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Surgical specimens of 80 ovarian tumors of borderline malignancy (OTBM) were investigated by scanning DNA cytophotometry. Diploid or euploid DNA histograms were found for 21 tumors, whereas 59 OTBM showed noneuploid or aneuploid DNA patterns. All patients were followed-up after surgery for at least 3 years (mean observation period, 6.7 years). Follow-up showed 11 cases of recurrent disease and 6 deaths. DNA findings and several other morphologic and clinical details (including patient age, histologic type and stage of disease, and extent of therapy) were correlated to the postoperative course. Statistical analyses disclosed that, of these parameters, only DNA content significantly affected prognosis. Recurrences and deaths resulting from tumor exclusively were observed among patients with noneuploid or aneuploid OTBM, whereas non of the diploid or euploid tumors recurred (P < 0.05). DNA cytophotometry thus might be regarded as an effective complementary means to assess the prognosis of individual OTBM cases. Cancer 1992; 69:2510-2514.

Tumor mutational burden (TMB) is a quantitative assessment of the number of somatic mutations within a tumor genome. Immunotherapy benefit has been associated with TMB assessed by whole-exome sequencing (wesTMB) and gene panel sequencing (psTMB). The initiatives of Quality in Pathology (QuIP) and Friends of Cancer Research have jointly addressed the need for harmonization among TMB testing options in tissues. This QuIP study identifies critical sources of variation in psTMB assessment.

The classification of neuroendocrine neoplasms (NENs) differs between organ systems and currently causes considerable confusion. A uniform classification framework for NENs at any anatomical location may reduce inconsistencies and contradictions among the various systems currently in use. The classification suggested here is intended to allow pathologists and clinicians to manage their patients with NENs consistently, while acknowledging organ-specific differences in classification criteria, tumor biology, and prognostic factors. The classification suggested is based on a consensus conference held at the International Agency for Research on Cancer (IARC) in November 2017 and subsequent discussion with additional experts. The key feature of the new classification is a distinction between differentiated neuroendocrine tumors (NETs), also designated carcinoid tumors in some systems, and poorly differentiated NECs, as they both share common expression of neuroendocrine markers. This dichotomous morphological subdivision into NETs and NECs is supported by genetic evidence at specific anatomic sites as well as clinical, epidemiologic, histologic, and prognostic differences. In many organ systems, NETs are graded as G1, G2, or G3 based on mitotic count and/or Ki-67 labeling index, and/or the presence of necrosis; NECs are considered high grade by definition. We believe this conceptual approach can form the basis for the next generation of NEN classifications and will allow more consistent taxonomy to understand how neoplasms from different organ systems inter-relate clinically and genetically.

The authors of this multicenter retrospective study assess whether the evolutionary distance measured from genome-wide single nucleotide polymorphism (SNP) data between tumor nodules and the cirrhotic liver may be a prognostic marker of survival after liver transplantation for multifocal hepatocellular carcinoma. Supplemental digital content is available in the text.

The numbers of diagnostic, prognostic, and predictive immunohistochemistry (IHC) tests are increasing; the implementation and validation of new IHC tests, revalidation of existing tests, as well as the on-going need for daily quality assurance monitoring present significant challenges to clinical laboratories. There is a need for proper quality tools, specifically tissue tools that will enable laboratories to successfully carry out these processes. This paper clarifies, through the lens of laboratory tissue tools, how validation, verification, and revalidation of IHC tests can be performed in order to develop and maintain high quality “fit-for-purpose” IHC testing in the era of precision medicine. This is the final part of the 4-part series “Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine.”

Validation of immunohistochemistry (IHC) assays is a subject that is of great importance to clinical practice as well as basic research and clinical trials. When applied to clinical practice and focused on patient safety, validation of IHC assays creates objective evidence that IHC assays used for patient care are “fit-for-purpose.” Validation of IHC assays needs to be properly informed by and modeled to assess the purpose of the IHC assay, which will further determine what sphere of validation is required, as well as the scope, type, and tier of technical validation. These concepts will be defined in this review, part 3 of the 4-part series “Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine.”

All laboratory tests have test performance characteristics (TPCs), whether or not they are explicitly known to the laboratorian or the pathologist. TPCs are thus also an integral characteristic of immunohistochemistry (IHC) tests and other in situ, cell-based molecular assays such as DNA or RNA in situ hybridization or aptamer-based testing. Because of their descriptive, in situ, cell-based nature, IHC tests have a limited repertoire of appropriate TPCs. Although only a few TPCs are relevant to IHC, proper selection of informative TPCs is nonetheless essential for the development of and adherence to appropriate quality assurance measures in the IHC laboratory. This paper describes the TPCs that are relevant to IHC testing and emphasizes the role of TPCs in the validation of IHC tests. This is part 2 of the 4-part series “Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine.”

Technical progress in immunohistochemistry (IHC) as well as the increased utility of IHC for biomarker testing in precision medicine avails us of the opportunity to reassess clinical IHC as a laboratory test and its proper characterization as a special type of immunoassay. IHC, as used in current clinical applications, is a descriptive, qualitative, cell-based, usually nonlinear, in situ protein immunoassay, for which the readout of the results is principally performed by pathologists rather than by the instruments on which the immunoassay is performed. This modus operandi is in contrast to other assays where the instrument also performs the readout of the test result (eg, nephelometry readers, mass spectrometry readers, etc.). The readouts (results) of IHC tests are used either by pathologists for diagnostic purposes or by treating physicians (eg, oncologists) for patient management decisions, the need for further testing, or follow-up. This paper highlights the distinction between the original purpose for which an IHC test is developed and its subsequent clinical uses, as well as the role of pathologists in the analytical and postanalytical phases of IHC testing. This paper is the first of a 4-part series, under the general title of “Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine.”

