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1.  The homogeneous mutation status of a 22 gene panel justifies the use of serial sections of colorectal cancer tissue for external quality assessment 
Virchows Archiv  2015;467(3):273-278.
Testing for treatment related biomarkers in clinical care, like Ras mutation status in colorectal cancer (CRC), has increased drastically over recent years. Reliable testing of these markers is pivotal for optimal treatment of patients. Participation in external quality assessment (EQA) programs is an important element in quality management and often obligatory to comply with regulations or for accreditation. Formalin-fixed paraffin-embedded (FFPE) clinical specimens would ideally form the basis for these assessments, as they represent the most common starting material for molecular testing. However, molecular heterogeneity of a lesion in a FFPE tissue block could potentially affect test results of participating laboratories, which might compromise reliability of the quality assessment results. To assess the actual impact of this potential problem, we determined the mutation status of 22 genes commonly mutated in colon cancer in four levels covering 360 μm of 30 FFPE tissue blocks, by Next Generation Sequencing. In each block, the genotype of these genes was identical at all four levels, with only little variation in mutation load. This result shows that the mutation status of the selected 22 genes in CRC specimens is homogeneous within a 360 μm segment of the tumor. These data justify the use of serial sections, within a defined segment of a CRC tissue block, for external quality assessment of mutation analysis.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-015-1789-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4552773  PMID: 26047774
Colon; RAS; Reproducibility; Mutant allele frequency
2.  HER2 gene and protein expression status of breast carcinoma can be reliably tested on a single slide 
Virchows Archiv  2015;467(2):169-175.
Human epidermal growth factor receptor 2 (HER2) status in breast carcinomas serves as a predictor of benefit from anti-HER2 therapy. In providing clinicians with the information necessary to decide whether or not to treat with targeted therapy, it might be necessary to choose between methods assessing HER2 protein overexpression or gene amplification. A new diagnostic approach could be a combination of both tests on the same slide. If accurate and reproducible, this approach might optimize patient stratification for therapy. In this study, formalin-fixed paraffin-embedded tumor samples from 77 breast cancer patients were examined for HER2 by immunohistochemistry (IHC) and silver in situ hybridization (SISH) using HER2 IHC (clone 4B5), HER2/CEN17SISH, and combined IHC and SISH assay, called gene protein (GP). Cases were selected to ensure a sufficient number of borderline cases on the basis of IHC scores (0, 1+, 2+, 3+), obtained during diagnostic histopathological workup. The concordance between the HER2 IHC score obtained during diagnostic histopathological workup and GP was 93 %. Discordances had no influence on therapy decisions. The concordance between ISH results using dual ISH (DISH) and GP was 96 %. Of the 77 cases studied by GP, three cases with a ratio close to 2 would have been called amplified by DISH. The use of GP reduced the time for slide reading for a trained pathologist by up to 25 %, relative to sequential reading of IHC followed by SISH. For cases with an IHC score of 2+, the final result was obtained in 1 day, while the sequential technique would have required retesting by ISH on a second day. In conclusion, assessment of HER2 status by GP is an improvement for pathologists and facilitates clinical decision-making for breast cancer management.
PMCID: PMC4529453  PMID: 25985875
HER2; Breast carcinomas; 4B5; SISH; Gene; Protein expression; Simultaneous
3.  A national population-based study provides insight in the origin of malignancies metastatic to the ovary 
Virchows Archiv  2015;467(1):79-86.
A significant proportion of ovarian malignancies consists of metastatic tumors, with a wide variety in site of origin. Differentiating between a primary and metastatic malignancy of the ovaries can be difficult and misdiagnosis might have considerable impact on both treatment and prognosis. To further examine the origin of malignancies metastatic to the ovary, we performed a large-scale, nationwide search for ovarian metastases in the Dutch Pathology Registry (PALGA). All pathology reports concerning malignancies metastatic to the ovary and associated primary tumors in the Netherlands between 2000 and 2010 were collected. Age, year of diagnosis, tumor type, location of the primary tumor, and side of the ovarian tumor were extracted from the database. We identified 2312 patients fulfilling our selection criteria. The most common primary malignancy sites were colon (33.2 %), endometrium (17.1 %), breast (14.3 %), appendix (7.3 %), and stomach (4.5 %). The metastases were most frequently bilateral (46.3 %) followed by unilateral metastases in the right (26.7 %) and left ovary (19.8 %), while side was unknown in 7.2 % of cases. Of colorectal carcinomas, only 40.2 % metastasized bilaterally, compared to 63.9 % of breast, 62.9 % of gastric, and 58.9 % of appendix carcinomas. Left-sided colorectal carcinomas most often metastasized to the left ovary (p < 0.0001). We found colon carcinomas to be most frequently responsible for metastases to the ovaries, followed by endometrial and breast carcinomas. Metastases from breast, stomach, and appendix carcinomas were mostly bilateral, whereas metastases from colorectal carcinomas were mostly unilateral. The mechanisms underlying preferred sites for metastasis or side remain unclear.
