Guido Sauter

Trastuzumab improves survival in the adjuvant treatment of HER-positive breast cancer, although combined therapy with anthracycline-based regimens has been associated with cardiac toxicity. We wanted to evaluate the efficacy and safety of a new nonanthracycline regimen with trastuzumab.We randomly assigned 3222 women with HER2-positive early-stage breast cancer to receive doxorubicin and cyclophosphamide followed by docetaxel every 3 weeks (AC-T), the same regimen plus 52 weeks of trastuzumab (AC-T plus trastuzumab), or docetaxel and carboplatin plus 52 weeks of trastuzumab (TCH). The primary study end point was disease-free survival. Secondary end points were overall survival and safety.At a median follow-up of 65 months, 656 events triggered this protocol-specified analysis. The estimated disease-free survival rates at 5 years were 75% among patients receiving AC-T, 84% among those receiving AC-T plus trastuzumab, and 81% among those receiving TCH. Estimated rates of overall survival were 87%, 92%, and 91%, respectively. No significant differences in efficacy (disease-free or overall survival) were found between the two trastuzumab regimens, whereas both were superior to AC-T. The rates of congestive heart failure and cardiac dysfunction were significantly higher in the group receiving AC-T plus trastuzumab than in the TCH group (P<0.001). Eight cases of acute leukemia were reported: seven in the groups receiving the anthracycline-based regimens and one in the TCH group subsequent to receiving an anthracycline outside the study.The addition of 1 year of adjuvant trastuzumab significantly improved disease-free and overall survival among women with HER2-positive breast cancer. The risk-benefit ratio favored the nonanthracycline TCH regimen over AC-T plus trastuzumab, given its similar efficacy, fewer acute toxic effects, and lower risks of cardiotoxicity and leukemia. (Funded by Sanofi-Aventis and Genentech; BCIRG-006 ClinicalTrials.gov number, NCT00021255.).

The goal of this review is to systematically address a number of issues raised in the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) guidelines on testing for the human epidermal growth factor receptor 2 (HER-2) alteration. A group of investigators who are experienced in the conduct and interpretation of HER-2 assay methods reviewed the ASCO-CAP guidelines and address several areas of the HER-2 testing guidelines with a particular emphasis on biologic and methodologic considerations. Although HER-2 status determined by immunohistochemistry (IHC) and the status determined by fluorescent in situ hybridization (FISH) are significantly correlated, we feel that standard considerations of laboratory testing, including test accuracy, reproducibility, and precision, as well as the current data favor FISH over IHC assay methods for determining HER-2 status. These considerations are clearly important in clinical practice because HER2 amplification is directly linked to protein expression levels in breast cancer. However, this protein is not consistently analyzed in formalin-fixed tissues as a result of variability in fixation methods and times and the impact of fixation on HER-2 protein antigenicity. Conversely, gene amplification and FISH are significantly less dependent on tissue fixation methods, making this assay more reproducible between central and peripheral laboratories than IHC. Moreover, review of the existing data demonstrate that FISH is more strongly correlated with responsiveness to either trastuzumab or lapatinib treatment. Until other methods achieve similar test accuracy, reproducibility, and predictive value, we suggest FISH as the primary HER-2 testing modality for women with breast cancer who are candidates for HER-2-targeted therapies.

Epithelial cell adhesion molecule (Ep-CAM; CD326) is used as a target by many immunotherapeutic approaches, but little data are available about Ep-CAM expression in major human malignancies with respect to level, frequency, tumour stage, grade, histologic tumour type and impact on survival. We analysed by immunohistochemical staining tissue microarrays with 4046 primary human carcinoma samples from colon, stomach, prostate and lung cancers for both frequency and intensity of Ep-CAM expression under highly standardised conditions. A total of 3360 samples were analysable. High-level Ep-CAM expression was observed in 97.7% (n=1186) of colon, 90.7% of gastric (n=473), and 87.2% of prostate cancers (n=414), and in 63.9% of lung cancers (n=1287). No detectable Ep-CAM staining was found with only 0.4% of colon, 2.5% of gastric, 1.9% of prostate cancers, and 13.5% of lung cancers. The only significant correlation of Ep-CAM expression with tumour grading was observed in colon cancer where high-level Ep-CAM expression on grade 3 tumours was down to 92.1% (P<0.0001). Adenosquamous and squamous carcinomas of the lung had a lower percentage of high-level Ep-CAM expression compared to adenocarcinomas with 35.4 and 53.6%, respectively, and with 45.5 and 17.3% of tumours being Ep-CAM negative. With the exception of moderately differentiated colon carcinoma, where patients not expressing Ep-CAM on their tumours showed an inferior survival (P=0.0014), correlation of Ep-CAM expression with survival did not reach statistical significance for any of the other cancer indications and subgroups. In conclusion, the data strongly support the notion that Ep-CAM is a prime target for immunotherapies in major human malignancies. This is because the most common human cancers show (i) a low frequency of Ep-CAM-negative tumours, (ii) a high frequency of Ep-CAM expression on cells of a given tumour, and (iii) for most cancers, an insignificant influence of tumour staging, grading and histology on Ep-CAM expression.

To critically assess the accuracy and reproducibility of human epidermal growth factor receptor type 2 (HER-2) testing in outside/local community-based hospitals versus two centralized reference laboratories and its effect on selection of women for trastuzumab (Herceptin)-based clinical trials.Breast cancer specimens from 2,600 women were prospectively evaluated by fluorescence in situ hybridization (FISH) for entry into Breast Cancer International Research Group (BCIRG) clinical trials for HER-2-directed therapies.HER-2 gene amplification by FISH was observed in 657 of the 2,502 (26%) breast cancers successfully analyzed. Among 2,243 breast cancers with central laboratory immunohistochemistry (10H8-IHC) analysis, 504 (22.54%) showed overexpression (2+ or 3+). Outside/local laboratories assessed HER-2 status by immunohistochemistry in 1,536 of these cases and by FISH in 131 cases. Overall, the HER-2 alteration status determined by outside/local immunohistochemistry showed a 79% agreement rate [kappa statistic, 0.56; 95% confidence interval (95% CI), 0.52-0.60], with FISH done by the central laboratories. The agreement rate comparing BCIRG central laboratory 10H8-IHC and outside/local laboratory immunohistochemistry was 77.5% (kappa statistic, 0.51; 95% CI, 0.46-0.55). Finally, HER-2 status, determined by unspecified FISH assay methods at outside/local laboratories, showed a 92% agreement rate (kappa statistic, 0.83; 95% CI, 0.73-0.93), with FISH done at the BCIRG central laboratories.Compared with the HER-2 status determined at centralized BCIRG reference laboratories, these results indicate superiority of FISH to accurately and reproducibly assess tumors for the HER-2 alteration at outside/local laboratories for entry to clinical trials.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is often altered in prostate cancer. To determine the prevalence and clinical significance of the different mechanisms of PTEN inactivation, we analyzed PTEN deletions in TMAs containing 4699 hormone-naïve and 57 hormone-refractory prostate cancers using fluorescence in situ hybridization analysis. PTEN mutations and methylation were analyzed in subsets of 149 and 34 tumors, respectively. PTEN deletions were present in 20.2% (458/2266) of prostate cancers, including 8.1% heterozygous and 12.1% homozygous deletions, and were linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), presence of lymph node metastasis (P = 0.0002), hormone-refractory disease (P < 0.0001), presence of ERG gene fusion (P < 0.0001), and nuclear p53 accumulation (P < 0.0001). PTEN deletions were also associated with early prostate-specific antigen recurrence in univariate (P < 0.0001) and multivariate (P = 0.0158) analyses. The prognostic impact of PTEN deletion was seen in both ERG fusion-positive and ERG fusion-negative tumors. PTEN mutations were found in 4 (12.9%) of 31 cancers with heterozygous PTEN deletions but in only 1 (2%) of 59 cancers without PTEN deletion (P = 0.027). Aberrant PTEN promoter methylation was not detected in 34 tumors. The results of this study demonstrate that biallelic PTEN inactivation, by either homozygous deletion or deletion of one allele and mutation of the other, occurs in most PTEN-defective cancers and characterizes a particularly aggressive subset of metastatic and hormone-refractory prostate cancers.

Early-onset prostate cancer (EO-PCA) represents the earliest clinical manifestation of prostate cancer. To compare the genomic alteration landscapes of EO-PCA with “classical” (elderly-onset) PCA, we performed deep sequencing-based genomics analyses in 11 tumors diagnosed at young age, and pursued comparative assessments with seven elderly-onset PCA genomes. Remarkable age-related differences in structural rearrangement (SR) formation became evident, suggesting distinct disease pathomechanisms. Whereas EO-PCAs harbored a prevalence of balanced SRs, with a specific abundance of androgen-regulated ETS gene fusions including TMPRSS2:ERG, elderly-onset PCAs displayed primarily non-androgen-associated SRs. Data from a validation cohort of > 10,000 patients showed age-dependent androgen receptor levels and a prevalence of SRs affecting androgen-regulated genes, further substantiating the activity of a characteristic “androgen-type” pathomechanism in EO-PCA.

Abstract Purpose: About 50% of prostate cancers have TMPRSS2–ERG fusions with concurrent ERG overexpression. The aim of this study was to determine whether clinical differences exist between ERG-positive and ERG-negative cancers in surgically treated patients not exposed to antihormonal therapy. A secondary aim was to search for differences between these tumor classes. Experimental Design: A tissue microarray containing samples from more than 2,800 prostate cancers with clinical data was analyzed for ERG alterations by immunohistochemistry and FISH. Results were compared with tumor phenotype, biochemical recurrence, and molecular features considered important for prostate cancer. The effect of ERG on androgen receptor (AR)-dependent transcription was analyzed in cell lines. Results: ERG expression was found in 52.4% of 2,805 cancers with a 95% concordance between ERG expression and ERG gene rearrangement detected by FISH. ERG expression was unrelated to clinical outcome and tumor phenotype. Differences in AMACR, Annexin A3, Bcl2, CD10, ALCAM, chromogranin A, epidermal growth factor receptor, HER2, mTOR, p53, and synaptophysin status were significant but minimal in absolute numbers. The most striking difference was found for AR expression, which was markedly higher in ERG-positive cancers. In vitro studies showed ERG-dependent impairment of AR-mediated transcriptional activity. Conclusions: The striking similarities between these two types of prostate cancers rules out a major impact of ERG on tumor aggressiveness in early, not hormonally treated cancer. The marked difference in AR levels between ERG-positive and -negative cancers supports a systematic difference in potential response to hormonal therapy as previously observed in clinical trials. Clin Cancer Res; 17(18); 5878–88. ©2011 AACR.