Immunohistochemistry of the PD-L1 protein may be predictive for anti-PD-1 and anti-PD-L1 immunotherapy in pulmonary adenocarcinoma and in clinically unselected cohorts of so-called non-small-cell lung cancer. Several PD-L1 immunohistochemistry assays with custom reagents and scoring-criteria are developed in parallel. Biomarker testing and clinical decision making would profit from harmonized PD-L1 diagnostics. To assess interobserver concordance and PD-L1 immunohistochemistry staining patterns, 15 pulmonary carcinoma resection specimens (adenocarcinoma: n=11, squamous-cell carcinoma: n=4) were centrally stained with the assays 28-8, 22C3, SP142, and SP263 according to clinical trial protocols. The slides were evaluated independently by nine pathologists. Proportions of PD-L1-positive carcinoma cells and immune cells were scored according to a 6-step system that integrates the criteria employed by the four PD-L1 immunohistochemistry assays. Proportion scoring of PD-L1-positive carcinoma cells showed moderate interobserver concordance coefficients for the 6-step scoring system (Light's kappa=0.47–0.50). The integrated dichotomous proportion cut-offs (≥1, ≥5, ≥10, ≥50%) showed good concordance coefficients (κ=0.6–0.8). Proportion scoring of PD-L1-positive immune cells yielded low interobserver concordance coefficients both for the 6-step-score (κ<0.2) and the dichotomous cut-offs (κ=0.12–0.25). The assays 28-8 and 22C3 stained similar proportions of carcinoma cells in 12 of 15 cases. SP142 stained fewer carcinoma cells compared to 28-8, 22C3, and SP263 in four cases, whereas SP263 stained more carcinoma cells in nine cases. SP142 and SP263 stained immune cells more intensely. The data indicate that carcinoma cells can be reproducibly scored in PD-L1 immunohistochemistry for pulmonary adenocarcinoma and squamous-cell carcinoma. No differences in interobserver concordance were noticed among the tested assays. The scoring of immune cells yielded low concordance rates and might require specific standardization. The four tested PD-L1 assays did not show comparable staining patterns in all cases. Thus, studies that correlate staining patterns and response to immunotherapy are required to test the significance of the observed differences.

The mutated BRAFoncogene represents a therapeutic target in malignant melanoma. Because BRAFmutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAFmutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAFV600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAFV600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology.

We evaluated the prognostic value of the mitosis-associated marker phosphorylated histone H3 (PHH3) and Ki-67 in prostate cancer with respect to ERG status and androgen receptor (AR) expression. PHH3 and Ki-67 expression was immunohistochemically detected and digitally quantitated in a radical prostatectomy cohort (n=640). The results were correlated to clinicopathological parameters including biochemical recurrence times. Prognostic values of PHH3 and Ki-67 were analysed by Cox regression and Kaplan-Meier statistics. In prostate cancer, mean Ki-67 and PHH3 rates were 3.40% (95%CI 3.16–3.63%) and 0.0152% (95%CI 0.0112–0.0191%), respectively. Ki-67 showed a significant correlation with Gleason scores, pTstatus, margin status, and AR expression, while PHH3 showed a significant correlation with Gleason scores and pT status. Univariate analyses for biochemical recurrence times demonstrated a significant prognostic value for median Ki-67 rate and for the PHH3 rate of the 90th percentile. Of importance, in patient subgroups stratified according to AR expression and ERG translocation, the prognostic power of proliferation markers PHH3 and Ki-67 was markedly enhanced in ERG translocation negative and high-level AR expressing ERG translocation positive prostate cancers. As expected, the proliferation markers PHH3 and Ki-67 predict adverse outcome of prostate cancer and have a particularly pronounced prognostic value in specific molecular subsets of prostate cancer (ERG− or AR+).

Currently, few prognostic factors are available to predict the emergence of castration-resistant prostate cancer and no curative options are available. Epigenetic gene regulation has been shown to trigger prostate cancer metastasis and androgen independence. Histone lysine demethylases (KDMs) are epigenetic enzymes that can remove both repressive and activating histone marks. KDM5 family members are capable of removing the histone H3 lysine 4 dimethylation–activating mark, rendering them potential players in the down-regulation of tumor suppressors and suggesting that their activity could repress oncogenes. Here, we systematically investigated KDM5C expression patterns in two independent radical prostatectomy cohorts (822 prostate tumors in total) by immunohistochemistry. Positive nuclear KDM5C staining was significantly associated with a reduced prostate-specific antigen relapse-free survival. Our study confirmed that nuclear KDM5C expression is an independent prognostic parameter. Most strikingly, the prognostic value of nuclear KDM5C expression for progression-free survival was exclusively pronounced for the Gleason group 7. In addition, KDM5C knockdown resulted in growth retardation of prostate cancer cells in vitroand induced regulation of several proliferation-associated genes. Our data indicate that KDM5C is functionally involved in proliferation control of prostate cancer cells and might represent a novel attractive therapy target. Moreover, overexpression of KDM5C is an independent new predictive marker for therapy failure as determined by biochemical recurrence in patients after prostatectomy.

Mutations in the KRASand P53genes belong to the most frequently observed genetic alterations in pancreatic ductal adenocarcinoma. The aim of this study was to examine the frequency and prognostic impact of KRASmutations. In addition, we attempted to define molecular subgroups with distinct biologic behavior by combination of KRASsequencing data with p53 protein expression data.

The ubiquitin-like protein interferon-stimulated gene, 15 kDa (ISG15) plays an ambiguous role in the progression and response to chemotherapy of solid cancers. We aimed to investigate the prognostic impact of ISG15 and its link to the nuclear factor B pathway in ovarian high-grade serous carcinoma. Immunohistochemistry was performed in a cohort of 128 primary ovarian high-grade serous carcinomas treated with standard surgery and adjuvant chemotherapy using tissue microarrays. In addition, 28 matched relapsed carcinomas were investigated. ISG15 protein expression was significantly increased in relapsed carcinomas as compared to primary tumors (P=0.027). In primary carcinoma, ISG15 was positively associated with total inhibitor of B (IB) (P=0.001) as well as nuclear and cytoplasmic phospho-IB (p-IB) expression (P=0.039 and P=0.002, respectively). Patients with ISG15-positive carcinomas had a significantly longer overall survival in univariate analysis (P=0.002), and in multivariate analysis hazard ratio=0.35 (95 confidence interval, 0.14–0.84, P=0.019). ISG15 is a potential prognostic marker in high-grade serous carcinoma of the ovary. Its impact on survival might be explained by its tight link to the nuclear factor B pathway, and the further evaluation of the interplay between ISGylation machinery and nuclear factor B, particularly with regard to response to chemotherapy, would be desirable.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit (PIK3CA) is a central element of a signaling pathway involved in cell proliferation, survival, and growth. Certain mutations in this pathway result in enhanced PI3K signaling, which is associated with oncogenic cellular transformation and cancer. The aims of this study were to characterize different types of PIK3CA mutations in exons 9 and 20 in a series of primary breast carcinomas and to correlate the results with clinicopathologic parameters and survival. We used frozen tissue samples and sequenced exons 9 and 20 for a series of 241 patients with a diagnosis of breast carcinoma. We found that 15.8 of the primary breast carcinomas possessed PIK3CA mutations in either exon 9 or exon 20. The rate of PIK3CA mutations was increased in HRHER2−tumors (18.6), but this difference did not reach a statistical significance. The lowest rate of mutations was observed in HRHER2tumors (5.3). No statistically significant association was found between the presence of PIK3CA mutations and the prognosticclinical features of breast cancer, including histologic subtype, Her2 status, axillary lymph node involvement, tumor grade, and tumor stage. However, the presence of the H1047R mutation in 10 samples was associated with a statistically significantly worse overall survival. PIK3CA mutation was found to be a frequent genetic change in all breast cancer subtypes but occurred with the highest rate in HRHER2−tumors. Further studies are needed to validate the prognostic impact of different PIK3CA mutations.