PMCID: PMC4491101  PMID: 25894432
Ovarian cancer; Mucinous ovarian carcinoma; Metastasis; Epidemiology
4.  Recommendations for reporting histopathology studies: a proposal 
Virchows Archiv  2015;466(6):611-615.
PMCID: PMC4460276  PMID: 25846513
5.  Morphological and molecular characteristics of HER2 amplified urothelial bladder cancer 
Virchows Archiv  2015;466(6):703-710.
Several (pre-) clinical trials are currently investigating the benefit of HER2-targeted therapy in urothelial bladder cancer (UBC). Patients with HER2 amplified UBC could potentially profit from these therapies. However, little is known about histomorphology, HER2 protein expression patterns and occurrence of alterations in the HER2 gene in their tumors. Among 150 metastasizing primary UBC, 13 HER2 amplified tumors were identified. Their histopathological features were compared with 13 matched, non-amplified UBC. HER2 protein expression was determined by immunohistochemistry. The 26 tumors were screened for mutations in exons 19 and 20 of the HER2 gene. UBC with HER2 amplification presented with a broad variety of histological variants (median 2 vs. 1), frequently featured micropapillary tumor components (77 % vs. 8 %) and demonstrated a high amount of tumor associated inflammation. Immunohistochemically, 10 of 13 (77 %) HER2 amplified tumors were strongly HER2 protein positive. Three tumors (23 %) were scored as HER2 negative. One of the HER2 amplified tumors harbored a D769N mutation in exon 19 of the HER2 gene; all other tested tumors were wild type. In conclusion, HER2 amplified UBC feature specific morphological characteristics. They frequently express the HER2 protein diffusely and are, therefore, promising candidates for HER2 targeted therapies. The detection of mutations at the HER2 locus might add new aspects to molecular testing of UBC.
PMCID: PMC4460277  PMID: 25809292
HER2; Amplification; Bladder cancer; Histopathology
6.  Improved cytodiagnostics and quality of patient care through double reading of selected cases by an expert cytopathologist 
Virchows Archiv  2015;466(6):617-624.
Double reading may be a valuable tool for improving the quality of patient care by restoring diagnostic errors before final sign-out, but standard double reading would significantly increase costs of pathology. The aim of this study was to assess the added value of routine double reading of defined categories of clinical cytology specimens by specialized cytopathologists. Specialized cytopathologists routinely re-diagnosed blinded defined categories of clinical cytology specimens that had been signed out by routine pathologists from January 2012 up to December 2013. Major and minor discordance rates between initial and expert diagnoses were determined, and both diagnoses were validated by comparison with same-site histological follow-up. Initial and expert diagnoses were concordant in 131/218 specimens (60.1 %). Major and minor discordances were present in 28 (12.8 %) and 59 (27.1 %) specimens, respectively. Pleural fluid, thyroid and urine specimens showed the highest major discordance rates (19.4, 19.2 and 16.7 %, respectively). Histological follow-up (where possible) supported the expert diagnosis in 95.5 % of specimens. Our implemented double reading strategy of defined categories of cytology specimens showed major discordance in 12.8 % of specimens. The expert diagnosis was supported in 95.5 % of discordant cases where histological follow-up was available. This indicates that this double reading strategy is worthwhile and contributes to better cytodiagnostics and quality of patient care, especially for suspicious pleural fluid, thyroid and urine specimens. Our results emphasize that cytopathology is a subspecialization of pathology and requires specialized cytopathologists.
PMCID: PMC4460278  PMID: 25775953
Cytopathology; Diagnostic accuracy; Patient safety; Quality assurance; Subspecialization
7.  Methodological requirements for valid tissue-based biomarker studies that can be used in clinical practice 
Paralleling the growth of ever more cost efficient methods to sequence the whole genome in minute fragments of tissue has been the identification of increasingly numerous molecular abnormalities in cancers - mutations, amplifications, insertions and deletions of genes and patterns of differential gene expression, i.e. overexpression of growth factors and under-expression of tumor suppressor genes. These can be translated into assays to be used in clinical decision making. In general terms the result of such an assay is subject to a large number of variables regarding the characteristics of the available sample, particularities of the used assay and the interpretation of the results. This review discusses the effects of these variables on assays of tissue-based biomarkers, classified by macromolecule - DNA, RNA (including micro RNA, messenger RNA, long noncoding RNA, protein and phosphoprotein). As presently the majority of clinically applicable biomarkers concern immunohistochemically detectable proteins the review focuses on this area. However, the principles outlined are mostly applicable to any other analyte. A variety of pre-analytical variables impacts on the results obtained, including analyte stability (which is different for different analytes i.e. DNA, RNA or protein), period of warm and of cold ischemia, fixation time, tissue processing, sample storage time and storage conditions. In addition, assay variables play an important role, including reagent specificity (notably but not uniquely an issue concerning antibodies used in immunohistochemistry), technical components of the assay, quantitation and assay interpretation. Finally, appropriateness of an assay for clinical application is an important issue. Reference is made to publicly available guidelines to improve on biomarker development in general and requirements for clinical use in particular. Strategic goals are formulated in order to improve on the quality of biomarker reporting, including issues of analyte quality, experimental detail, assay efficiency and precision and assay appropriateness.