Abstract BACKGROUND Prostate specific membrane antigen (PSMA) is a suggested target for antibody‐based therapy of prostate cancer potentially involved in the regulation of cell migration. This study was undertaken, to gain more insight on the role of PSMA in early prostate cancer and its distribution in various normal tissues. METHODS A total of 1,700 different prostate cancers treated by radical prostatectomy and 612 samples from 76 different normal tissue types were successfully analyzed by immunohistochemistry (IHC) in a tissue microarray (TMA) format. PSMA immunostaining in cancers was also compared with clinical follow‐up, preexisting HER2 expression and Ki67 labeling index data. RESULTS PSMA staining was only found in prostate epithelium and expression was higher in cancer cells than in benign tissue. PSMA staining was found in 94.1% of cancers and was significantly associated with tumor stage, high Gleason grade, preoperative PSA, and HER2 expression ( P &lt; 0.0001 each). Tumors with strong PSMA expression had a higher risk of biochemical recurrence than cancers with only weak PSMA staining intensity ( P = 0.0483). There was no significant association between PSMA expression and Ki67 labeling index ( P = 0.442). CONCLUSIONS Based on the high frequency of PSMA overexpression in all stages and grades of prostate cancer and the high prevalence of PSMA overexpression, it can be speculated that increased PSMA expression may be related with prostate cancer development rather than progression. The known function of PSMA activating cell migration would be in line with the suggested role in cancer progression and the missing association between PSMA overexpression and tumor cell proliferation. Prostate 71:281–288, 2011. © 2010 Wiley‐Liss, Inc.

Purpose Docetaxel-trastuzumab (TH) is effective therapy for HER2-amplified metastatic breast cancer (MBC). Preclinical findings of synergy between docetaxel, carboplatin, and trastuzumab (TCH) prompted a phase III randomized trial comparing TCH with TH in patients with HER2-amplified MBC. Patients and Methods Two hundred sixty-three patients were randomly assigned to receive eight 3-week cycles of TH (trastuzumab plus docetaxel 100 mg/m 2 ) or TCH (trastuzumab plus carboplatin at area under the serum concentration-time curve 6 and docetaxel 75 mg/m 2 ). Trastuzumab was given at 4 mg/kg loading dose followed by a 2 mg/kg dose once per week during chemotherapy, and then 6 mg/kg once every 3 weeks until progression. Results Patient characteristics were balanced between groups. There was no significant difference between TH and TCH in terms of the primary end point, time to progression (medians of 11.1 and 10.4 months, respectively; hazard ratio, 0.914; 95% CI, 0.694 to 1.203; P = .57), response rate (72% for both groups), or overall survival (medians of 37.1 and 37.4 months, respectively; P = .99). Rates of grades 3 or 4 adverse effects for TH and TCH, respectively, were neutropenic-related complications, 29% and 23%; thrombocytopenia, 2% and 15%; anemia, 5% and 11%; sensory neuropathy, 3% and 0.8%; fatigue, 5% and 12%; peripheral edema, 3.8% and 1.5%; and diarrhea, 2% and 10%. Two patients given TCH died of sepsis, and one patient given TH experienced sudden cardiac death. Absolute left ventricular ejection fraction decline &gt; 15% was seen in 5.5% of patients on the TH arm and 6.7% of patients on the TCH arm. Conclusion Adding carboplatin did not enhance TH antitumor activity.TH (docetaxel, 100 mg/m 2 ) and TCH (docetaxel, 75 mg/m 2 ) demonstrated efficacy with acceptable toxicity in women with HER2-amplified MBC.

Gleason grading is the strongest prognostic parameter in prostate cancer. Gleason grading is categorized as Gleason ≤ 6, 3 + 4, 4 + 3, 8, and 9-10, but there is variability within these subgroups. For example, Gleason 4 components may range from 5-45% in a Gleason 3 + 4 = 7 cancer.To assess the clinical relevance of the fractions of Gleason patterns.Prostatectomy specimens from 12823 consecutive patients and of 2971 matched preoperative biopsies for which clinical data with an annual follow-up between 2005 and 2014 were available from the Martini-Klinik database.To evaluate the utility of quantitative grading, the fraction of Gleason 3, 4, and 5 patterns seen in biopsies and prostatectomies were recorded. Gleason grade fractions were compared with prostatectomy findings and prostate-specific antigen recurrence.Our data suggest a striking utility of quantitative Gleason grading. In prostatectomy specimens, there was a continuous increase of the risk of prostate-specific antigen recurrence with increasing percentage of Gleason 4 fractions with remarkably small differences in outcome at clinically important thresholds (0% vs 5%; 40% vs 60% Gleason 4), distinguishing traditionally established prognostic groups. Also, in biopsies, the quantitative Gleason scoring identified various intermediate risk groups with respect to Gleason findings in corresponding prostatectomies. Quantitative grading may also reduce the clinical impact of interobserver variability because borderline findings such as tumors with 5%, 40%, or 60% Gleason 4 fractions and very small Gleason 5 fractions (with pivotal impact on the Gleason score) are disclaimed.Quantitative Gleason pattern data should routinely be provided in addition to Gleason score categories, both in biopsies and in prostatectomy specimens.Gleason score is the most important prognostic parameter in prostate cancer, but prone to interobserver variation. The results of our study show that morphological aspects that define the Gleason grade in prostate cancer represent a continuum. Quantitation of Gleason patterns provides clinically relevant information beyond the traditional Gleason grading categories ≤ 3 + 3, 3 + 4, 4 + 3, 8, 9 -1 0. Quantitative Gleason scoring can help to minimize variations between different pathologists and substantially aid in optimized therapy decision-making.

Intraoperative frozen-section analysis allows real-time histologic assessment of surgical margins (SMs) and identification of candidates for nerve-sparing (NS) procedures.To examine the efficacy and oncologic safety of a systematic neurovascular structure-adjacent frozen-section examination (NeuroSAFE) during NS radical prostatectomy (RP).From January 2002 to June 2011, 11 069 consecutive RPs were performed at the University Medical Center Hamburg-Eppendorf. Of these, 5392 (49%) were conducted with NeuroSAFE.Our NeuroSAFE approach included the whole laterorectal circumference of the prostate to determine the SM status of the complete neurovascular tissue-corresponding prostatic surface.The impact of NeuroSAFE on NS frequency, SM status, and biochemical recurrence (BCR) was analyzed by chi-square test, and by Kaplan-Meier analyses in propensity score-based matched cohorts.Positive SMs (PSMs) were detected in 1368 (25%) NeuroSAFE RPs, leading to a secondary resection of the ipsilateral neurovascular tissue. Secondary wide resection resulted in conversion to a definitive negative SM (NSM) status in 1180 (86%) patients. In NeuroSAFE RPs, frequency of NS was significantly higher (all stages: 97% vs 81%; pT2: 99% vs 92%; pT3a: 94% vs 72%; pT3b: 88% vs 40%; p<0.0001) and PSM rates were significantly lower (all stages: 15% vs 22%; pT2: 7% vs 12%; pT3a: 21% vs 32%; p<0.0001) than in the matched non-NeuroSAFE RPs. In propensity score-based comparisons, NeuroSAFE had no negative impact on BCR (pT2, p=0.06; pT3a, p=0.17, pT3b, p=0.99), and BCR-free survival of patients with conversion to NSM did not differ significantly from patients with primarily NSM (pT2, p=0.16; pT3, p=0.26). The accuracy of our NeuroSAFE approach was 97% with a false-negative rate of 2.5%. The major limitations of this study are its retrospective nature and relatively short follow-up.NeuroSAFE enables real-time histologic monitoring of the oncologic safety of a NS procedure. Systematic NeuroSAFE significantly increases NS frequencies and reduces PSMs. Patients with a NeuroSAFE-detected PSM could be converted to a prognostically more favorable NSM status by secondary wide resection.

<h2>Summary</h2> Despite much evidence on epigenetic abnormalities in cancer, it is currently unclear to what extent epigenetic alterations can be associated with tumors' clonal genetic origins. Here, we show that the prostate intratumor heterogeneity in DNA methylation and copy-number patterns can be explained by a unified evolutionary process. By assaying multiple topographically distinct tumor sites, premalignant lesions, and lymph node metastases within five cases of prostate cancer, we demonstrate that both DNA methylation and copy-number heterogeneity consistently reflect the life history of the tumors. Furthermore, we show cases of genetic or epigenetic convergent evolution and highlight the diversity in the evolutionary origins and aberration spectrum between tumor and metastatic subclones. Importantly, DNA methylation can complement genetic data by serving as a proxy for activity at regulatory domains, as we show through identification of high epigenetic heterogeneity at androgen-receptor-bound enhancers. Epigenome variation thereby expands on the current genome-centric view on tumor heterogeneity.

Abstract Deletions involving the chromosomal band 5q21 are among the most frequent alterations in prostate cancer. Using single-nucleotide polymorphism (SNP) arrays, we mapped a 1.3 megabase minimally deleted region including only the repulsive guidance molecule B (RGMB) and chromodomain helicase DNA-binding protein 1 (CHD1) genes. Functional analyses showed that CHD1 is an essential tumor suppressor. FISH analysis of 2,093 prostate cancers revealed a strong association between CHD1 deletion, prostate-specific antigen (PSA) biochemical failure (P = 0.0038), and absence of ERG fusion (P &amp;lt; 0.0001). We found that inactivation of CHD1 in vitro prevents formation of ERG rearrangements due to impairment of androgen receptor (AR)-dependent transcription, a prerequisite for ERG translocation. CHD1 is required for efficient recruitment of AR to responsive promoters and regulates expression of known AR-responsive tumor suppressor genes, including NKX3-1, FOXO1, and PPARγ. Our study establishes CHD1 as the 5q21 tumor suppressor gene in prostate cancer and shows a key role of this chromatin remodeling factor in prostate cancer biology. Cancer Res; 73(9); 2795–805. ©2013 AACR.

Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ≤55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.