Background: Human cancer cell lines grown in the presence of the cytotoxic agent mitoxantrone frequently develop resistance associated with a reduction in intracellular drug accumulation without increased expression of the known drug resistance transporters P-glycoprotein and multidrug resistance protein (also known as multidrug resistance-associated protein). Breast cancer resistance protein (BCRP) is a recently described adenosine triphosphate-binding cassette transporter associated with resistance to mitoxantrone and anthracyclines. This study was undertaken to test the prevalence of BCRP overexpression in cell lines selected for growth in the presence of mitoxantrone. Methods: Total cellular RNA or poly [A.sup.+] RNA and genomic DNA were isolated from parental and drug-selected cell lines. Expression of BCRP messenger RNA (mRNA) and amplification of the BCRP gene were analyzed by northern and Southern blot hybridization, respectively. Results: A variety of drug-resistant human cancer cell lines derived by selection with mitoxantrone markedly overexpressed BCRP mRNA; these cell lines included sublines of human breast carcinoma (MCF-7), colon carcinoma (S1 and HT29), gastric carcinoma (EPG85-257), fibrosarcoma (EPF86-079), and myeloma (8226) origins. Analysis of genomic DNA from BCRP-overexpressing MCF-7/MX cells demonstrated that the BCRP gene was also amplified in these cells. Conclusions: Overexpression of BCRP mRNA is frequently observed in multidrug-resistant cell lines selected with mitoxantrone, suggesting that BCRP is likely to be a major cellular defense mechanism elicited in response to exposure to this drug. It is likely that BCRP is the putative 'mitoxantrone transporter' hypothesized to be present in these cell lines. [J Natl Cancer Inst 1999;91:429-33]

A 19-yr-old woman with previously diagnosed clear cell adenocarcinoma was referred to the Charité for further treatment. Biopsies were taken from the cervix, the endometrium, pseudomembranes in the peritoneum, and sentinel lymph nodes. The morphologic picture of pseudomembranes and inflammation together with the provided information about plasminogen deficiency of the patients led to the hypothesis of ligneous cervicitis. The previously taken biopsies of the adenocarcinoma were reevaluated and showed a clear cell lesion. Further immunohistochemical examination with antibodies against p16, Ki67, CEA, and p53 could not prove its malignant character. As a result we diagnosed an atypical form of microglandular hyperplasia in a patient with ligneous cervicitis. Ligneous cervicitis is a rare inflammatory condition that might affect all mucus membranes in patients with plasminogen deficiency. This case shows the importance of correlating pathologic and clinical findings in the diagnosis of ligneous cervicitis because of the rarity of the disease and the heterogeneity at presentation.

Due to the approval of various new targeted therapies for the treatment of cancer, molecular pathology laboratories with a diagnostic focus have to meet new challenges: simultaneous handling of a large number of samples, small amounts of input material, and fragmentation of nucleic acids because of formalin fixation. As a consequence, fully automated systems for a fast and standardized extraction of high-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues are urgently needed. In this study, we tested the performance of a fully automated, high-throughput method for the extraction of nucleic acids from FFPE tissues. We investigated the extraction performance in sections of 5 different tissue types often analyzed in routine pathology laboratories (cervix, colon, liver, lymph node, and lung; n=340). Furthermore, we compared the quality, labor input, and applicability of the method for diagnostic purposes with those of a laboratory-validated manual method in a clinical setting by screening a set of 45 colorectal adenocarcinoma for the KRAS mutation. Automated extraction of both DNA and RNA was successful in 339 of 340 FFPE samples representing 5 different tissue types. In comparison with a conventional manual extraction protocol, the method showed an overall agreement of 97.7 (95 confidence interval, 88.2-99.9) for the subsequent mutational analysis of the KRAS gene in colorectal cancer samples. The fully automated system is a promising tool for a simple, robust, and rapid extraction of DNA and RNA from formalin-fixed tissue. It ensures a standardization of sample processing and can be applied to clinical FFPE samples in routine pathology.

Human epidermal growth factor receptor 2 (HER2, alias ERBB2)-targeted therapy in breast and gastric cancers depends on the reliable assessment of HER2 protein expression and (in equivocal cases) the quantitative evaluation of HER2gene amplification. Typically, HER2and centromere 17 gene copy numbers are evaluated using in situhybridization (ISH) to calculate ratios for which cutoff values dividing nonamplified and amplified cases have been proposed. Although several studies have investigated how laboratory procedures affect diagnostics, a rigorous quantitative assessment of the diagnostic guidelines for data analysis is still missing. Here, we analyze the dependence of the diagnosed HER2/chromosome 17 ratios on i) sample size (evaluated cells), ii) gene/chromosome signal distributions, and iii) the approach used for quotient calculation using Monte Carlo simulations. Our data show that the current recommendation may lead to statistical HER2/CHR17ratio variations of up to 0.94 and may therefore lead to incorrect HER2status diagnoses, given the ratio threshold of 2.0 defined by the Food and Drug Administration. Moreover, borderline cases may receive different amplification diagnoses, depending on the ratio calculation approach: Brightfield-silver ISH with aggregated signal counts may underestimate the HER2/CHR17ratio compared with two-color fluorescence ISH. Our results provide a basis for quantitative rationales behind HER2diagnostic guidelines that call for increased numbers of evaluated cells and emphasize the importance of well-designed data analysis methods in diagnostic pathology, especially for predictive clinical application.

