in a subset of pancreatic adenocarcinomas and high expression was significantly correlated with increased nuclear translocation of RelA/p65 (p = 0.024). The link of HDAC activity and RelA/p65 in this tumor entity was confirmed in vitro, where RelA/p65 nuclear translocation as well as RelA/p65 DNA binding activity could be markedly diminished by HDI treatment.
The RelA/p65 inhibitory effects of SAHA and VPA in vitro and the close relationship of class I HDACs and RelA/p65 in vivo suggest that treatment with HDIs could serve as a promising approach to suppress NF-κB activity which in turn may lead to enhanced apoptosis and chemosensitization of pancreatic cancers.
PMCID: PMC2779818  PMID: 19912635
24.  Identification of biology-based breast cancer types with distinct predictive and prognostic features: role of steroid hormone and HER2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy 
Reliable predictive and prognostic markers for routine diagnostic purposes are needed for breast cancer patients treated with neoadjuvant chemotherapy. We evaluated protein biomarkers in a cohort of 116 participants of the GeparDuo study on anthracycline/taxane-based neoadjuvant chemotherapy for operable breast cancer to test for associations with pathological complete response (pCR) and disease-free survival (DFS). Particularly, we evaluated if interactions between hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression might lead to a different clinical behavior of HR+/HER2+ co-expressing and HR+/HER2- tumors and whether subgroups of triple negative tumors might be identified by the help of Ki67 labeling index, cytokeratin 5/6 (CK5/6), as well as cyclooxygenase-2 (COX-2), and Y-box binding protein 1 (YB-1) expression.
Expression analysis was performed using immunohistochemistry and silver-enhanced in situ hybridization on tissue microarrays (TMAs) of pretherapeutic core biopsies.
pCR rates were significantly different between the biology-based tumor types (P = 0.044) with HR+/HER2+ and HR-/HER2- tumors having higher pCR rates than HR+/HER2- tumors. Ki67 labeling index, confirmed as significant predictor of pCR in the whole cohort (P = 0.001), identified HR-/HER- (triple negative) carcinomas with a higher chance for a pCR (P = 0.006). Biology-based tumor type (P = 0.046 for HR+/HER2+ vs. HR+/HER2-), Ki67 labeling index (P = 0.028), and treatment arm (P = 0.036) were independent predictors of pCR in a multivariate model. DFS was different in the biology-based tumor types (P < 0.0001) with HR+/HER2- and HR+/HER2+ tumors having the best prognosis and HR-/HER2+ tumors showing the worst outcome. Biology-based tumor type was an independent prognostic factor for DFS in multivariate analysis (P < 0.001).
Our data demonstrate that a biology-based breast cancer classification using estrogen receptor (ER), progesterone receptor (PgR), and HER2 bears independent predictive and prognostic potential. The HR+/HER2+ co-expressing carcinomas emerged as a group of tumors with a good response rate to neoadjuvant chemotherapy and a favorable prognosis. HR+/HER2- tumors had a good prognosis irrespective of a pCR, whereas patients with HR-/HER- and HR-/HER+ tumors, especially if they had not achieved a pCR, had an unfavorable prognosis and are in need of additional treatment options.
Trial registration identifier: NCT00793377
PMCID: PMC2790846  PMID: 19758440
25.  Down-regulation of the pro-apoptotic XIAP associated factor-1 (XAF1) during progression of clear-cell renal cancer 
BMC Cancer  2009;9:276.
Decreased expression of the interferon-stimulated, putative tumour suppressor gene XAF1 has been shown to play a role during the onset, progression and treatment failure in various malignancies. However, little is yet known about its potential implication in the tumour biology of clear-cell renal cell cancer (ccRCC).
This study assessed the expression of XAF1 protein in tumour tissue obtained from 291 ccRCC patients and 68 normal renal tissue samples, utilizing immunohistochemistry on a tissue-micro-array. XAF1 expression was correlated to clinico-pathological tumour features and prognosis.
Nuclear XAF1 expression was commonly detected in normal renal- (94.1%) and ccRCC (91.8%) samples, without significant differences of expression levels. Low XAF1 expression in ccRCC tissue, however, was associated with progression of tumour stage (p = 0.040) and grade (p < 0.001). Low XAF1 tumour levels were also prognostic of significantly shortened overall survival times in univariate analysis (p = 0.018), but did not provide independent prognostic information.
These data suggest down-regulation of XAF1 expression to be implicated in ccRCC progression and implies that its re-induction may provide a therapeutic approach. Although the prognostic value of XAF1 in ccRCC appears to be limited, its predictive value remains to be determined, especially in patients with metastatic disease undergoing novel combination therapies of targeted agents with Interferon-alpha.
PMCID: PMC3087333  PMID: 19664236

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