The cell cycle progression score is associated with prostate cancer outcomes in various clinical settings. However, previous studies of men treated with radical prostatectomy evaluated cell cycle progression scores generated from resected tumor tissue. We evaluated the prognostic usefulness of the score derived from biopsy specimens in men treated with radical prostatectomy.We evaluated the cell cycle progression score in cohorts of patients from the Martini Clinic (283), Durham Veterans Affairs Medical Center (176) and Intermountain Healthcare (123). The score was derived from simulated biopsy (Martini Clinic) or diagnostic biopsy (Durham Veterans Affairs Medical Center and Intermountain Healthcare) and evaluated for an association with biochemical recurrence and metastatic disease.In all 3 cohorts the cell cycle progression score was associated with biochemical recurrence and metastatic disease. The association with biochemical recurrence remained significant after adjusting for other prognostic clinical variables. On combined analysis of all cohorts (total 582 patients) the score was a strong predictor of biochemical recurrence on univariate analysis (HR per score unit 1.60, 95% CI 1.35-1.90, p=2.4×10(-7)) and multivariate analysis (HR per score unit 1.47, 95% CI 1.23-1.76, p=4.7×10(-5)). Although there were few events (12), the cell cycle progression score was the strongest predictor of metastatic disease on univariate analysis (HR per score unit 5.35, 95% CI 2.89-9.92, p=2.1×10(-8)) and after adjusting for clinical variables (HR per score unit 4.19, 95% CI 2.08-8.45, p=8.2×10(-6)).The cell cycle progression score derived from a biopsy sample was associated with adverse outcomes after surgery. These results indicate that the score can be used at disease diagnosis to better define patient prognosis and enable more appropriate clinical care.

Aims: To determine prostate‐specific membrane antigen (PSMA) expression in normal tissues and in 3161 benign and malignant tumours and subsequently to define its sensitivity and specificity in prostatic adenocarcinoma (PaC). Methods and results: Multiple tissue microarray sections were stained with a monoclonal antibody to PSMA. PaC was positive in 93/141 cases (66.0%) with various staining patterns including cytoplasmic, apical, apical/cytoplasmic and cytoplasmic with membranous accentuation. Of 2174 various tumour types, 154 expressed PSMA, including 59/346 (17.0%) urothelial carcinomas of the bladder (UBC). In those tumours, the staining pattern was always cytoplasmic. All 846 benign tumours were negative for PSMA. The sensitivity and specificity of PSMA in distinguishing PaC from any other type of malignancy is 65.9% and 94.5%, respectively. Furthermore, its sensitivity and specificity in differentiating PaC from urothelial cancer is 65.9% and 82.9%, respectively. Conclusions: Despite its expression by subsets of various types of malignancies, PSMA is still considered to be fairly sensitive and highly specific for PaC.

Her-2 is the molecular target for antibody-based treatment of breast cancer (trastuzumab). The potential benefit of anti-Her-2 therapy is currently investigated in several other HER-2-amplified cancers including gastric cancer. Although HER-2 amplification occurs in more than 10% of gastric cancers, potential heterogeneity of HER-2 amplification and overexpression could represent a major drawback for anti-Her-2 therapy. To address the potential applicability of trastuzumab in gastric cancer, tissue microarray sections of 166 gastric adenocarcinomas and 69 lymph node metastases were analyzed for Her-2 overexpression and amplification using Food and Drug Administration-approved reagents for immunohistochemistry and fluorescence in situ hybridization. HER-2 amplification was seen in 27 (16%) of 166 gastric adenocarcinomas. Amplification was typically high level with more than 20 HER-2 copies per tumor cell and a HER-2/centromere 17 ratio >3. Amplification was associated with intestinal tumor phenotype but unrelated to survival, grading, pT, pN, or pM. Identical HER-2 status was found in primary tumor and their matched lymph node metastases. Moreover, HER-2 and Topoisomerase IIalpha coamplification analysis of 3 to 16 large sections from 8 Her-2-positive gastric cancers did not reveal any heterogeneity of the amplicon site. The high level of HER-2 amplification in combination with the homogeneity of its expression in primary and metastatic tumors argues for a possible therapeutic utility of trastuzumab in HER-2-amplified gastric adenocarcinomas.

NKX3.1 is a prostate-specific homeobox gene located on chromosome 8p21. In the mouse, Nkx3.1 has growth-suppressive and differentiating effects on prostatic epithelium. Mutations of the coding region of NKX3.1 were not found in human prostate cancer, failing to support the notion that NKX3.1 was a tumor suppressor gene. To study the expression o NKX3.1 protein in human tissues and prostate cancer, we derived a rabbit antiserum against purified recombinant NKX3.1. Among normal human tissues, NKX3.1 expression was seen in testis, in rare pulmonary mucous glands, and in isolated regions of transitional epithelium of the ureter. NKX3.1 was uniformly expressed in nuclei of normal prostate epithelial cells in 61 histological sections from radical prostatectomy specimens. We analyzed 507 samples of neoplastic prostate epithelium, most of which were contained on a tissue microarray that contained samples from different stages of prostatic neoplasia. We observed complete loss of NKX3.1 expression in 5% of benign prostatic hyperplasias, 20% of high-grade prostatic intraepithelial neoplasias, 6% of T1a/b samples, 22% of T3/4 samples, 34% of hormone-refractory prostate cancers, and 78% of metastases. Our data show that NKX3.1 expression is highly, but not exclusively, specific for the prostate. Loss of NKX3.1 expression is strongly associated with hormone-refractory disease and advanced tumor stage in prostate cancer (P < 0.0001).

Extracellular matrix metalloproteinase inducer expressed by tumor cells stimulates peritumoral fibroblasts to produce matrix metalloproteinases, thus contributing to tumor invasion and metastasis. To assess its suitability as potential therapeutic target, the overall incidence of EMMPRIN expression in normal and neoplastic tissues was analyzed. EMMPRIN expression was detected immunohistochemically using monoclonal antibodies MEM-M6/1 and HIM6 and tissue microarrays with 2,348 and 608 tissue samples from 129 distinct tumor types and 76 different normal tissues, respectively. Expression and glycosylation state of EMMPRIN in human breast cancer cells were analyzed by Western blot analysis with monoclonal antibodies recognizing distinct carbohydrate structures and biochemical methods. EMMPRIN expression was found in 112 of 129 tumor entities analyzed with malignant tumors being EMMPRIN positive more frequently than benign tumors. A remarkable heterogeneity in EMMPRIN expression between tumor entities was observed. Among others, squamous-cell carcinomas (60-100%), pancreatic (87%), chromophobic kidney (83%), hepatocellular (83%) or medullary breast (83%) adenocarcinomas as well as glioblastoma multiforme (79%) presented with a particular high incidence of EMMPRIN expression. There were a limited number of EMMPRIN-positive normal cell types including proliferatively active and differentiating epithelial cells, germ cells, myocardial cells in the left heart ventricle or vascular endothelial cells of the brain. We could further demonstrate that breast cancer cells expressed EMMPRIN isoforms differing in the presence or absence of Lewis X glycan structures. Our results may assist in defining the suitability of EMMPRIN as therapeutic target and predicting negative side effects.

Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene encoding an enzyme that is a major target of anthracyclines. This study was designed to evaluate whether TOP2A gene alterations may predict incremental responsiveness to anthracyclines in some breast cancers.A total of 4,943 breast cancers were analyzed for alterations in TOP2A and HER2. Primary tumor tissues from patients with metastatic breast cancer treated in a trial of chemotherapy plus/minus trastuzumab were studied for amplification/deletion of TOP2A and HER2 as a test set followed by evaluation of malignancies from two separate, large trials for changes in these same genes as a validation set. Association between these alterations and clinical outcomes was determined.Test set cases containing HER2 amplification treated with doxorubicin and cyclophosphamide (AC) plus trastuzumab, demonstrated longer progression-free survival compared to those treated with AC alone (P = .0002). However, patients treated with AC alone whose tumors contain HER2/TOP2A coamplification experienced a similar improvement in survival (P = .004). Conversely, for patients treated with paclitaxel, HER2/TOP2A coamplification was not associated with improved outcomes. These observations were confirmed in a larger validation set, where HER2/TOP2A coamplification was again associated with longer survival when only anthracycline-containing chemotherapy was used for treatment compared with outcome in HER2-positive cancers lacking TOP2A coamplification.In a study involving nearly 5,000 breast malignancies, both test set and validation set demonstrate that TOP2A coamplification, not HER2 amplification, is the clinically useful predictive marker of an incremental response to anthracycline-based chemotherapy. Absence of HER2/TOP2A coamplification may indicate a more restricted efficacy advantage for breast cancers than previously thought.

Despite the high number of previous studies, the role of p53 alterations in prostate cancer is not clearly defined. To address the role of p53 alterations in prostate cancer biology, a total of 2514 cancers treated by radical prostatectomy were successfully analyzed by immunohistochemistry in a tissue microarray format. Overall a low rate of p53-positive tumors was found (2.5%). A significant underestimation of p53-positive cases was excluded by subsequent large section analyses and direct sequencing of the p53 gene in subsets of our patients. Large section analysis of 23 cases considered negative on the tissue microarray yielded only one weakly p53-positive tumor. Only 4 out of 64 (6.4%) high-grade tumors, that were considered negative for p53 by immunohistochemistry, presented exon 5–8 mutations. These data suggest a high sensitivity of our immunohistochemistry approach and confirm the overall low frequency of p53 alterations in clinically localized prostate cancer. A positive p53 immunostaining was strongly associated with presence of exon 5–8 mutations (P<0.0001), advanced pT-stage (P<0.0001), high Gleason grade (P<0.0001), positive surgical margins (P=0.03) and early biochemical tumor recurrence (P<0.0001). A higher rate of positive p53 immunostaining was detected in late-stage diseases including metastatic prostate cancer (P=0.0152) and hormone-refractory tumors (P=0.0003). Moreover, p53 expression was identified as an independent predictor of biochemical tumor recurrence in the subgroup of low- and intermediate-grade cancers. In summary, the results of this study show that p53 mutations characterize a small biologically aggressive subgroup of prostate cancers with a high risk of progression after prostatectomy. The rate of p53 alterations increases with prostate cancer progression.

HER-2 is the target for antibody based treatment of breast cancer (Herceptin). In order to evaluate the potential role of such a treatment in esophageal cancers, HER-2 amplification and overexpression was investigated in primary and metastatic cancers of the esophagus. A tissue microarray was constructed from 255 primary esophageal cancers (110 adenocarcinomas and 145 squamous cell carcinomas), 89 nodal and 33 distant metastases. Slides were analyzed by immunohistochemistry (HercepTest; DAKO) and fluorescence in situ hybridization (FISH; PathVysion; Vysis-Abbott) for HER-2 amplification and overexpression. Amplification was seen in 16/110 (15%) adenocarcinomas and in 7/145 (5%) squamous cell carcinomas. There was a strong association between HER-2 amplification and overexpression, especially in adenocarcinomas (P<0.0001, log rank). There was a 100% concordance of the HER-2 results in primary tumor and corresponding metastases in 84 analyzed pairs. Amplification was typically high-level with more than 10-15 HER-2 copies per tumor cell. Amplification was unrelated to survival, grading, pT, pN, pM or UICC stage. We conclude that esophageal adenocarcinomas belong to those cancer types with relevant frequency high-level HER-2 gene amplification clinical trials or individual case studies investigating the response of metastatic HER-2-positive esophageal cancers to Herceptin((R)) should be undertaken. The strong concordance of the HER-2 status in primary and metastatic cancers argues for a possible response of metastases from patients with HER-2-positive primary tumors to Herceptin.