PMCID: PMC4009398  PMID: 24487786
immunohistochemistry; preanalytical; tissue; variables; prognostic; predictive
8.  Mitochondrial microsatellite instability in patients with metastatic colorectal cancer 
Virchows Archiv  2015;466(5):495-502.
Mitochondrial microsatellite instability (mtMSI), a change in length in mtDNA microsatellite sequences between normal and tumor tissue, has been described as a frequent occurrence in colorectal cancer (CRC). We evaluated the prevalence and prognostic value of mtMSI and its relation to nuclear microsatellite instability (MSI) in patients with metastatic CRC (mCRC). At six loci (D310, D514, D16184, ND1, ND5, and COX1), the mitochondrial DNA sequence was analyzed in normal and tumor tissue, and the mtMSI status was determined. We evaluated the prevalence and outcome in terms of overall survival (OS) in 83 CRC patients with a MSI tumor (including 39 patients with Lynch syndrome) and in 99 mCRC patients with a microsatellite stable (MSS) tumor. A meta-analysis was performed to compare our findings with existing data. mtMSI at the D-loop region was found in 54.4 % (99 out of 182) of all patients. Prevalence of mtMSI was most pronounced at the D310 locus (50.5 %). Prevalence of mtMSI at the D-loop region was not different among patients with MSI compared to MSS tumors. There was no effect of mtMSI on prognosis in patients with MSI or MSS tumors. Prevalence of mtMSI was high in mCRC patients with both MSI and MSS tumors, but there was no correlation with prognosis. mtMSI was particularly present at the D310 locus.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-015-1733-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4422840  PMID: 25697538
Metastatic colorectal cancer; mtMSI
9.  The diagnostic value of 11q13 amplification and protein expression in the detection of nodal metastasis from oral squamous cell carcinoma: a systematic review and meta-analysis 
Virchows Archiv  2015;466(4):363-373.
Despite improvements in both diagnostic and therapeutic strategies, the prognosis of oral squamous cell carcinoma (OSCC) has not changed significantly over the last decades. Prognosis of OSCC particularly depends on the presence of nodal metastasis in the neck. Therefore, proper determination of the nodal status is pivotal for appropriate treatment. Unfortunately, current available imaging techniques (magnetic resonance imaging or ultrasound even with fine needle aspiration of suspected lymph nodes (LNs)) fail to detect occult nodal disease accurately. Clinicians in head and neck oncology urgently need new diagnostic tools to reliably determine the presence of nodal metastasis of the neck. Gain of the chromosomal region 11q13 is one of the most prominent genetic alterations in head and neck cancer and is associated with poor prognosis and metastasis. The aim of this systematic review and meta-analysis was to determine the diagnostic value of either 11q13 amplification or amplification/protein overexpression of individual genes located on 11q13 to detect nodal metastasis in OSCC. A search was conducted in Pubmed, EMBASE, and Cochrane, and 947 unique citations were retrieved. Two researchers independently screened all articles and only 18 were found to meet our inclusion criteria and were considered of sufficient quality for meta-analysis. Pooled results of those show that both amplification of CCND1 and protein overexpression of cyclin D1 significantly correlate with lymph node metastasis (LNM) in OSCC. In addition, amplification of CCND1 shows a negative predictive value of 80 % in the detection of LNM in early stage OSCCs which are clinically lymph node negative although this evidence is sparse and should be validated in a larger homogeneous cohort of T1-2 OSCC.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-015-1719-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4392171  PMID: 25663615
11q13; Oral cavity; Nodal metastasis; Systematic review; Meta-analysis
10.  Extensive lipoma-like changes of myxoid liposarcoma: morphologic, immunohistochemical, and molecular cytogenetic analyses 
Virchows Archiv  2015;466(4):453-464.
Myxoid liposarcomas (MLSs) with extensive lipoma-like changes (MLSLC) are rare, and it is often difficult to distinguish them from well-differentiated liposarcoma (LS)/dedifferentiated LS (WDLS/DDLS) with myxoid changes. For the characterization of these neoplasms, we studied 8 MLSLCs, 11 ordinary MLSs, 4 WDLSs, and 6 DDLSs. Cytogenetically, MLSLC and ordinary MLS were characterized by t(12;16)(q13;p11) and FUS-DDIT3 fusion gene, whereas WDLS/DDLS lacked the fusion gene but possessed giant marker/ring chromosomes. Both lipoma-like and myxoid components of the same MLSLC exhibited the identical FUS-DDIT3, as confirmed by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemically, MDM2 and CDK4 were positive in WDLS/DDLS but negative in MLSLC and ordinary MLS. PPARγ, C/EBPα, adipophilin, and perilipin were found in each type of LS. Adipophilin was expressed chiefly in tiny fat droplets of immature lipoblasts, whereas perilipin showed a strong positive staining in large fat vacuoles of signet ring and multivacuolated lipoblasts. The Ki-67 labeling index was lower in the lipoma-like component of MLSLC when compared with the myxoid component of the same tumors as well as ordinary MLS (p < 0.001). When compared with ordinary MLS, MLSLC may be less aggressive in clinical behavior (rare recurrences or metastases) after a wide surgical excision. In conclusion, the distinction between MLSLC and WDLS/DDLS is important, because of the differences of molecular cytogenetic features as well as clinical behaviors between these distinct sarcomas presenting similar morphologic features. In addition, the combined immunohistochemical detection of adipophilin and perilipin may provide a useful ancillary tool for identification of lipoblastic cells in soft tissue sarcomas.