Abstract: 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.

Previously, we identified the calcium-activated nucleotidase 1 (CANT1) transcript as up-regulated in prostate cancer. Now, we studied CANT1 protein expression in a large cohort of nearly 1000 prostatic tissue samples including normal tissue, prostatic intraepithelial neoplasia (PIN), primary carcinomas, metastases, and castrate-resistant carcinomas, and further investigated its functional relevance. CANT1 displayed predominantly a Golgi-type immunoreactivity with additional and variable cytoplasmic staining. In comparison to normal tissues, the staining intensity was significantly increased in PIN lesions and cancer. In cancer, high CANT1 levels were associated with a better prognosis, and castrate-resistant carcinomas commonly showed lower CANT1 levels than primary carcinomas. The functional role of CANT1 was investigated using RNA interference in two prostate cancer cell lines with abundant endogenous CANT1 protein. On CANT1knockdown, a significantly diminished cell number and DNA synthesis rate, a cell cycle arrest in G1phase, and a strong decrease of cell transmigration rate and wound healing capacity of CANT1knockdown cells was found. However, on forced CANT1overexpression, cell proliferation and migration remained unchanged. In summary, CANT1is commonly overexpressed in the vast majority of primary prostate carcinomas and in the precursor lesion PIN and may represent a novel prognostic biomarker. Moreover, this is the first study to demonstrate a functional involvement of CANT1 in tumor biology.

The development of optimized therapy strategies against malignant tumors is critically dependent on the assessment of tissue-based biomarkers in routine diagnostic tissue samples. We investigated a novel, fully automated, and xylene-free method for RNA isolation and biomarker determination using formalin-fixed paraffin-embedded (FFPE) tissue. The aim was to show that this approach is feasible and gives results that are comparable to the current gold standards. Expression of the breast cancer biomarkers ESR1, PGR, and HER2 was measured in a total of 501 FFPE tissue samples from 167 breast carcinomas, which had been stored for up to 21 years. Total RNA was extracted from tissue sections and biomarker expression was measured by kinetic RT-PCR (RT-kPCR). The results of the new method were compared with immunohistochemistry as the current gold standard.

Abstract: The behavior of Incidental prostate cancer (IPC) cannot be reliably predicted by means of conventional histomorphology. MUC1 (episialin) expression has been linked to poor outcome in peripheral prostate cancer (PC). We aimed to determine the so far neglected prognostic role of MUC1 expression in IPC which most commonly represents transition zone cancer. Using Tissue microarray (TMA), we assessed the association between MUC1 expression recorded in transurethral resection specimens of the prostate (TURP chips) and histopathologic outcome parameters (Gleason scores and histologic staging) performed on the subsequent radical prostatectomies (RPs) in a study cohort of 54 patients. Due to tissue loss during arraying and sectioning, a total of 44 (81.5%) tumor samples remained available for immunostaining which was dichotomized by two independent clinical pathologists as being absent or present. MUC1 expression was present in 7 (15.9%) of the 44 IPC immunohistochemically investigated with a striking over-representation in high stage tumors, and was significantly correlated with histopathologic staging (ρ = 0.4; p = 0.02) and Gleason scores (ρ = 0.3; p = 0.03) performed on the corresponding RPs. These data were confirmed by means of the McNemar test (staging: p = 0.01; grading: p = 0.04). Our findings suggest that MUC1 might become a valuable adjunct to enable individual prognostic ramification prior to radical surgery in prostate cancer histologically detected in TURP chips. This interesting observation clearly awaits validation by larger studies surveying clinical follow-up data.

The majority of prostate cancers harbor recurrent gene fusions between the hormone-regulated TMPRSS2and members of the ETS family of transcription factors, most commonly ERG. Prostate cancer with ERGrearrangements represent a distinct sub-class of tumor based on studies reporting associations with histomorphologic features, characteristic somatic copy number alterations, and gene expression signatures. This study describes the frequency of ERGrearrangement prostate cancer and three 5 prime (5′) gene fusion partners (ie, TMPRSS2, SLC45A3,and NDRG1) in a large prostatectomy cohort. ERGgene rearrangements and mechanism of rearrangement, as well as rearrangements of TMPRSS2, SLC45A3,and NDRG1,were assessed using fluorescence in situhybridization (FISH) on prostate cancer samples from 614 patients treated using radical prostatectomy. ERGrearrangement occurred in 53% of the 540 assessable cases. TMPRSS2and SLC45A3were the only 5′ partner in 78% and 6% of these ERGrearranged cases, respectively. Interestingly, 11% of the ERG rearranged cases showed concurrent TMPRSS2and SLC45A3rearrangements. TMPRSS2or SLC45A3rearrangements could not be identified for 5% of the ERGrearranged cases. From these remaining cases we identified one case with NDRG1rearrangement. We did not observe any associations with pathologic parameters or clinical outcome. This is the first study to describe the frequency of SLC45A3–ERGfusions in a large clinical cohort. Most studies have assumed that all ERGrearranged prostate cancers harbor TMPRSS2–ERGfusions. This is also the first study to report concurrent TMPRSS2and SLC45A3rearrangements 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 rearranged prostate cancer. Incorporation of these less common ERGrearranged prostate cancer fusion assays could further increase the sensitivity of the current PCR-based approaches.

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 sub-class of tumor based on studies reporting associations with histomorphologic features, characteristic somatic copy number alterations, and gene expression signatures. This study describes the frequency of ERG rearrangement prostate cancer and three 5 prime (5′) gene fusion partners (ie, 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 using 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 showed 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 rearranged 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 rearranged prostate cancer. Incorporation of these less common ERG rearranged prostate cancer fusion assays could further increase the sensitivity of the current PCR-based approaches.

KRASmutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRASmutations, and the different methods reported concordant results (κ values 0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRASmutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis.