Prostate cancer is driven by a combination of genetic and/or epigenetic alterations. Epigenetic alterations are frequently observed in all human cancers, yet how aberrant epigenetic signatures are established is poorly understood. Here we show that the gene encoding BAZ2A (TIP5), a factor previously implicated in epigenetic rRNA gene silencing, is overexpressed in prostate cancer and is paradoxically involved in maintaining prostate cancer cell growth, a feature specific to cancer cells. BAZ2A regulates numerous protein-coding genes and directly interacts with EZH2 to maintain epigenetic silencing at genes repressed in metastasis. BAZ2A overexpression is tightly associated with a molecular subtype displaying a CpG island methylator phenotype (CIMP). Finally, high BAZ2A levels serve as an independent predictor of biochemical recurrence in a cohort of 7,682 individuals with prostate cancer. This work identifies a new aberrant role for the epigenetic regulator BAZ2A, which can also serve as a useful marker for metastatic potential in prostate cancer.

TMPRSS2-ERG gene fusions occur in about 50% of all prostate cancer cases and represent promising markers for molecular subtyping. Although TMPRSS2-ERG fusion seems to be a critical event in prostate cancer, the precise functional role in cancer development and progression is still unclear.We studied large-scale gene expression profiles in 47 prostate tumor tissue samples and in 48 normal prostate tissue samples taken from the non-suspect area of clinical low-risk tumors using Affymetrix GeneChip Exon 1.0 ST microarrays.Comparison of gene expression levels among TMPRSS2-ERG fusion-positive and negative tumors as well as benign samples demonstrated a distinct transcriptional program induced by the gene fusion event. Well-known biomarkers for prostate cancer detection like CRISP3 were found to be associated with the gene fusion status. WNT and TGF-β/BMP signaling pathways were significantly associated with genes upregulated in TMPRSS2-ERG fusion-positive tumors.The TMPRSS2-ERG gene fusion results in the modulation of transcriptional patterns and cellular pathways with potential consequences for prostate cancer progression. Well-known biomarkers for prostate cancer detection were found to be associated with the gene fusion. Our results suggest that the fusion status should be considered in retrospective and future studies to assess biomarkers for prostate cancer detection, progression and targeted therapy.

6q12-22 is the second most commonly deleted genomic region in prostate cancer. Mapping studies have described a minimally deleted area at 6q15, containing MAP3K7/TAK1, which was recently shown to have tumor suppressive properties. To determine prevalence and clinical significance of MAP3K7 alterations in prostate cancer, a tissue microarray containing 4699 prostate cancer samples was analyzed by fluorescence in situ hybridization. Heterozygous MAP3K7 deletions were found in 18.48% of 2289 interpretable prostate cancers. MAP3K7 deletions were significantly associated with advanced tumor stage (P<0.0001), high Gleason grade (P<0.0001), lymph node metastasis (P<0.0108) and early biochemical recurrence (P<0.0001). MAP3K7 alterations were typically limited to the loss of one allele as homozygous deletions were virtually absent and sequencing analyses revealed no evidence for MAP3K7 mutations in 15 deleted and in 14 non-deleted cancers. There was a striking inverse association of MAP3K7 deletions and TMPRSS2:ERG fusion status with 26.7% 6q deletions in 1125 ERG-negative and 11.1% 6q deletions in 1198 ERG-positive cancers (P<0.0001). However, the strong prognostic role of 6q deletions was retained in both ERG-positive and ERG-negative cancers (P<0.0001 each). In summary, our study identifies MAP3K7 deletion as a prominent feature in ERG-negative prostate cancer with strong association to tumor aggressiveness. MAP3K7 alterations are typically limited to one allele of the gene. Together with the demonstrated tumor suppressive function in cell line experiments and lacking evidence for inactivation through hypermethylation, these results indicate MAP3K7 as a gene for which haploinsufficency is substantially tumorigenic.

Abstract Prostate cancer is the second most common cancer among men worldwide. Alterations in the DNA methylation pattern can be one of the leading causes for prostate cancer formation. This study is the first high-throughput sequencing study investigating genome-wide DNA methylation patterns in a large cohort of 51 tumor and 53 benign prostate samples using methylated DNA immunoprecipitation sequencing. Comparative analyses identified more than 147,000 cancer-associated epigenetic alterations. In addition, global methylation patterns show significant differences based on the TMPRSS2–ERG rearrangement status. We propose the hypermethylation of miR-26a as an alternative pathway of ERG rearrangement-independent EZH2 activation. The observed increase in differential methylation events in fusion–negative tumors can explain the tumorigenic process in the absence of genomic rearrangements. Significance: In contrast to TMPRSS2–ERG-rearranged tumors, the pathomechanism for gene fusion–negative tumors is completely unclear. Using a sequencing-based approach, our work uncovers significant global epigenetic alterations in TMPRSS2–ERG gene fusion–negative tumors and provides a mechanistic explanation for the tumor formation process. Cancer Discov; 2(11); 1024–35. ©2012 AACR. Read the Commentary on this article by Alumkal and Herman, p. 979. This article is featured in Highlights of This Issue, p. 961

Number of intratumoral mast cells predicts survival in various cancers. The prognostic significance of such mast cells in surgically treated prostate cancer is unknown.Mast cell densities were determined in prostate cancer samples of more than 2,300 hormone-naïve patients using a tissue microarray format in correlation with clinical follow-up data. Mast cells were visualized immunohistochemically (c-kit). All patients were homogeneously treated by radical prostatectomy at a single institution.Mast cells were present in 95.9% of the tumor samples. Median mast cell number on the tissue spot was 9 (range: 0-90; median density: 31 mast cells/mm(2)). High mast cell densities were significantly associated with more favorable tumors having lower preoperative prostate-specific antigen (P = 0.0021), Gleason score (P < 0.0001) and tumor stage (P < 0.0001) than tumors with low mast cell densities. Prostate-specific antigen recurrence-free survival significantly (P = 0.0001) decreased with decline of mast cell density showing poorest outcome for patients without intratumoral mast cells. In multivariate analysis mast cell density narrowly missed to add independent prognostic information (P = 0.0815) for prostate-specific antigen recurrence.High intratumoral mast cell density is associated with favorable tumor characteristics and good prognosis in prostate cancer. This finding is consistent with a role of mast cells in the immunological host-defense reaction on prostate cancer. Triggering mast cell activity might expand immunotherapeutic strategies in prostate cancer.

Deletions of 8p and gains of 8q belong to the most frequent cytogenetic alterations in prostate cancer. The target genes of these alterations and their biological significance are unknown.To determine the relationship between chromosome 8 changes, and prostate cancer phenotype and prognosis, a set of 1.954 fully annotated prostate cancers were analyzed in a tissue microarray format by fluorescence in situ hybridization.Both 8p deletions and 8q gains increased in number during different stages of prostate cancer progression. 8p deletions/8q gains were found in 26.1%/4.8% of 1,239 pT(2) cancers, 38.5%/9.8% of 379 pT(3a) cancers, 43.5%/8.9% of 237 pT(3b) cancers, 40.7%/14.8% of 27 pT(4) cancers, 39.1%/34.8% of 23 nodal metastases, 51.9%/33.3% of 27 bone metastases, and 45.5%/59.9% of 22 hormone refractory cancers (P < 0.0001 each). Both 8p deletions and 8q gains were also significantly associated with high Gleason grade and with each other (P < 0.0001 each). In primary tumors, 8p deletions were seen in only 27.3% of 1,882 cancers without 8q gain but in 57.4% of 122 tumors with 8q gain (P < 0.0001). Among cancers treated with radical prostatectomy, 8p deletions (P = 0.003) and 8q gains (P = 0.02) were associated with biochemical tumor recurrence. However, multivariate analysis (including prostate-specific antigen, pT/pN stage, Gleason score, and surgical margin status) did not reveal any statistically independent effect of 8p or 8q alterations on biochemical tumor recurrence.8p deletions and 8q gains are relatively rare in early stage prostate cancer but often develop during tumor progression. The prognostic effect does not seem to be strong enough to warrant clinical application.

Abstract Purpose: The HER2 oncogene is involved in the biology of many different tumor types and serves as a prognostic marker and a therapeutic target in breast cancer. In contrast to breast cancer, studies on Her2 overexpression and gene amplification in prostate cancer have yielded different results. The purpose of this study was to learn more on the prevalence and clinical significance of HER2 amplification and overexpression in prostate cancer. Experimental Design: A tissue microarray containing &amp;gt;2,000 prostate cancers with follow-up data was used. Tissue microarray sections were analyzed on protein and DNA level using two different antibodies (HercepTest, DAKO; Novocastra NCL-CB11) and fluorescence in situ hybridization. Results: Immunohistochemical analyses showed highly similar results for both antibodies. Detectable Her2 immunostaining was observed in 17.2% for the HercepTest and in 22.5% for the Novocastra antibody with the vast majority of cases showing 1+ or 2+ staining. For both antibodies (HercepTest/Novocastra), significant associations were found between positive staining and high Gleason grade (P &amp;lt; 0.0001, both), advanced pT stage (P &amp;lt; 0.0001/P = 0.0015), rapid tumor cell proliferation (P = 0.0004/P = 0.0071), and tumor recurrence (P &amp;lt; 0.0001, both). HER2 amplification was only found in 1 of 2,525 analyzable cases (0.04%). Conclusions: Low-level Her2 overexpression occurs at relevant frequency in prostate cancer and in the absence of gene amplification. Increased Her2 expression may potentially lead to an aggressive behavior of tumor cells through the stimulation of tumor cell proliferation because Her2 staining was shown to be significantly associated with Ki67 labeling index. These data argue for reconsidering anti-Her2 therapy, possibly with modified approaches. Clin Cancer Res; 16(5); 1553–60