PMCID: PMC4392166  PMID: 25650275
Myxoid liposarcoma; Dedifferentiated liposarcoma; Lipoma-like liposarcoma; FUS-DDIT3; Fusion gene
11.  Mammary analogue secretory carcinoma of salivary glands: a new entity associated with ETV6 gene rearrangement 
Virchows Archiv  2014;466(3):245-254.
Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumour that harbours the recurrent ETV6-NTRK3 translocation. This is the first series of MASC cases identified in the historic cohort of carcinomas of salivary glands with clinical/pathological correlation and follow-up data. We reviewed 183 primary carcinomas of major and minor salivary glands resected at the Medical University of Gdańsk, Poland, between 1992 and 2012. Based on morphology and immunohistochemistry, cases suspicious for MASC were selected, and the diagnosis was confirmed by fluorescence in situ hybridization (FISH) for ETV6 rearrangement and by RT-PCR for the ETV6-NTRK3 fusion transcript. Seven carcinomas met the criteria of MASC, as they exhibited a typical appearance with solid/microcystic and papillary architecture and intraluminal secretions, and cells completely devoid of basophilic cytoplasmic zymogen granules indicative of true acinar differentiation. The only paediatric case was an unencapsulated tumour composed of macrocystic structures covered by a mostly single but, focally, double layer of cells with apocrine morphology. In all cases, the neoplastic cells revealed immunoreactivity for S100, mammaglobin, cytokeratin CK7, CK8, STAT5a and vimentin. FISH for ETV6 gene rearrangement was positive in six out of seven cases, and RT-PCR was positive in three cases. MASC is a new entity of malignant epithelial salivary gland tumours not included in the 2005 WHO Classification of Head and Neck Tumours. There is a growing body of evidence that it is not as rare as was assumed, as is also indicated by our series (3.8 %). In most cases, MASC shares some microscopic features with AciCC, adenocarcinoma/cystadenocarcinoma NOS and low-grade MEC. In rare cases, MASC with high-grade transformation may mimic the morphological appearances of high-grade salivary gland malignancies, such as salivary duct carcinoma.
PMCID: PMC4353861  PMID: 25503077
Mammary analogue secretory carcinoma; MASC; Salivary gland; ETV6-NTRK3 fusion; Translocation t(12;15)
12.  Isocitrate dehydrogenase 1 mutations (IDH1) and p16/CDKN2A copy number change in conventional chondrosarcomas 
Virchows Archiv  2014;466(2):217-222.
To determine whether IDH1 mutations are present in primary and relapsed (local and distal) conventional central chondrosarcomas; and secondly, to assess if loss of p16/CDKN2A is associated with tumour grade progression, 102 tumour samples from 37 patients, including material from presenting and relapse events, were assessed. All wild-type cases for IDH1 R132 substitutions were also tested for IDH2 R172 and R140 alterations. The primary tumour and the most recent relapse sample were tested for p16/CDKN2A by interphase fluorescence in situ hybridisation. An additional 120 central cartilaginous tumours from different patients were also tested for p16/CDKN2A copy number. The study shows that if an IDH1 mutation were detected in a primary central chondrosarcoma, it is always detected at the time of presentation, and the same mutation is detected in local recurrences and metastatic events. We show that p16/CDKN2A copy number variation occurs subsequent to the IDH1 mutation, and confirm that p16/CDKN2A copy number variation occurs in 75 % of high grade central chondrosarcomas, and not in low grade cartilaginous tumours. Finally, p16/CDKN2A copy number variation is seen in both the IDH1 wild-type and mutant cartilaginous central tumours.
PMCID: PMC4325180  PMID: 25432631
Chondrosarcomas; IDH1; p16/CDKN2A; Sarcoma
13.  CEACAM6 as detected by the AP11 antibody is a marker notable for mucin-producing adenocarcinomas 
Virchows Archiv  2014;466(2):151-159.