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. ClinicalTrials.gov identifier: NCT00793377

The insulin-like growth factor-II mRNA-binding protein IMP3 plays an important role in embryogenesis and recent reports suggest an involvement in tumorigenesis. Although IMP3 expression has been well studied in mouse and human fetal and adult gonads, its role in ovarian cancer is unknown. We investigated the expression of IMP3 at protein and mRNA levels in a cohort of primary ovarian carcinomas and in 11 ovarian cancer cell lines. Western blot analysis revealed an expression of IMP3 in all ovarian cancer cell lines and immunohistochemistry demonstrated a positive cytoplasmic staining in 32 of 68 carcinomas (47). In contrast, epithelium of borderline tumors, as well as, benign ovarian lesions and normal ovaries exhibited only weak or no IMP3 expression. In univariate Kaplan-Meier analysis, IMP3 protein expression was significantly associated with better overall survival (P0.048). To confirm these findings, we further determined IMP3 mRNA expression in 43 ovarian cancer specimens by real time quantitative reverse transcription-polymerase chain reaction. A significant correlation between protein and mRNA levels (r0.414, P0.006), as well as a correlation of IMP3 mRNA expression with patient overall survival (P0.044), was observed. Our results demonstrate that IMP3 is expressed in a subpopulation of ovarian cancer and a marker of good prognosis.

Topoisomerase IIα(Top IIα) is a nuclear enzyme that plays a central role in DNA metabolism, and is a molecular target for a variety of chemotherapeutic agents. Top IIαhas recently gained attention as a biomarker for therapy response and patient survival. In this study, we attempted to assess the feasibility of measuring Top IIαgene expression in RNA, isolated from archival formalin-fixed paraffin-embedded tissue specimens, which are used routinely in pathology laboratories. We have employed a new technique on the basis of magnetic particles' separation and purification of nucleic acids, and evaluated both protein and mRNA expressions from the same routinely processed tissue blocks. We investigated the expression of Top IIαmRNA and protein by real-time reverse transcription polymerase chain reaction and immunohistochemistry, in a cohort of 133 primary ovarian carcinomas, and evaluated the association between Top IIαexpression and clincopathological variables as well as patient outcome. Elevated Top IIαmRNA expression was observed in high-grade tumors (P=0.003) and advanced stage disease (P=0.011). In univariate Kaplan–Meier analysis, patients with higher expression of Top IIαnuclear protein had a significantly decreased overall survival (P=0.045). Interestingly, we detected cytoplasmic protein expression of Top IIαin a subset of samples. Cytoplasmic expression of Top IIαwas associated with the expression of chromosomal region maintenance/exportin 1 (CRM1)—a nuclear export protein (P=0.008). Our study suggests that Top IIαoverexpression is involved in the progression of ovarian cancer in a subset of the patients. Our results encourage the further evaluation of the prognostic and predictive values of Top IIαexpression in ovarian carcinoma, which might help to assess the patients' risk profile, and the planning of an individualized therapy.

CD146 is a potentially metastasis promoting cell adhesion molecule and its expression has been described in various solid tumours. We aimed to evaluate the expression of CD146 in prostate cancer by immunohistochemistry in a clinically characterised study cohort to evaluate its prognostic properties.

Abstract: Gene products of the A disintegrin and metalloprotease (ADAM) family are critically involved in carcinogenesis and tumor progression of various solid tumors. Little is known about ADAM8 in prostate cancer. In our quest for novel diagnostic tissue markers of prostate cancer, we aimed to evaluate the expression of ADAM8 in prostate cancer and to correlate it with clinicopathological parameters. One hundred twenty-eight clinicopathologically characterized prostate cancer patients, with available follow-up data, were immunostained for ADAM8. Additionally, ADAM8 mRNA expression was quantified by real-time reverse transcription polymerase chain reaction (n = 59). ADAM8 protein expression was significantly associated with higher pT status, positive nodal status, and higher Gleason scores. Still, a significant prognostic value for the prostate-specific antigen relapse-free survival of ADAM8 could not be demonstrated. The differentiality of ADAM8 expression on protein and on mRNA level was low and partially inconclusive. Therefore, despite of its significant association with conventional parameters of an unfavorable prognosis, ADAM8 adds only limited information to the conventional histopathological assessment of prostate cancer.

Creutzfeldt-Jakob disease and other prion diseases are diseases with yet not well-defined routes of transmission and infection. The safe processing of potentially contaminated tissue material remains a challenge for histologic laboratories. Formic acid pretreatment is considered to be effective in prion inactivation. We evaluated the c-erbB2 and the hormone receptor-status in potentially prion infectious breast cancer tissue after pretreatment with formic acid. Paired breast cancer tissue samples were immunostained with commercially available antibodies against c-erbB2, estrogen receptor, and progesterone receptor with 1 tissue sample of each pair being pretreated with 98 formic acid. Staining was evaluated either according to the HercepTest score or using an immunoreactive score. Additionally, fluorescence in situ hybridization (FISH) analyses were performed for 7 of these cases. Untreated tissues showed strong circumferential staining for c-erbB2 (HercepTest score 3), whereas the membranous staining of the tissues pretreated with formic acid was significantly weaker. FISH analyses showed no differences in both groups. The hormone receptor expression was not significantly influenced and positivity was maintained in all cases. In breast cancer patients, the pretreatment of tissue with formic acid for prion-decontamination in the case of suspected Creutzfeldt-Jakob disease or other prion diseases can lead to underestimation of the immunohistologically determined c-erbB2 status. In these cases, a c-erbB2-FISH analysis should be performed. For the immunostaining of hormone receptors in breast cancer, formic acid pretreatment can be applied without negative effects on the sensitivity or specificity of the assay.

Abstract: Primary, as well as secondary, lymphomas of the breast are rare diseases and might, in some cases, be misdiagnosed as breast cancer on routine hematoxylin/eosin stainings. We report a case of an anaplastic large cell lymphoma in a 72-year-old woman with a history of breast cancer treated with breast-ablative surgery and a subsequent silicon implant 32 years ago. Clinically, she presented with an ulceration of the skin, which had developed within a few months. On conventional histology, the tumor cells were mimicking poorly differentiated invasive ductal carcinoma with a prominent leukocytic infiltrate. The immunoprofile of the tumor showed negativity for cytokeratins and led to the diagnosis of a CD30-positive anaplastic large cell lymphoma.