Deletion of 3p13 has been reported from about 20% of prostate cancers. The clinical significance of this alteration and the tumour suppressor gene(s) driving the deletion remain to be identified. We have mapped the 3p13 deletion locus using SNP array analysis and performed fluorescence in situ hybridization (FISH) analysis to search for associations between 3p13 deletion, prostate cancer phenotype and patient prognosis in a tissue microarray containing more than 3200 prostate cancers. SNP array analysis of 72 prostate cancers revealed a small deletion at 3p13 in 14 (19%) of the tumours, including the putative tumour suppressors FOXP1, RYBP and SHQ1. FISH analysis using FOXP1-specific probes revealed deletions in 16.5% and translocations in 1.2% of 1828 interpretable cancers. 3p13 deletions were linked to adverse features of prostate cancer, including advanced stage (p < 0.0001), high Gleason grade (p = 0.0125), and early PSA recurrence (p = 0.0015). In addition, 3p13 deletions were linked to ERG+ cancers and to PTEN deletions (p < 0.0001 each). A subset analysis of ERG+ tumours revealed that 3p13 deletions occurred independently from PTEN deletions (p = 0.3126), identifying tumours with 3p13 deletion as a distinct molecular subset of ERG+ cancers. mRNA expression analysis confirmed that all 3p13 genes were down regulated by the deletion. Ectopic over-expression of FOXP1, RYBP and SHQ1 resulted in decreased colony-formation capabilities, corroborating a tumour suppressor function for all three genes. In summary, our data show that deletion of 3p13 defines a distinct and aggressive molecular subset of ERG+ prostate cancers, which is possibly driven by inactivation of multiple tumour suppressors. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Robot-assisted laparoscopic radical prostatectomy (RALP) using the da Vinci Surgical System (Intuitive Surgical, Sunnyvale, CA, USA) is now in widespread use for the management of localised prostate cancer (PCa). Many reports of the safety and efficacy of this procedure have been published. However, there are few specific reports of the limitations and complications of RALP.The primary purpose of this review is to ascertain the downsides of RALP by focusing on complications and limitations of this approach.A Medline search of the English-language literature was performed to identify all papers published since 2001 relating to RALP. Papers providing data on technical failures, complications, learning curve, or other downsides of RALP were considered. Of 412 papers identified, 68 were selected for review based on their relevance to the objective of this paper.RALP has the following principal downsides: (1) device failure occurs in 0.2-0.4% of cases; (2) assessment of functional outcome is unsatisfactory because of nonstandardised assessment techniques; (3) overall complication rates of RALP are low, although higher rates are noted when complications are reported using a standardised system; (4) long-term oncologic data and data on high-risk PCa are limited; (5) a steep learning curve exists, and although acceptable operative times can be achieved in <20 cases, positive surgical margin (PSM) rates may require experience with >80 cases before a plateau is achieved; (6) robotic assistance does not reduce the difficulty associated with obese patients and those with large prostates, middle lobes, or previous surgery, in whom outcomes are less satisfactory than in patients without such factors; (7) economic barriers prevent uniform dissemination of robotic technology.Many of the downsides of RALP identified in this paper can be addressed with longer-term data and more widespread adoption of standardised reporting measures. The significant learning curve should not be understated, and the expense of this technology continues to restrict access for many patients.

Abstract Purpose: Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involved in other types of cancer we investigated the expression of MMSET protein in different types of human tumors. Experimental Design: A monoclonal antibody against MMSET was developed and immunohistochemical staining of tissue microarrays (TMA) containing a large number of tumor samples (n = 3774) and corresponding normal tissues (n = 904) was carried out. Further validations of MMSET expression were carried out on independent, tumor-specific sets of TMAs for urinary bladder (n = 1293) and colon cancer (n = 1206) with corresponding clinicopathological data and long-term follow-up. Results: MMSET protein was highly expressed in different tumor types compared to normal counterparts. Particular frequent and/or high MMSET expression was found in carcinomas of the gastrointestinal tract (stomach, colon, anal canal), small cell lung carcinoma, tumors of the urinary bladder, female genitals, and skin. In bladder cancer, MMSET expression correlated with tumor aggressiveness. In contrast, MMSET expression was associated with good prognostic factors in colon cancer and was more pronounced in early stages of colon carcinogenesis (dysplasias) than in adenocarcinomas. However, colon cancer patients with high MMSET levels showed a worse 5-year survival. Conclusions: Our data suggest that MMSET has a broader role in cancer than previously anticipated, and further analysis might qualify it as a prognostic marker and a target for the development of therapy against several types of cancer. Clin Cancer Res; 17(9); 2919–33. ©2011 AACR.

Purpose ASCO and the College of American Pathologists (ASCO-CAP) recently recommended further changes to the evaluation of human epidermal growth factor receptor 2 gene (HER2) amplification by fluorescent in situ hybridization (FISH). We retrospectively assessed the impact of these new guidelines by using annotated Breast Cancer International Research Group (BCIRG) -005, BCIRG-006, and BCIRG-007 clinical trials data for which we have detailed outcomes. Patients and Methods The HER2 FISH status of BCIRG-005/006/007 patients with breast cancers was re-evaluated according to current ASCO-CAP guidelines, which designates five different groups according to HER2 FISH ratio and average HER2 gene copy number per tumor cell: group 1 (in situ hybridization [ISH]-positive): HER2-to-chromosome 17 centromere ratio ≥ 2.0, average HER2 copies ≥ 4.0; group 2 (ISH-positive): ratio ≥ 2.0, copies < 4.0; group 3 (ISH-positive): ratio < 2.0, copies ≥ 6.0; group 4 (ISH-equivocal): ratio < 2.0, copies ≥ 4.0 and < 6.0; and group 5 (ISH-negative): ratio < 2.0, copies < 4.0. We assessed correlations with HER2 protein, clinical outcomes by disease-free survival (DFS) and overall survival (OS) and benefit from trastuzumab therapy (hazard ratio [HR]). Results Among 10,468 patients with breast cancers who were successfully screened for trial entry, 40.8% were in ASCO-CAP ISH group 1, 0.7% in group 2; 0.5% in group 3, 4.1% in group 4, and 53.9% in group 5. Distributions were similar in screened compared with accrued subpopulations. Among accrued patients, FISH group 1 breast cancers were strongly correlated with immunohistochemistry 3+ status (P < .0001), whereas groups 2, 3, 4, and 5 were not; however, groups 2, 4 and, 5 were strongly correlated with immunohistochemistry 0/1+ status (all P < .0001), whereas group 3 was not. Among patients accrued to BCIRG-005, group 4 was not associated with significantly worse DFS or OS compared with group 5. Among patients accrued to BCIRG-006, only group 1 showed a significant benefit from trastuzumab therapy (DFS HR, 0.71; 95% CI, 0.60 to 0.83; P < .0001; OS HR, 0.69; 95% CI, 0.55 to 0.85; P = .0006), whereas group 2 did not. Conclusion Our findings support the original categorizations of HER2 by FISH status in BCIRG/Translational Research in Oncology trials.

Intra-tumor heterogeneity is a potential cause for failure of targeted therapy in gastric cancer, but the extent of heterogeneity of established (HER2) or potential (EGFR, CCND1) target genes and prognostic gene alterations (MYC) had not been systematically studied.To study heterogeneity of these genes in a large patient cohort, a heterogeneity tissue microarray was constructed containing 0.6 mm tissue cores from 9 different areas of the primary gastric cancers of 113 patients and matched lymph node metastases from 61 of these patients. Dual color fluorescence in-situ hybridization was performed to assess amplification of HER2, EGFR, CCND1 and MYC using established thresholds (ratio ≥ 2.0). Her2 immunohistochemistry (IHC) was performed in addition.Amplification was found in 17.4% of 109 interpretable cases for HER2, 6.4% for EGFR, 17.4% for CCND1, and 24.8% for MYC. HER2 amplification was strongly linked to protein overexpression by IHC in a spot-by-spot analysis (p < 0.0001). Intra-tumor heterogeneity was found in the primary tumors of 9 of 19 (47.3%) cancers with HER2, 8 of 17 (47.0%) cancers with CCND1, 5 of 7 (71.4%) cancers with EGFR, and 23 of 27 (85.2%) cancers with MYC amplification. Amplification heterogeneity was particularly frequent in case of low-level amplification (<10 gene copies). While the amplification status was often different between metastases, unequivocal intra-tumor heterogeneity was not found in individual metastases.The data of our study demonstrate that heterogeneity is common for biomarkers in gastric cancer. Given that both TMA tissue cores and clinical tumor biopsies analyze only a small fraction of the tumor bulk, it can be concluded that such heterogeneity may potentially limit treatment decisions based on the analysis of a single clinical cancer biopsy.

To correlate Ki67 expression with outcome in colorectal cancer (CRC).Ki67 labelling index (Ki67LI) was analysed by immunohistochemistry on a tissue microarray containing 1800 CRCs. The results were compared with clinicopathological and molecular parameters.Ki67LI was considered low in 26.3%, moderate in 56.7% and high in 17.0% of 1653 interpretable CRCs. High Ki67 expression was associated with low tumour stage (p<0.0001) and nodal status (p=0.0315), but not with tumour grade (p=0.8639), histological tumour type (p=0.1542) or tumour localisation, and was an independent prognosticator of favourable survival (p=0.0121). High Ki67 expression was also significantly associated with high-level nuclear β-catenin and p53 expression (p<0.0001 and p=0.0095, respectively).In summary, our data show that high Ki67 expression in CRCs is associated with good clinical outcome. Ki67, p53 and β-catenin overexpression seem to be linked to CRC, and indicate a cellular state of high proliferative activity. Finally, our findings strongly argue for a clinical utility of Ki67 immunostaining as an independent prognostic biomarker in CRC.

Cell-mediated immunity may impact prostate cancer progression and has great therapeutic potential. Here, we investigated the clinical significance of the numeric density of regulatory T cells (Tregs) in prostate cancer as the presence of such cells in the tumour microenvironment has been linked to clinical outcome in other tumour entities. We detected Tregs by FOXP3 immunohistochemistry in 88.8% of 2002 prostate cancer specimens in tissue microarray format, the largest cohort so far in which Tregs have been quantified. The density of Tregs in tumour tissue was compared with pathological parameters and clinical outcome. The number of Tregs identified per 0.6mm tissue spot ranged from 1 to 10 in normal and 1 to 103 FOXP3+ cells in tumour samples. Prostate-specific antigen (PSA) recurrence-free survival was significantly reduced in patients with higher numbers of Tregs (p=0.0151). Further, a higher number of intratumoural FOXP3+ Tregs was associated with a more advanced tumour stage (p=0.0355) and higher Ki67 labelling index (p<0.0001). The tissue density of Tregs was unrelated to other clinical parameters such as spread to lymph nodes, preoperative PSA level and Gleason score. Our study suggests that the intratumoural presence of regulatory T cells may have substantial functional impact and may confer an adverse clinical course in prostate cancer.

Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses.