A new monoclonal antibody recognizing CEACAM6, which we named AP11, was generated by immunizing BALB/c mice with phytohemagglutinin-activated human peripheral blood mononuclear cells. This study aims to evaluate whether CEACAM6 can serve as a tumor marker using AP11. We examined the expression of CEACAM6 with AP11 in 11 human carcinoma cell lines by flow cytometry and 439 human tissues including 282 tumor tissues and 157 normal tissues by immunohistochemistry. CEACAM6 epitope recognized by AP11 was well preserved in formalin-fixed and paraffin-embedded tissues. Adenocarcinomas of the stomach (86 %), colorectum (95 %), pancreas (100 %), and lung (83 %), urinary bladder (100 %), and mucinous ovarian tumors (88 %) had a high rate of CEACAM6 immunoreactivity. We observed a variable expression of CEACAM6 in hepatocellular carcinomas (35 %), squamous cell carcinomas of the lung (60 %), renal cell carcinomas (14 %), urothelial carcinomas (13 %), serous carcinomas of the ovary (17 %), and breast carcinomas (11 %). Small-cell carcinomas of the lung, prostatic adenocarcinomas, papillary thyroid carcinomas, malignant melanomas, giant cell tumors, and osteosarcomas were negative for CEACAM6. All normal tissues of various organs were negative for CEACAM6. In conclusion, CEACAM6 as detected by AP11, may serve as a marker for mucin-producing adenocarcinomas of the gastrointestinal tract and ovary as well as non-small cell lung cancer. Thus, AP11 represents a valuable diagnostic tool for detecting CEACMA6-positive cancers.
PMCID: PMC4325187  PMID: 25427744
CEACMA6; AP11; Monoclonal antibody; Tumor marker
14.  SATB1 is an independent prognostic factor in radically resected upper gastrointestinal tract adenocarcinoma 
Virchows Archiv  2014;465(6):649-659.
Gastric cancer is the second most common cause of cancer-related death worldwide, and the incidence of esophageal adenocarcinoma is rising. While some progress has been made in treatment strategies, overall survival remains very poor for patients with adenocarcinoma in the upper gastrointestinal tract. Special AT-rich sequence binding protein 1 (SATB1) is a global genome organizer that has been demonstrated to promote aggressive tumor behavior in several different types of cancer, including gastric cancer. The prognostic value of SATB1 expression in esophageal cancer has, however, not yet been described. In this study, expression of SATB1 was examined by immunohistochemistry on tissue microarrays prepared from tissue samples from 175 patients with adenocarcinoma of the esophagus, cardia, or stomach and containing normal tissue, intestinal metaplasia, primary tumors, and metastases. A well-validated antibody was used. We found SATB1 to be an independent prognostic factor in patients with a radically resected tumor, correlating with shorter overall survival as well as with shorter recurrence-free survival. SATB1 expression was also found to be significantly lower in primary tumors associated with intestinal metaplasia than those without intestinal metaplasia. This observation is of potential biological interest as it has been proposed that intestinal metaplasia-associated tumors constitute a less aggressive phenotype.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-014-1667-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4245492  PMID: 25326863
SATB1; SATB2; Gastric adenocarcinoma; Esophageal adenocarcinoma; Prognosis
15.  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
16.  Changes of placental syndecan-1 expression in preeclampsia and HELLP syndrome 
Preeclampsia is characterized by maternal systemic anti-angiogenic and pro-inflammatory states. Syndecan-1 is a cell surface proteoglycan expressed by the syncytiotrophoblast, which plays an important role in angiogenesis and resolution of inflammation. Our aim was to examine placental syndecan-1 expression in preeclampsia with or without HELLP syndrome.
Placentas were obtained from women in the following groups: (1) late-onset preeclampsia (n=8); (2) early-onset preeclampsia without (n=7) and (3) with HELLP syndrome (n=8); (4) preterm controls (n=5); and (5) term controls (n=9). Tissue microarrays (TMAs) were constructed from paraffin-embedded placentas. TMA slides were immunostained for syndecan-1 and evaluated using microscopy, virtual microscopy, and semi-automated image analysis. Maternal sera from patients with preeclampsia (n=49) and controls (n=32) were immunoassayed for syndecan-1. BeWo cells were treated with Forskolin or Latrunculin-B, or kept in ischemic conditions. SDC1 expression and syndecan-1 production were investigated with qRT-PCR, confocal microscopy, and immunoassays.
Syndecan-1 was localized to the syncytiotrophoblast apical membrane in normal placentas. Syndecan-1 immunoscores were higher in late-onset preeclampsia (p=0.0001) and early-onset preeclampsia with or without HELLP syndrome (p=0.02 for both) than in controls. Maternal serum syndecan-1 concentration was lower in preeclampsia (median: 673ng/ml, interquartile range: 459-1161ng/ml) than in controls (1158ng/ml, 622-1480ng/ml). SDC1 expression and syndecan-1 immunostainings in BeWo cells and syndecan-1 concentrations in supernatants increased during cell differentiation. Disruption of the actin cytoskeleton with Latrunculin-B decreased syndecan-1 release, while ischemic conditions increased it.