Due to continuous technical developments and new insights into the high complexity of many diseases, molecular pathology is a rapidly growing field gaining center stage in the clinical management of tumors as well as in the pharmaceutical development of new anti-cancer drugs. The application of novel compounds in clinical trials has revealed promising results; however, the current diagnostic procedures available for determining which patients will primarily benefit from rational tumor therapy are insufficient. To read a patient’s tissue as “deeply” as possible, in the future, gaining information on the morphology and on genetic, proteomic, and epigenetic alterations will be the upcoming task of surgical pathologists experienced in molecular diagnostics to provide the clinicians with information relevant for an individualized medicine. Among the different high-throughput technologies, DNA microarrays are now the first array approach close to enter routine diagnostics. Technically advanced and well-established microarray platforms can nowadays be evaluated by distinct bioinformatic tools capable of identifying both novel genes associated with disease development and clusters of genes predicting clinical outcome of an individual tumor. The automatic, highly parallel analysis of proteins and complex proteins lysates for early detection of cancers such as breast, prostate and ovary as proteomic patterns in the serum also appears at the horizon. In addition, an improved analysis of tumor samples via antibody or reverse-phase protein arrays is likely to provide the pathologist in the future with information about activated oncogenic signaling pathways and other cell functions, such as drug response or the potential to metastasize. While expression microarrays and proteomic analysis rely on relatively unstable material incompatible with paraffin-embedded tissue samples, an investigation of DNA methylation using specialized high-throughput platforms has revealed the potential of being used in future diagnostics. Each of these approaches on its own might not suffice to extract all information required for an efficient individualized diagnostics. Therefore, a “multiplex approach” combining the different biological levels DNA, RNA, and protein, may be necessary to functionally classify malignant tumors. This appears to become a major challenge for diagnostic pathologists.Due to continuous technical developments and new insights into the high complexity of many diseases, molecular pathology is a rapidly growing field gaining center stage in the clinical management of tumors as well as in the pharmaceutical development of new anti-cancer drugs. The application of novel compounds in clinical trials has revealed promising results; however, the current diagnostic procedures available for determining which patients will primarily benefit from rational tumor therapy are insufficient. To read a patient’s tissue as “deeply” as possible, in the future, gaining information on the morphology and on genetic, proteomic, and epigenetic alterations will be the upcoming task of surgical pathologists experienced in molecular diagnostics to provide the clinicians with information relevant for an individualized medicine. Among the different high-throughput technologies, DNA microarrays are now the first array approach close to enter routine diagnostics. Technically advanced and well-established microarray platforms can nowadays be evaluated by distinct bioinformatic tools capable of identifying both novel genes associated with disease development and clusters of genes predicting clinical outcome of an individual tumor. The automatic, highly parallel analysis of proteins and complex proteins lysates for early detection of cancers such as breast, prostate and ovary as proteomic patterns in the serum also appears at the horizon. In addition, an improved analysis of tumor samples via antibody or reverse-phase protein arrays is likely to provide the pathologist in the future with information about activated oncogenic signaling pathways and other cell functions, such as drug response or the potential to metastasize. While expression microarrays and proteomic analysis rely on relatively unstable material incompatible with paraffin-embedded tissue samples, an investigation of DNA methylation using specialized high-throughput platforms has revealed the potential of being used in future diagnostics. Each of these approaches on its own might not suffice to extract all information required for an efficient individualized diagnostics. Therefore, a “multiplex approach” combining the different biological levels DNA, RNA, and protein, may be necessary to functionally classify malignant tumors. This appears to become a major challenge for diagnostic pathologists.

High cytoplasmic expression of maspin was described in ovarian cancers of shorter survival rates. Until now, no relationship has been described between expression of maspin and sensitivity to cisplatin in ovarian cancers. This study aimed at examining the relationship between expression of maspin, detected by immunohistochemistry and clinical response to cisplatin in ovarian cancer cases as well as the in vitrosensitivity to cisplatin of 11 ovarian cancer cell lines. The analyzes were performed on 73 samples of ovarian cancer and on A2780P, A2780RCIS, CAOV-3, EFO 21, EFO 27, ES-2, Mdah 2774, OAW 42, OVCAR-3, PA-1, and SKOV-3 ovarian cancer cells. Cytoplasmic maspin expression in studied cells significantly correlated with cisplatin sensitivity. A significantly shorter overall survival and progression-free survival was associated with lower cytoplasmic maspin expression at first-look laparotomies and nuclear maspin expression and secondary cytoreductions. Higher nuclear maspin at first-look laparotomies expression was specific for cases of complete response. In the study, the elevated expression of maspin was shown to be typical for cisplatin-sensitive ovarian cancers.

Background and aims: Recent cDNA expression profiling analyses indicate that within specific organ cancers Cytokeratins (CKs) dysregulation may identify subgroups with distinct biological phenotypes. Our objectives in this study were (1) to test whether cytokeratins were also distinct on the protein level, (2) to evaluate these biomarkers in a series of well-characterised CRCs, (3) to apply hierarchical cluster analysis to immunohistochemical data. Methods: Tissue microarrays (TMA) comprising 468 CRC specimens from 203 patients were constructed to evaluate CK5, CK7, CK8, CK13, CK14, CK16, CK17, CK18, CK19 and CK20. In total, 2919 samples were analyzed. Results: Unsupervised hierarchical clustering discovered subgroups represented by reduced CK8 and CK20 expression, that differed by a shorter patients survival. The evaluation of the specific biomarkers by Kaplan–Meier analysis showed that reduced CK8 expression (p<0.01) was significantly associated with shorter patients' survival, but was not an independent factor correlated with tumour stage (pT), grading (G) and nodal stage (pN). Conclusions: Reduced coexpression of CK8 and CK20 may indicate an epithelial-mesenchymal transition (EMT) representing an important step in the development of more aggressive CRCs. In addition, multiplex analysis of TMAs together with immunohistochemistry (IHC) supplemented by hierarchical clustering are a useful, promising and very powerful tool for the identification of tumour subgroups with diagnostic and prognostic signatures.