Abstract RAD51 is the central protein in the homologous recombination pathway and is therefore of great relevance in terms of both therapy resistance as well as genomic stability. By using a tissue microarray analysis of 1,213 biopsies taken from colorectal adenocarcinomas (CRCs), we investigated whether RAD51 expression can be used as a prognostic marker as well as potential associations between this and the expression of other proteins known to be related to CRC. Strong RAD51 expression was observed in 1% of CRC, moderate in 11%, weak in 34% and no expression in 44%. No correlation was found between RAD51 expression and clinicopathological parameters. RAD51 expression correlated significantly ( p = 0.001) with overall survival, with a median survival of 11 months for patients with strong, 46 with moderate, 76 with weak and 68 with negative expression. Multivariate analyses revealed that in addition to tumor stage ( p &lt; 0.0001) and nodal status ( p &lt; 0.0001), RAD51 expression is also an independent prognostic parameter ( p = 0.011). Strong RAD51 expression was found to be associated with the loss of the two DNA mismatch repair proteins MSH ( p = 0.0003), MLH ( p = 0.002) and β‐catenin ( p = 0.012) as well as with elevated p21 ( p = 0.003) and EGFR expression ( p = 0.0001). However, a correlation with overall survival could only be found for EGFR expression ( p = 0.008), although no added benefit in risk stratification could be determined when evaluated together with RAD51. Overexpression of RAD51 is a predictor of poor outcome in CRC. This finding indicated the promise of future studies using RAD51 as a prognostic marker and therapeutic target.

Nuclear mutations are well known to drive tumor incidence, aggression and response to therapy. By contrast, the frequency and roles of mutations in the maternally inherited mitochondrial genome are poorly understood. Here we sequence the mitochondrial genomes of 384 localized prostate cancer patients, and identify a median of one mitochondrial single-nucleotide variant (mtSNV) per patient. Some of these mtSNVs occur in recurrent mutational hotspots and associate with aggressive disease. Younger patients have fewer mtSNVs than those who diagnosed at an older age. We demonstrate strong links between mitochondrial and nuclear mutational profiles, with co-occurrence between specific mutations. For example, certain control region mtSNVs co-occur with gain of the MYC oncogene, and these mutations are jointly associated with patient survival. These data demonstrate frequent mitochondrial mutation in prostate cancer, and suggest interplay between nuclear and mitochondrial mutational profiles in prostate cancer.In prostate cancer, the role of mutations in the maternally-inherited mitochondrial genome are not well known. Here, the authors demonstrate frequent, age-dependent mitochondrial mutation in prostate cancer. Strong links between mitochondrial and nuclear mutational profiles are associated with clinical aggressivity.

The benefit of intraoperative neurovascular structure-adjacent frozen section examination (NeuroSAFE) of the prostate was demonstrated in open radical prostatectomy. In da Vinci robot-assisted prostatectomy (DVP), this approach is often avoided due to suspected difficulties in harvesting the prostate, loss in pneumoperitoneum, increased blood loss, and prolonged operating room (OR) time.To provide a detailed description of the technique, feasibility, and impact of the NeuroSAFE technique on OR time, blood loss, frequency of nerve sparing (NS), and positive surgical margins (PSMs) in DVP.We analyzed 1570 consecutive patients undergoing DVP from 2004 to 2012. NeuroSAFE was performed in 1178 patients.The prostate was intraoperatively harvested via an extension of the camera trocar incision without undocking the robotic arms. Blood spillage from the dorsal vein complex due to the loss of pneumoperitoneum was avoided by upward traction on the transurethral catheter. After prostate removal, pneumoperitoneum was reestablished by closing the extended incision with running sutures and repositioning the optical trocar. The NeuroSAFE procedure consisted of intraoperative bilateral frozen sections covering the entire neurovascular bundles adjacent prostate surface.We compared OR time, blood loss, NS frequency, and PSMs in non-NeuroSAFE versus NeuroSAFE DVP.There was no significant difference in blood loss (253.5 ± 204.4 ml vs 265.8 ± 246.7 ml; p=0.49) and OR time (220 min ± 51 vs 224 min ± 64; p=0.22). No complications associated with specimen harvesting occurred. NS rate increased significantly with versus without NeuroSAFE (overall 97% vs 81%; pT2 99% vs 90%, pT3a 94% vs 74%, pT3b 91% vs 30). PSM rate dropped significantly with NeuroSAFE (overall 16% vs 24%; pT2 8% vs 15%, pT3a 22% vs 39%, pT3b 49% vs 67%; all p<0.05).We demonstrate a time-efficient and safe adaption of the NeuroSAFE technique to DVP.We describe a feasible and secure adaption of the neurovascular structure-adjacent frozen section examination (NeuroSAFE) procedure for da Vinci robot-assisted prostatectomy. We showed that there was no increased blood loss and operating room time. We maximized the nerve-sparing frequency and could reduce positive surgical margins even in non-organ-confined tumors.

Despite a multitude of p53 immunohistochemistry (IHC) studies, data on the combined effect of nuclear p53 protein accumulation and TP53 genomic inactivation are lacking for prostate cancer. A tissue microarray including 11,152 prostate cancer samples was analyzed by p53 IHC and fluorescence in situ hybridization. Nuclear p53 accumulation was found in 10.1% of patients including 1.4% with high-level and 8.7% with low-level immunostaining. TP53 sequencing revealed that 17 of 22 (77%) cases with high-level p53 immunostaining, but only 3% (1 of 31) low-level p53 cases carried putative dominant-negative mutations. TP53 deletions occurred in 14.8% of cancers. Both deletions and protein accumulation were linked to unfavorable tumor phenotype and prostate specific antigen (PSA) recurrence (p < 0.0001 each). The combination of both methods revealed subgroups with remarkable differences in their clinical course. Tumors with either TP53 deletion (14%) or low-level p53 positivity (8.7%) had identical risks of PSA recurrence, which were markedly higher than in cancers without p53 alterations (p < 0.0001). Tumors with both p53 deletion and low-level p53 positivity (1.5%) had a worse prognosis than patients with only one of these alterations (p < 0.0001). Tumors with strong p53 immunostaining or homozygous inactivation through deletion of one allele and disrupting translocation involving the second allele had the worst outcome, independent from clinical and pathological parameters. These data demonstrate a differential clinical impact of various TP53 alterations in prostate cancer. Strong p53 immunostaining—most likely accompanying dominant negative or oncogenic p53 mutation—has independent prognostic relevance and may thus represent a clinical useful molecular feature of prostate cancer.

Abstract Background The clinical course of prostate cancer (PCa) is highly variable, demanding an individualized approach to therapy. Overtreatment of indolent PCa cases, which likely do not progress to aggressive stages, may be associated with severe side effects and considerable costs. These could be avoided by utilizing robust prognostic markers to guide treatment decisions. Results We present a random forest-based classification model to predict aggressive behaviour of prostate cancer. DNA methylation changes between PCa cases with good or poor prognosis (discovery cohort with n = 70) were used as input. DNA was extracted from formalin-fixed tumour tissue, and genome-wide DNA methylation differences between both groups were assessed using Illumina HumanMethylation450 arrays. For the random forest-based modelling, the discovery cohort was randomly split into a training (80%) and a test set (20%). Our methylation-based classifier demonstrated excellent performance in discriminating prognosis subgroups in the test set (Kaplan-Meier survival analyses with log-rank p value &lt; 0.0001). The area under the receiver operating characteristic curve (AUC) for the sensitivity analysis was 95%. Using the ICGC cohort of early- and late-onset prostate cancer ( n = 222) and the TCGA PRAD cohort ( n = 477) for external validation, AUCs for sensitivity analyses were 77.1% and 68.7%, respectively. Cancer progression-related DNA hypomethylation was frequently located in ‘partially methylated domains’ (PMDs)—large-scale genomic areas with progressive loss of DNA methylation linked to mitotic cell division. We selected several candidate genes with differential methylation in gene promoter regions for additional validation at the protein expression level by immunohistochemistry in &gt; 12,000 tissue micro-arrayed PCa cases. Loss of ZIC2 protein expression was associated with poor prognosis and correlated with significantly shorter time to biochemical recurrence. The prognostic value of ZIC2 proved to be independent from established clinicopathological variables including Gleason grade, tumour stage, nodal stage and prostate-specific-antigen. Conclusions Our results highlight the prognostic relevance of methylation loss in PMD regions, as well as of several candidate genes not previously associated with PCa progression. Our robust and externally validated PCa classification model either directly or via protein expression analyses of the identified top-ranked candidate genes will support the clinical management of prostate cancer.

Mesothelin (MSLN) represents an attractive molecule for targeted cancer therapies. To identify tumors that might benefit from such therapies, tissue microarrays including 15,050 tumors from 122 different tumor types and 76 healthy organs were analyzed for MSLN expression by immunohistochemistry. Sixty-six (54%) tumor types showed at least occasional weak staining, including 50 (41%) tumor types with at least one strongly positive sample. Highest prevalence of MSLN positivity had ovarian carcinomas (serous 97%, clear cell 83%, endometrioid 77%, mucinous 71%, carcinosarcoma 65%), pancreatic adenocarcinoma (ductal 75%, ampullary 81%), endometrial carcinomas (clear cell 71%, serous 57%, carcinosarcoma 50%, endometrioid 45%), malignant mesothelioma (69%), and adenocarcinoma of the lung (55%). MSLN was rare in cancers of the breast (7% of 1138), kidney (7% of 807), thyroid gland (1% of 638), soft tissues (0.3% of 931), and prostate (0 of 481). High expression was linked to advanced pathological tumor (pT) stage (p &lt; 0.0001) and metastasis (p &lt; 0.0001) in 1619 colorectal adenocarcinomas, but unrelated to parameters of malignancy in 1072 breast-, 386 ovarian-, 174 lung-, 757 kidney-, 171 endometrial-, 373 gastric-, and 925 bladder carcinomas. In summary, numerous important cancer types with high-level MSLN expression might benefit from future anti-MSLN therapies, but MSLN’s prognostic relevance appears to be limited.

Cyclin E amplification and overexpression have recently been described in several tumour types. However, many tumour entities have never been examined for cyclin E alterations. Numerous and time-consuming experiments were previously required to determine the significance of potential oncogenes across different tumour types. To overcome this problem, tissue microarrays (TMAs) consisting of 3670 primary tumours from 128 different tumour types, 709 metastases, and 354 normal tissues were generated. Cyclin E alterations were then analysed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Cyclin E gene amplification was observed in 15 different tumour types and subtypes, ie rhabdomyosarcoma, urinary bladder cancer (three subtypes), ovarian cancer (two subtypes), malignant fibrous histiocytoma, adenocarcinoma of the small intestine, medullary breast cancer, gall bladder adenocarcinoma, phaeochromocytoma, gastric adenocarcinoma, squamous cell carcinoma of the uterine cervix, colonic adenocarcinoma, and endometrial carcinoma. Cyclin E protein accumulation was found in 48 different tumour types. The use of TMA technology has enabled us to expand considerably our knowledge of cyclin E alterations in human tumours. The occurrence of amplification and overexpression in many different tumour types suggests that cyclin E plays an important role in tumour biology.