Syncytiotrophoblastic syndecan-1 expression depends on the differentiation of villous trophoblasts, and trophoblastic syndecan-1 release is decreased in preeclampsia and HELLP syndrome. This phenomenon may be related to the disturbed syncytiotrophoblastic cortical actin cytoskeleton, and associated with maternal anti-angiogenic and pro-inflammatory states in these syndromes.
PMCID: PMC3939785  PMID: 23807541
cell signaling; pregnancy; proteoglycan; syncytiotrophoblast; systemic inflammation; virtual microscopy
17.  Neuropeptide S receptor 1 (NPSR1) activates cancer-related pathways and is widely expressed in neuroendocrine tumors 
Virchows Archiv  2014;465(2):173-183.
Neuroendocrine tumors (NETs) arise from disseminated neuroendocrine cells and express general and specific neuroendocrine markers. Neuropeptide S receptor 1 (NPSR1) is expressed in neuroendocrine cells and its ligand neuropeptide S (NPS) affects cell proliferation. Our aim was to study whether NPS/NPSR1 could be used as a biomarker for neuroendocrine neoplasms and to identify the gene pathways affected by NPS/NPSR1. We collected a cohort of NETs comprised of 91 samples from endocrine glands, digestive tract, skin, and lung. Tumor type was validated by immunostaining of chromogranin-A and synaptophysin expression and tumor grade was analyzed by Ki-67 proliferation index. NPS and NPSR1 expression was quantified by immunohistochemistry using polyclonal antibodies against NPS and monoclonal antibodies against the amino-terminus and carboxy-terminus of NPSR1 isoform A (NPSR1-A). The effects of NPS on downstream signaling were studied in a human SH-SY5Y neuroblastoma cell line which overexpresses NPSR1-A and is of neuroendocrine origin. NPSR1 and NPS were expressed in most NET tissues, with the exception of adrenal pheochromocytomas in which NPS/NPSR1 immunoreactivity was very low. Transcriptome analysis of NPSR1-A overexpressing cells revealed that mitogen-activated protein kinase (MAPK) pathways, circadian activity, focal adhesion, transforming growth factor beta, and cytokine–cytokine interactions were the most altered gene pathways after NPS stimulation. Our results show that NETs are a source of NPS and NPSR1, and that NPS affects cancer-related pathways.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-014-1602-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4116602  PMID: 24915894
Neuropeptide S; Neuroendocrine tumor; Neuroendocrine marker; Immunohistochemistry
18.  Risk factor analysis for bone marrow histiocytic hyperplasia with hemophagocytosis: an autopsy study 
Virchows Archiv  2014;465(1):109-118.
The excessive release of inflammatory cytokines occasionally induces life-threatening hemophagocytosis referred to as hemophagocytic syndrome (HPS). A similar condition, histiocytic hyperplasia with hemophagocytosis (HHH), is often seen in bone marrow collected during autopsy. Unlike HPS, the pathogenesis of HHH remains unclear. Therefore, we performed a clinicopathological analysis of HHH from 70 autopsy cases at the University of Fukui Hospital. HHH was detected in 29 of 70 autopsies (41.4 %) and was significantly complicated with hematological diseases (p < 0.05) and sepsis (p < 0.05). The percentage of macrophages in bone marrow (BM) nucleated cells was significantly increased in HHH (p < 0.001). Data from medical records indicated no significant changes, except for the minimum values of white blood cell counts (p < 0.05) and platelet counts (p < 0.05) in HHH patients as compared with non-HHH patients. Concentrations of inflammatory mediators including IL-1β, IL-6, and IL-8 were significantly increased in HHH patients. Multivariate risk factor analysis identified hematological diseases (odds ratio (OR), 11.71), ≥15 % BM macrophages (OR, 9.42), sepsis (OR, 7.77), and high serum IL-6 levels (OR, 1.00) as independent risk factors for HHH. HHH with hypocellular BM, the most aggressive form of HHH, was recognized in 8 of 29 HHH patients and was associated with ≥25 % BM macrophages (p < 0.001), leukocytopenia (p < 0.05), and high IL-8 levels (p < 0.05). None of the HHH patients fulfilled the diagnostic criteria of HPS. These findings suggest that HHH is a different entity from HPS and that it preferentially develops under conditions of excessive inflammation and its associated risks, such as hematological diseases and sepsis.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-014-1592-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4077255  PMID: 24852692
Bone marrow; Hematological diseases; Histiocytic hyperplasia with hemophagocytosis; Inflammation; Inflammatory cytokine; Sepsis
19.  Measurements of cancer extent in a conservatively treated prostate cancer biopsy cohort 
Virchows Archiv : an international journal of pathology  2010;457(5):10.1007/s00428-010-0971-z.