Introduction Recent reports suggest that expression of the cyclooxygenase 2 (COX-2) enzyme may up-regulate expression of MDR1/P-glycoprotein (MDR1/P-gp), an exponent of resistance to cytostatic drugs. The present study aimed at examining the relationship between the expression of COX-2 and of MDR1/P-gp in a group of breast cancer cases.Methods Immunohistochemical reactions were performed using monoclonal antibodies against COX-2 and MDR1/P-gp on samples originating from 104 cases of primary invasive breast cancer.Results COX-2-positive cases were shown to demonstrate higher expression of MDR1/P-gp (P < 0.0001). The studies also demonstrate that COX-2 expression was typical for cases of a higher grade (P = 0.01), a shorter overall survival time (P < 0.0001) and a shorter progression-free time (P < 0.0001). In the case of MDR1/P-gp, its higher expression characterised cases of a higher grade (P < 0001), with lymph node involvement (P < 0001), and shorter overall survival (P < 0.0001) and progression-free time (P < 0.0001).Conclusion Our studies confirmed the unfavourable prognostic significance of COX-2 and MDR1/P-gp. We also document a relationship between COX-2 and MDR1/P-gp, which suggests that COX-2 inhibitors should be investigated in trials as a treatment supplementary to chemotherapy of breast cancers.

Resistance to cis- or carboplatin represents the principal cause of therapeutic failures in ovarian carcinoma. The phenomenon of resistance to platinum-based drugs is partly related to expression of metallothionein (MT) and of glutathione S-transferase pi (GST-pi), but opinion on the subject is discordant. Documentation of a negative predictive effect of MT and GST-pi expression for the therapy employing platinum-based drugs would permit to select resistant cases in which other therapeutic approaches could be employed. The present study aimed at examining the relation between intensities of MT and GST-pi expressions in ovarian carcinomas and dynamics of the clinical course in the neoplastic disease in a group of cisplatin-treated patients. The analyses were performed on samples of ovarian carcinoma originating from 43 first-look laparotomies (FLLs) and, in 30 cases, from second-look laparotomies (SLL) from the same patients. Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies to MT and GST-pi. The calculations showed that in cases with augmented expression of MT, mortality was higher. On the other hand, augmented expression of GST-pi predisposed to more frequent relapses, deaths and progression of the tumor. Kaplan–Meier analysis showed that a significantly shorter survival time was linked to cases of higher expression of MT at FLL and of higher expression of GST-pi at FLL, whereas a shorter progression-free time was manifested by cases with higher expression of GST-pi at FLL. The performed investigations indicate that augmented expressions of MT at FLL and GST-pi at FLL in ovarian cancer represent an unfavourable predictive factor in cisplatin-treated patients.

The present study is based on the initiative for quality assurance in pathology of the German Society of Pathology and the Professional Association of German Pathologists. Four panel laboratories with experience and expertise in polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis were selected to establish the prerequisites for continuous external laboratory trials, in particular, by providing pre-tested specimens and evaluation criteria for participating institutes. In the first step, the four panel laboratories performed an internal trial to test their own reliability and reproducibility. Paraffin sections and DNA preparations from 34 tissues (25 clinical specimens and 9 controls) totalling to 66 samples were evaluated by each panel institute according to their own protocols. The methodologies differed and are described in detail. Despite these differences, a high degree of inter-laboratory reliability was achieved. In this report, we summarise our results including the correlation with the histology and provide recommendations for applying PCR-based methodology for the detection of mycobacterial DNA in surgical specimens. Supplementary data are available online at http://www.charite.de/ch/patho (rubric “Forschung”). Pre-tested specimens are now available for the external trial and can be ordered from the steering institute via Oligene (http://www.oligene.com/). All molecular pathology laboratories are invited to participate in this quality assurance initiative.

Polo-like kinase (PLK) family members are known to be functionally involved in mitotic signaling and in cytoskeletal reorganization in both normal and malignant cells. In this study, expression of PLK1 and PLK3 was determined immunohistochemically in tissue specimens of 135 breast carcinomas, and expression was correlated with clinicopathological parameters and patient prognosis. Strong PLK isoform overexpression was observed in 42.2% (PLK1) and 47.4% (PLK3) of breast carcinomas when compared with non-transformed breast tissue. A positive correlation of PLK isoform expression with tumor grade, vascular invasion, erbB2/HER-2 expression and markers of proliferative activity was evident. Additionally, an inverse correlation of PLK isoform expression and estrogen receptor status was observed. Overexpression of PLK3 but not of PLK1 was significantly linked to reduced median overall (P<0.001) and relapse-free (P=0.021) survival time. PLK3 expression remained an independent prognostic factor for overall (RR=3.2, P=0.002) and relapse-free (RR=2.9, P=0.049) survival in multivariate survival analysis. These results suggest PLK3 as a novel independent prognostic marker in breast cancer and hint toward a role for PLK isoform overexpression in disease progression. Therefore, in vivo inhibition of PLK family members might represent a rewarding approach in the development of new anticancer drugs for this tumor entity.

The mitogen-activated protein kinase phosphatase-1, MKP-1 (CL100) is involved in inactivation of MAP-kinase pathways, regulation of stress-responses and suppression of apoptosis. We investigated expression of MKP-1 in 90 cases of primary ovarian tumors, 11 normal ovaries as well as 4 ovarian carcinoma cell lines. Immunohistochemical expression of MKP-1 protein was reduced in tissue from LMP tumors and invasive ovarian carcinomas compared to normal ovaries and cystadenomas. A moderate to strong expression of MKP-1 was detected in 57.6% of invasive ovarian carcinomas. In a descriptive univariate survival analysis, MKP-1 expression was a prognostic marker for shorter progression-free survival of patients with invasive ovarian carcinomas. Patients with carcinomas positive for MKP-1 had a median progression-free survival of only 18.3 months compared to 40.6 months for patients with carcinomas negative for MKP-1 (log-rank test, p = 0.019). Other prognostic parameters for progression-free survival were FIGO stage, grade and pT stage. In an exploratory multivariate analysis, we found that MKP-1 expression as well as FIGO stage and grade were independent prognostic factors for progression-free survival. In contrast to progression-free survival, we did not find any influence of MKP-1 expression on patient overall survival. We investigated expression and regulation of MKP-1 mRNA by Northern Blot in vitro using 4 ovarian carcinoma cell lines (SKOV-3, OVCAR-3, CAOV-3, OAW-42). MKP-1 mRNA was inducible by interleukin-1β and tumor necrosis factor-α in SKOV-3 and OVCAR-3 cells, whereas CAOV-3 and OAW-42 expressed MKP-1 mRNA constitutively. In OVCAR-3 cells MKP-1 mRNA levels were strongly inducible upon treatment of cells with cisplatin. Our data indicate that MKP-1 is expressed in a subset of ovarian carcinomas and regulated by inflammatory mediators. Expression of MKP-1 may be associated with shorter progression-free survival times. Further studies are needed to determine whether MKP-1 expression is a clinically useful marker to estimate patient prognosis as well as the response to chemotherapy. © 2002 Wiley-Liss, Inc.