Abstract Purpose: EMMPRIN (extracellular matrix metalloprotease inducer) is a glycosylated member of the immunoglobulin superfamily known to stimulate the production of matrix metalloproteases (MMPs) 1, 2, and 3 and MT1-MMP in peritumoral fibroblasts. We here evaluated whether EMMPRIN expression is related to tumor progression in human breast cancer. Experimental Design: An immunohistochemical study using high-density tissue microarrays (n = 2222 breast cancer samples) and EMMPRIN-specific antibodies HIM6 and MEM-M6/1 was performed, and staining results were statistically correlated with various clinicopathological parameters. To analyze the putative association between EMMPRIN expression and bone marrow (BM) micrometastasis, an additional set of 55 breast tumors from patients with or without micrometastatic cells as determined with anti-cytokeratin antibody A45-B/B3 were included in our study. Cytokeratin-positive cells in BM were costained with EMMPRIN-specific antibody 1G6.2. Results: Positive EMMPRIN staining correlated significantly with various histopathological risk factors (higher tumor grade, increased tumor size, negative estrogen receptor status and progesterone receptor status, and higher mitotic index) as well as decreased tumor-specific survival (log-rank, P = 0.0027). In particular, in patients &amp;gt; 50 years (i.e., postmenopausal women), EMMPRIN expression was an independent prognosticator as shown by Cox regression analysis (relative risk = 1.7, 95% confidence interval 1.4–4.3, P = 0.036). An involvement of EMMPRIN in tumor progression was also supported by the fact that it was expressed on ∼90% of micrometastatic cells in BM. Conclusions: EMMPRIN expression in primary tumor predicts an unfavorable prognosis in breast cancer, suggesting a crucial role of EMMPRIN in progression of human mammary carcinomas.

Tissue microarrays (TMAs) are potentially suited to find associations between molecular features and clinical outcome. Enhanced cell proliferation, as measured by Ki67 immunohistochemistry, is related to poor patient prognosis in many different tumor types. Ki67 expression shows considerable intratumoral heterogeneity. It is unclear if the TMA format is suitable for the analysis of potentially heterogeneous markers because of the small size of TMA spots. We have analyzed a breast cancer TMA containing 2,517 breast tissues, including 2,222 neoplastic and 295 normal or premalignant samples, for Ki67 labeling index (Ki67 LI) and additional markers with a known relationship to Ki67 LI by immunohistochemistry (ER, PR, Bcl-2, Egfr, p16, p53) and Fluorescence in situ hybridization (HER2, MDM2, CCND1, MYC). A high Ki67 LI was linked to tumor phenotype including grade (p < 0.0001), stage (p < 0.0001), nodal stage (p = 0.0018), and patient prognosis (p < 0.0001), elevated protein levels of p53, p16 and Egfr, reduced levels of Bcl2, ER, and PR (p < 0.0001 each), as well as amplifications of HER2, MYC, CCND1 and MDM2 (p < 0.0001 each). In summary, all expected associations between Ki67 and the analyzed molecular markers could be reproduced with high statistical significance using a TMA containing only one tissue sample per tumor, measuring 0.6 mm in diameter. We conclude that associations with cell proliferation can be reliably analyzed in a TMA format.

By differential quantitative protein expression, it has previously been shown that annexin A3 (ANXA3) expression is associated with prostate cancer. However little is known about the role and biology of ANXA3 in the human prostate. The aim of this study was to thoroughly analyze ANXA3 expression patterns and its potential as a prognostic marker in a large set of benign, preneoplastic, and neoplastic prostate tissue samples. Immunohistochemistry-based ANXA3 protein expression was analyzed for 1589 prostate cancers as well as smaller subsets of benign epithelium and high-grade prostatic intraepithelial neoplasia (PIN) in a tissue microarray format. All samples of benign prostatic epithelium and PIN showed ANXA3 protein expression, with PIN lesions showing a decreased staining intensity compared with benign epithelium (p < 0.0001). In cancer, ANXA3 protein expression was essentially reduced, resulting in a negative staining rate of 27.2%, which correlated with increasing pT stage and Gleason score (p < 0.0001). ANXA3 status in cancer was shown to be an independent adverse prognostic factor and enabled substratification of the large group of intermediate-risk patients (n = 969) into high- and low-risk subgroups. ANXA3 represents a promising candidate tissue marker, and when combined with the standard prognostic parameters, is suggested to provide a more precise prediction of prognosis in the individual patient, therefore harboring the potential to contribute to future patient management.

We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.

Study Type – Therapy (case series) Level of Evidence 4 OBJECTIVE To examine the long‐term rates of biochemical recurrence (BCR)‐free survival, cancer‐specific mortality (CSM)‐free survival, and overall survival (OS) in patients with prostate cancer treated with open radical prostatectomy (RP) in the prostate‐specific antigen (PSA) era. PATIENTS AND METHODS The study comprised 436 patients who were treated with RP between 1992 and 1997 at our institution. None received adjuvant/salvage therapy in the absence of BCR. The BCR‐free, CSM‐free and OS rates were defined using the Kaplan‐Meier method. Multivariable Cox‐regression models were used to test the effect of age, preoperative PSA level, neoadjuvant hormonal therapy, pT stage, lymph node status, RP Gleason sum and surgical margin status on BCR. RESULTS The median follow‐up of censored patients was 122, 128, and 132 months for, respectively, BCR‐free, CSM‐free and OS estimates. The 10‐year event‐free survival rates for the same endpoints were 60%, 94% and 86%, respectively. Preoperative PSA level, RP Gleason sum, pT stage, lymph node status, and surgical margin status were independent predictors of BCR (all adjusted P &lt; 0.05). CONCLUSIONS This study is the first to evaluate the long‐term cancer control outcomes after RP from a European country in the PSA era. Our data indicate that RP provides excellent long‐term survival rates in patients with clinically localized prostate cancer. Although ≈40% of patients have BCR after 10 years of follow‐up, the CSM rate after 10 years is as low as 6%.

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+)-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.

A positive surgical margin after radical prostatectomy is considered an adverse prognostic feature. However, few groups have explored the potential interaction between surgical margin status and other cancer characteristics, specifically pathological stage. We addressed the first degree of interaction between positive surgical margins and other established adverse predictors of biochemical recurrence after radical prostatectomy.We used univariate and multivariate analysis to test the effect of surgical margin status on biochemical recurrence in 4,490 patients treated at a single institution between 1992 and 2008. We systematically tested all first-degree interactions between surgical margin status, and pretreatment prostate specific antigen, pT and pN stage, and radical prostatectomy Gleason sum. If interactions were significant, we quantified the effect on the biochemical recurrence rate.Overall 850 patients (18.9%) had positive surgical margins. In those with negative vs positive surgical margins the 5-year biochemical recurrence-free survival rate was 95% vs 83%, 74% vs 62% and 47% vs 29% for pT2, pT3a and pT3b disease, respectively. In multivariate models only the pT stage-surgical margin status interaction achieved independent predictor status (p = 0.003). Negative vs positive surgical margin multivariate HRs were 1 vs 2.9, 2.3 vs 4.3 and 4.1 vs 5.6 in pT2, pT3a and pT3b cases, respectively.Compared to negative surgical margins, positive surgical margins increase the absolute biochemical recurrence 5-year rate by 12% to 18%. More importantly, positive surgical margins may substantially worsen the prognosis beyond that of the original pathological disease stage.

HER-2 is the molecular target for antibody-based treatment of breast cancer (trastuzumab). The potential benefit of anti–HER-2 therapy is currently investigated in several other HER-2 amplified cancers. For example, trastuzumab was recently shown to be effective in HER-2 positive gastric cancer. To address the potential applicability of anti–HER-2 therapy in colorectal cancer, tissue microarray sections and colorectal resection specimens of 1851 colorectal cancers were analyzed for HER-2 overexpression and amplification using FDA approved reagents for immunohistochemistry and fluorescence in situ hybridization. HER-2 amplification was seen in 2.5% and HER-2 overexpression in 2.7% of 1439 interpretable colorectal cancers. Amplification was often high level with HER-2 copies ranging from 4 to 60 per tumor cell and was strongly related to protein overexpression. HER-2 amplification and overexpression were unrelated to histological tumor type, tumor localization, grading, pT, pN, pM or survival. As heterogeneity of drug target expression could represent a major drawback for targeted cancer therapy we next studied HER-2 heterogeneity in selected cases. Extensive evaluation of all available large sections from patients with HER-2 positive colorectal cancer revealed heterogenous findings in 3 of 4 cases. In summary, high-level HER-2 amplification occurs in a small fraction of colorectal cancers. Heterogeneity of amplification may limit the utility of anti- HER-2 therapy in some of these tumors and therefore, adequate clinical trials are needed to further evaluate this approach.

HER2 is a target for antibody-based treatment of breast and gastric carcinoma which is highly successful in advanced disease as well as in the adjuvant setting. To determine the potential applicability of such therapies, salivary gland tumours were analysed for HER2 overexpression/amplification in this study.A tissue microarray (TMA) was constructed from 994 carcinomas and 205 adenomas of the salivary gland. Slides were analysed for HER2 overexpression and gene amplification using FDA approved reagents for immunohistochemistry (IHC; Hercep-Test; Dako) and fluorescence in situ hybridisation (FISH; PathVysion; Vysis-Abbott).HER2 was found overexpressed in 39 of 915 (4.26%) and amplified in nine of 915 interpretable salivary gland carcinomas (0.98%). HER2 overexpression was mostly found in salivary duct carcinoma. All other entities were mainly HER2 negative. HER2 was overexpressed in 34 of 319 (10.65%) mucoepidermoid carcinomas, one of 170 (0.59%) acinic cell adenocarcinomas and three of 14 (21.43%) salivary duct carcinomas. HER2 amplification was seen in three of 294 (1.01%) mucoepidermoid carcinomas, three of 14 (21.43%) salivary duct carcinomas, one of 81 (1.23%) adenocarcinomas (NOS), one of 12 (8.33%) cystadenocarcinomas and one of 19 (2.08%) myoepithelial carcinomas. HER2 amplification was found in seven of 39 immunohistochemically HER2 positive (2+ and 3+) tumours (17.95%) but in only two of 849 (0.24%) IHC negative tumours (p < 0.0001). No amplification/overexpression was found in adenoid cystic carcinoma, epithelial-myoepithelial carcinoma, polymorphous low grade carcinoma, basal cell carcinoma, myoepithelial carcinoma, cystadenocarcinoma and oncocytic carcinoma.HER2 overexpression caused by gene amplification was observed in about 20% of patients with salivary duct cancers but was rare in salivary adenomas and other carcinomas. Therefore, anti-HER2 therapy may represent a therapeutic option only in a small subgroup of salivary gland tumours.