The optimal method for measuring cancer extent in prostate biopsy specimens is unknown. Seven hundred forty-four patients diagnosed between 1990 and 1996 with prostate cancer and managed conservatively were identified. The clinical end point was death from prostate cancer. The extent of cancer was measured in terms of number of cancer cores (NCC), percentage of cores with cancer (PCC), total length of cancer (LCC) and percentage length of cancer in the cores (PLC). These were correlated with prostate cancer mortality, in univariate and multivariate analysis including Gleason score and prostate-specific antigen (PSA). All extent of cancer variables were significant predictors of prostate cancer death on univariate analysis: NCC, hazard ration (HR)=1.15, 95% confidence interval (CI)=1.04–1.28, P=0.011; PPC, HR=1.01, 95% CI=1.01–1.02, P<0.0001; LCC, HR=1.02, 95% CI=1.01–1.03, P=0.002; PLC, HR=1.01, 95% CI=1.01–1.02, P=0.0001. In multivariate analysis including Gleason score and baseline PSA, PCC and PLC were both independently significant P=0.004 and P=0.012, respectively, and added further information to that provided by PSA and Gleason score, whereas NNC and LCC were no longer significant (P=0.5 and P=0.3 respectively). In a final model, including both extent of cancer variables, PCC was the stronger, adding more value than PLC (χ2 (1df)=7.8, P=0.005, χ2 (1df)=0.5, P=0.48 respectively). Measurements of disease burden in needle biopsy specimens are significant predictors of prostate-cancer-related death. The percentage of positive cores appeared the strongest predictor and was stronger than percentage length of cancer in the cores.
PMCID: PMC3853376  PMID: 20827488
Prostate biopsy; Prostate cancer; Biopsy; prognostic factors; Tumour extent
20.  CD105 is a more appropriate marker for evaluating angiogenesis in urothelial cancer of the upper urinary tract than CD31 or CD34 
Virchows Archiv  2013;463(5):673-679.
Angiogenesis plays an important role in cancer progression in many types of cancer. Evaluation of angiogenesis is often performed, but the optimal methodology for human cancer has not been agreed upon. As adequate evaluation of angiogenesis in cancer tissues might be important for prediction of prognosis and treatment decisions, we evaluated angiogenesis semiquantitatively by assessing microvessel density (MVD) in urothelial cancer of the upper urinary tract (UC-UUT). We compared the performance of three endothelial cell markers (CD31, CD34, and CD105) on formalin-fixed tissues from 122 patients diagnosed with UC-UUT without metastasis. Vascular endothelial growth factor (VEGF)-A expression was also evaluated immunohistochemically. Correlations between MVD with each marker and pT stage, grade, survival, and VEGF-A expression were investigated. Mean (standard deviation) MVD as estimated by immunohistochemical staining with anti-CD31, anti-CD34, and anti-CD105 were 47.1 (17.9)/high-power field (HPF), 70.9 (19.5)/HPF, and 31.2 (16.7)/HPF, respectively. Although all MVDs were significantly associated with pT stage and grade, CD105-MVD showed the strongest association. Similarly, CD105-MVD showed the strongest correlation with VEGF-A expression (r = 0.530, p < 0.001). Although all MVDs were associated with metastasis-free survival and cause-specific survival on univariate analysis, only CD105-MVD was retained as an independent predictor in multivariate analysis including pT stage and grade. CD105-MVD may be the preferred marker for semiquantitative assessment of angiogenesis in patients with UC-UUT.
PMCID: PMC3825622  PMID: 23975255
Angiogenesis; CD105; CD31; CD34; Urothelial cancer of the upper urinary tract
21.  [No title available] 
PMCID: PMC3644192  PMID: 23536281
22.  Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing 
Virchows Archiv  2013;462(4):409-419.
Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS is associated with TKI resistance. Accordingly, EGFR mutation status is routinely examined in NSCLC specimens, but the employed methods may have a dramatic impact on the interpretation of results and, consequently, therapeutic decisions. Specimens with low tumour cell content are at particular risk for false-negative EGFR mutation reporting by sequencing with Sanger chemistry. To improve reliability of detecting clinically relevant mutant variants of EGFR and KRAS, we took full advantage of 454 deep sequencing and developed a two-step amplification protocol for the analysis of EGFR exons 18–21 and KRAS exons 2 and 3. We systematically addressed the sensitivity, reproducibility and specificity of the developed assay. Mutations could be reliably identified down to an allele frequency of 0.2–1.5 %, as opposed to 10–20 % detection limit of Sanger sequencing. High reproducibility (0–2.1 % variant frequency) and very low background level (0.4–0.8 % frequency) further complement the reliability of this assay. Notably, re-evaluation of 16 NSCLC samples with low tumour cell content ≤40 % and EGFR wild type status according to Sanger sequencing revealed clinically relevant EGFR mutations at allele frequencies of 0.9–10 % in seven cases. In summary, this novel two-step amplification protocol with 454 deep sequencing is superior to Sanger sequencing with significantly increased sensitivity, enabling reliable analysis of EGFR and KRAS in NSCLC samples independent of the tumour cell content.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-013-1376-6) contains supplementary material, which is available to authorized users.