High risk human papillomaviruses (HPVs) are central to the development of cervical cancer and the deregulated expression of high risk HPV oncogenes is a critical event in this process. Here, we find that the cell protein nucleolin binds in a sequence-specific manner to the HPV18 enhancer. The DNA binding activity of nucleolin is primarily S phase specific, much like the transcription of the E6 and E7 oncoproteins of HPV18 in cervical cancer cells. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18+ cervical cancer cell growth. Furthermore, nucleolin controls the chromatin structure of the HPV18 enhancer. In contrast, HPV16 oncogene transcription and proliferation rates of HPV16+ SiHa cervical cancer cells are independent of nucleolin activity. Moreover, nucleolin expression is altered in HPV18+ precancerous and cancerous tissue from the cervix uteri. Whereas nucleolin was homogeneously distributed in the nuclei of normal epithelial cells, it showed a speckled nuclear phenotype in HPV18+ carcinomas. Thus, the host cell protein nucleolin is directly linked to HPV18-induced cervical carcinogenesis.

Abstract Purpose. Drug-resistant phenotypes of cancer cells may be caused by complex multimodal mechanisms of resistance. In order to gain further insighte into these mechanisms, a P-glycoprotein-mediated multidrug-resistant phenotype induced by daunorubicin-selection and an alternative drug resistance due to treatment with mitoxantrone were investigated in pancreatic carcinoma-derived cells. Methods. For assessing cross-resistance against various drugs, cell proliferation assays were performed. Drug accumulation was measured by flow cytometry. Messenger RNA expression was analyzed by Northern blot and RT-PCR, whereas protein expression was determined by Western blot. Catalytic activity of DNA-topoisomerases (Topo) II was determined by the decatenation assay. Results. In mitoxantrone-selected EPP85-181RNOV cells a decreased accumulation of mitoxantrone and daunorubicin was observed in the absence of P-glycoprotein, multidrug resistance protein or breast cancer resistance protein over-expression. An approximately twofold decrease of DNA topoisomerase II catalytic activity could be observed in both drug-resistance-exhibiting cell lines. The reduction of Topo II catalytic activity was reflected by decreased expression of Topo IIα and IIβ mRNAs and proteins. Conclusions. The decreased drug accumulation in EPP85-181RNOV cells indicates that alternative transport events are occurring. The decreased catalytic activity and expression of Topo II indicate that modulation of Topo II catalytic activity contributes to both drug-resistant phenotypes in pancreatic carcinoma cells.

Quantitative analyses of renal allograft tissue using immunohistochemical double-staining could be a useful tool to extend the existing knowledge on renal allograft immunopathology. Due to technical reasons, this method has been only rarely applied in the past. The use of indirect immunohistochemistry for double-staining bears the risk of nonspecific cross reactions between the two staining sequences. To date, various procedures have been refined to avoid such cross reactions. Here we assessed the validity of three different protocols for indirect immunohistochemical double-staining on frozen sections of renal transplant biopsies (n=12). Both colocalized antigens and antigens with a non-overlapping distribution were stained according to each of the three protocols. Differentiation between the two staining sequences was achieved by employing different colored substrates of alkaline phosphatase (protocol 1), different enzymes (peroxidase and alkaline phosphatase) together with the use of 3,3′-diaminobenzidine-tetrahydrochloride substrate in the first staining sequence (protocol 2), or primary antibodies from different species (protocol 3). Sensitivity and specificity of each protocol were determined by quantitative comparison with control single-stainings of adjacent sections. Sensitivity of the first staining sequence was about 100% with each of the three protocols investigated. In the second staining sequence, sensitivities of protocols 1 (50%) and 2 (54–66%) were much lower than of protocol 3 (100%). Specificity of the second staining sequence was only 44% with protocol 1 compared with 98% with protocol 2 and 100% with protocol 3. In conclusion, protocols 1 and 2 are not recommended for quantitative double-staining analyses. In contrast, protocol 3 provided maximum sensitivity and specificity, even for antigens that are colocalized on the same cell type. Thus, the use of primary antibodies from different species is by far the most reliable technique for quantitative double-staining analyses in renal allograft tissue.

The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication. We show that YB-1 facilitates E2 gene expression through the E2 late promoter thus controlling E2 gene activity at later stages of infection. The role of YB-1 for adenovirus replication was demonstrated with an E1-minus adenovirus vector containing a YB-1 transgene. In infected cells, AdYB-1 efficiently replicated and produced infectious progeny particles. Thus, adenovirus E1B-55kDa protein and the host cell factor YB-1 act jointly to facilitate adenovirus replication in the late phase of infection.

The impact of the ABC transporters breast cancer resistance protein/mitoxantrone resistance associated transporter (BCRP/MXR), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance gene-1/P-glycoprotein (MDR1/PGP) on the multidrug resistance (MDR) phenotype in chemoresistance and thermoresistance was investigated in the parental human gastric carcinoma cell line EPG85-257P, the atypical MDR subline EPG85-257RNOV, the classical MDR subline EPG85-257RDB and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RNOV-TR and EPG85-257RDB-TR. Within the atypical MDR subline EPG85-257RNOV expression of BCRP/MXR and of MRP1 were clearly enhanced (vs. parental and classical MDR lines). MDR1/PGP expression was distinctly elevated in the classical MDR subline EPG85-257RDB (vs. parental and atypical MDR sublines). In all thermoresistant counterparts basal expression of BCRP/MXR, MRP1 and MDR1/PGP was increased relative to thermosensitive sublines. Although it could be shown that the overexpressed ABC transporters were functionally active, however, no decreased drug accumulations of doxorubicin, mitoxantrone and rhodamine 123 were observed. Thus, expression of BCRP/MXR, MRP1 and MDR1/PGP was found to be dependent on the appropriate type of chemoresistance; correlating with a classical or atypical MDR phenotype. Within the thermoresistant variants, however, the increase in ABC transporter expression did obviously not influence the MDR phenotype. © 2001 Wiley-Liss, Inc.