PMCID: PMC3624006  PMID: 23468066
NSCLC; EGFR; KRAS; 454 Deep sequencing; Sensitivity
23.  Sample parameters affecting the clinical relevance of RNA biomarkers in translational breast cancer research 
Virchows Archiv  2012;462(2):141-154.
In the frame of translational breast cancer research, eligibility criteria for formalin-fixed paraffin-embedded tissue (FFPE) material processing for gene expression studies include tumor cell content (TCC) and sample site (primary vs metastatic tumors). Herein we asked whether the observed differences in gene expression between paired samples with respect to TCC and sample site also have different clinical significance. We assessed ESR1, ERBB2, MAPT, MMP7, and RACGAP1 mRNA expression with real time PCR in paired samples before (NMD) and after macrodissection (MD) from 98 primary tumors (PMD, PNMD) and 72 metastatic lymph nodes (LNMD, LNNMD), as well as from 93 matched P (mP) and LN (mLN). TCC range was 2.5–75 % in the NMD series and 28–98 % in the MD and in the mP/mLN series. The prognostic effect of these markers, individually or in clusters, remained stable between paired PMD/NMD. In comparison, cluster classification failed in the LNNMD group with lower TCC. In the mP/mLN cohort, RACGAP1 mRNA expression was of prognostic significance when tested in mLN samples (p < 0.001). Similarly, luminal B, HER2, and triple negative tumors were of dismal prognosis when classified in the LN component of the same series (mLN, overall survival: p = 0.013, p = 0.034, and p = 0.007, respectively). In conclusion, the clinical relevance of the RNA markers examined may be affected by TCC in metastatic LN samples but not in primary tumors, while it differs between primary tumors and matched metastases. These data will facilitate the design of translational studies involving FFPE sample series.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-012-1357-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3568476  PMID: 23262785
Macrodissection; Tumor cell content; Gene expression; FFPE; Primary tumor; Metastatic lymph node; Breast cancer; Translational study
24.  Absence of TCL1A expression is a useful diagnostic feature in splenic marginal zone lymphoma 
Virchows Archiv  2012;461(6):677-685.
Splenic marginal zone lymphoma (SMZL) is a low-grade lymphoma showing a rather nonspecific immunophenotype. Gene expression profiling studies suggested that TCL1A could be a marker of SMZL, but reported data are conflicting. We evaluated TCL1A expression in a series of spleen and bone marrow samples involved by SMZL and correlated the findings with other immunophenotypical, morphological, and clinical data. In addition, we evaluated the expression of TCL1A in a series of spleens and lymph nodes involved by lymphomas that might mimic SMZL (13 nodal marginal zone lymphomas (NMZL), 39 follicular lymphomas (FL), 30 B-cell chronic lymphocytic leukemias (B-CLL), 31 mantle cell lymphomas (MCL), 1 lymphoplasmacytic lymphoma) and 15 bone marrow specimens involving hairy cell leukemia (HCL). TCL1A staining was negative in 24/31 cases of SMZL (77 %); 27/31 MCL and all B-CLL were positive for TCL1A; 32/34 cases of nodal FL (96 %) and all five splenic FL were positive for TCL1A, although at a lower intensity. Eight of 13 NMZL were positive for TCL1A, often showing a heterogeneous staining pattern. All HCL samples were strongly positive for TCL1A. No correlation was found between the pattern of splenic infiltration, TCL1A expression, and the clinical course. TCL1A-positive SMZL showed a higher rate of DBA44 staining compared to the negative ones, and this difference was statistically significant (Fisher test, single-tailed, p = 0.0397). Our data support the use of TCL1A in the panel of diagnostic markers used in the differential diagnosis of splenic low-grade B-cell lymphoma; a possible prognostic value, however, needs a larger series to be established.
PMCID: PMC3506200  PMID: 23064660
Splenic marginal zone lymphoma; TCL1A; Immunohistochemistry; Differential diagnosis
25.  Tumor architecture exerts no bias on nuclear grading in breast cancer diagnosis 
Virchows Archiv  2012;461(4):399-403.
We recently reported that nuclear grading in prostate cancer is subject to a strong confirmation bias induced by the tumor architecture. We now wondered whether a similar bias governs nuclear grading in breast carcinoma. An unannounced test was performed at a pathology conference. Pathologists were asked to grade nuclei in a PowerPoint presentation. Circular high power fields of 27 invasive ductal carcinomas were shown, superimposed over low power background images of either tubule-rich or tubule-poor carcinomas. We found (a) that diagnostic reproducibility of nuclear grades was poor to moderate (weighed kappa values between 0.07 and 0.54, 27 cases, 44 graders), but (b) that nuclear grades were not affected by the tumor architecture. We speculate that the categorized grading in breast cancer, separating tubule formation, nuclear pleomorphism, and mitotic figure counts in a combined three tier score, prevents the bias that architecture exerts on nuclear grades in less well-controlled situations.
PMCID: PMC3461209  PMID: 22936350
Confirmation bias; Cancer grading; Nuclear pleomorphism; Architecture; Cognitive psychology

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