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.

TMPRSS2:ERG fusions, in combination with deletion of the phosphatase and tensin homolog (PTEN) tumor suppressor, have been suggested to cooperatively drive tumor progression in prostate cancer. We utilized a novel heterogeneity tissue microarray containing samples from 10 different tumor blocks of 189 prostatectomy specimens to study heterogeneity of genomic PTEN alterations in individual foci. PTEN alterations were found in 48/123 (39%) analyzable individual tumor foci, including 40 foci with deletions, 7 with deletion and rearrangement, and 1 focus with rearrangement only. PTEN was homogeneously aberrant in only 4 (8%) and heterogeneously in 44 (92%) of the foci. We found a specific sequence of molecular events from PTEN breakage followed by deletion of DNA sequences flanking the breakpoint, resulting in homozygous deletion. The observation that 16 of 19 foci with homogeneous ERG positivity had focal PTEN alterations but none of 10 foci with PTEN alterations had focal ERG positivity (P<0.0001) suggests that PTEN alterations typically develop subsequent to ERG fusions. We demonstrate a high level of intratumoral heterogeneity of PTEN alterations with deletions and rearrangements that challenges potential PTEN routine diagnosis testing in biopsies. The observation that PTEN alterations develop subsequent to ERG fusion strongly suggests that ERG expression may directly drive development of PTEN aberrations.

Objective: The aim of this study was to assess oncological outcome in patients after R0/R1 resections in a homogenous cohort suffering from ductal adenocarcinoma (PDAC) of the pancreatic head.

Approximately 50% of prostate cancers are characterized by TMPRSS2 (transmembrane protease serine 2)-ERG (avian v-ets erythroblastosis virus E26 oncogene homolog) gene fusions resulting in an androgen-regulated overexpression of the transcription factor ERG. Some studies have suggested prognostic or predictive relevance of ERG status in prostate cancer. Such concepts could be impaired by extensive ERG heterogeneity in analyzed tumors. The aim of this study was to analyze the extent of heterogeneity for TMPRSS2-ERG fusion in prostate cancer. To enable large-scale studies on the extent of heterogeneity of biomarkers in prostate cancer, a heterogeneity tissue microarray containing samples from 10 different tumor blocks of 190 large prostate cancers selected from a consecutive series of 480 radical prostatectomies was developed. ERG expression was analyzed by immunohistochemistry. Positive ERG immunostaining was found in arrayed cancer-containing samples from 103 of the 178 analyzable patients (58%). ERG immunostaining was homogeneously positive in 29 prostate cancers (16%), whereas heterogeneous ERG positivity was seen in 74 cancers (42%). ERG heterogeneity was within one tumor focus (intrafocal heterogeneity) in 69 cases (93% of heterogeneous cases) and between different tumor foci (interfocal heterogeneity) in 5 cases (7%). Marked intrafocal heterogeneity challenges the concept of TMPRSS2-ERG fusion always representing an early step in prostate cancer development. Marked heterogeneity also compromises the concept of analyzing ERG status for treatment decisions in diagnostic needle core biopsies.

// Tamara L. Lotan 1, 2 , Asmus Heumann 3 , Sebastian Dwertmann Rico 3 , Jessica Hicks 1 , Kristen Lecksell 1 , Christina Koop 3 , Guido Sauter 3 , Thorsten Schlomm 4, 5 and Ronald Simon 3 1 Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA 2 Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA 3 Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4 Martini-Klinik Prostate Cancer Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 5 Department of Urology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to: Tamara L. Lotan, email: tlotan1@jhmi.edu Keywords: prostatic carcinoma, PTEN, fluorescence in situ hybridization, immunohistochemistry, biomarker Received: April 04, 2017 Accepted: April 27, 2017 Published: July 10, 2017 ABSTRACT PTEN deletion is an established prognostic biomarker in prostate cancer. We compared PTEN immunohistochemistry (IHC) and PTEN fluorescence in situ hybridization (FISH) in the largest existing radical prostatectomy cohort with clinical follow-up data. There was high concordance between IHC and FISH: 93% (3098/3330) of tumors with intact PTEN IHC showed absence of PTEN gene deletion and 66% (720/1087) of cases with PTEN protein loss by IHC showed PTEN gene deletion by FISH. 84% (447/533) of cases with PTEN homozygous gene deletion had PTEN protein loss by IHC. PTEN loss by IHC was associated with reduced PSA recurrence-free survival (RFS) in multivariable models (HR=1.3; 95% CI: 1.16-1.47). Among cases with either PTEN deletion or absence of PTEN deletion by FISH, PTEN loss by IHC was strongly associated with reduced RFS on univariable analysis (p=0.0005 and p<0.0001 respectively). Among cases with intact PTEN by IHC, homozygous (p=0.04) but not heterozygous (p=0.10) PTEN gene deletion was weakly associated with reduced RFS. Among cases with PTEN loss by IHC, both homozygous (p=0.0044) and heterozygous (p=0.0017) PTEN gene deletion were associated with reduced RFS. These data support the utility of PTEN IHC and PTEN FISH as complementary screening tools for PTEN loss in prostate cancer.

Biglycan (BGN), a proteoglycan of the extracellular matrix, is included in mRNA signatures for prostate cancer aggressiveness. To understand the impact of BGN on prognosis and its relationship to molecularly defined subsets, we analyzed BGN expression by immunohistochemistry on a tissue microarray containing 12,427 prostate cancers. Seventy-eight percent of 11,050 interpretable cancers showed BGN expression, which was considered as low intensity in 47.7% and as high intensity in 31.1% of cancers. BGN protein expression rose with increasing pathological tumor stage, Gleason grade, lymph node metastasis and early PSA recurrence (P<.0001 each). Comparison with our molecular database attached to the TMA revealed that BGN expression was linked to presence of TMPRRS2:ERG fusion and PTEN deletion (P<.0001 each). In addition, BGN was strongly linked to androgen-receptor (AR) levels (P<.0001), suggesting a hormone-depending regulation of BGN. BGN up-regulation is a frequent feature of prostate cancer that parallels tumor progression and may be useful to estimate tumor aggressiveness particularly if combined with other molecular markers.

Microtubules are multifunctional cytoskeletal proteins that are involved in crucial cellular roles including maintenance of cell shape, intracellular transport, meiosis, and mitosis. Class III beta-tubulin (βIII-tubulin, also known as TUBB3) is a microtubule protein, normally expressed in cells of neuronal origin. Its expression was also reported in various other tumor types, such as several types of lung cancer, ovarian cancer, and esophageal cancer. TUBB3 is of clinical relevance as overexpression has been linked to poor response to microtubule-targeting anti-cancer drugs such as taxanes. To systematically investigate the epidemiology of TUBB3 expression in normal and neoplastic tissues, we used tissue microarrays for analyzing the immunohistochemically detectable expression of TUBB3 in 3911 tissue samples from 100 different tumor categories and 76 different normal tissue types. At least 1 tumor with weak expression could be found in 93 of 100 (93%) different tumor types, and all these 93 entities also had at least 1 tumor with strong positivity. In normal tissues, a particularly strong expression was found in neurons of the brain, endothelium of blood vessels, fibroblasts, spermatogenic cells, stroma cells, endocrine cells, and acidophilic cells of the pituitary gland. In tumors, strong TUBB3 expression was most frequently found in various brain tumors (85%-100%), lung cancer (35%-80%), pancreatic adenocarcinoma (50%), renal cell carcinoma (15%-80%), and malignant melanoma (77%). In summary, these results identify a broad spectrum of cancers that can at least sporadically express TUBB3. Testing of TUBB3 in cancer types eligible for taxane-based therapies could be helpful to identify patients who might best benefit from this treatment.

TIGIT is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies. Here, we analysed two different types of tissue microarrays of healthy lymphatic and various inflamed tissues, colorectal and lung cancers, as well as &gt;1700 tumour samples from 86 different tumour entities for TIGIT and/or PD-1 by bright field and/or multiplex fluorescence immunohistochemistry. TIGIT was detected in CD8 + cytotoxic T cells, CD4 + T helper cells, FOXP3 + regulatory T cells, and NK cells, but not in CD11c + dendritic cells, CD68 + macrophages, and CD20 + B lymphocytes. TIGIT expression paralleled that of PD-1. More than 70% of TIGIT + cells were PD-1 + , and more than 90% of the PD-1 + cells were TIGIT + . Expression varied between different tissue compartments. TIGIT expression in tonsil gradually increased from the interfollicular area over the marginal/mantle zone to the germinal centre in all T cell subtypes. In inflammatory diseases, the strongest expression of TIGIT/PD-1 was found in Hashimoto thyroiditis. TIGIT + lymphocytes were seen in all 86 different tumour entities with considerable high variability of TIGIT positivity within and between different cancer entities. Particularly, high densities of TIGIT + lymphocytes were, for example, seen in squamous cell cancers of various origins. In summary, the variable expression levels of TIGIT and PD-1 in cell types and tissue compartments illustrate the high complexity of immune microenvironments. The high frequency of TIGIT (and PD-1) expressing lymphocytes in cancers highlights considerable opportunities for cotargeting with checkpoint inhibitors.

Mismatch repair deficient (dMMR) gastro-oesophageal adenocarcinomas (GOAs) show better outcomes than their MMR-proficient counterparts and high immunotherapy sensitivity. The hypermutator-phenotype of dMMR tumours theoretically enables high evolvability but their evolution has not been investigated. Here we apply multi-region exome sequencing (MSeq) to four treatment-naive dMMR GOAs. This reveals extreme intratumour heterogeneity (ITH), exceeding ITH in other cancer types >20-fold, but also long phylogenetic trunks which may explain the exquisite immunotherapy sensitivity of dMMR tumours. Subclonal driver mutations are common and parallel evolution occurs in RAS, PIK3CA, SWI/SNF-complex genes and in immune evasion regulators. MSeq data and evolution analysis of single region-data from 64 MSI GOAs show that chromosome 8 gains are early genetic events and that the hypermutator-phenotype remains active during progression. MSeq may be necessary for biomarker development in these heterogeneous cancers. Comparison with other MSeq-analysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies.

Abstract Background Cytokeratin 18 (CK18) is an intermediate filament protein of the cytokeratin acidic type I group and is primarily expressed in single-layered or “simple” epithelial tissues and carcinomas of different origin. Methods To systematically determine CK18 expression in normal and cancerous tissues, 11,952 tumor samples from 115 different tumor types and subtypes (including carcinomas, mesenchymal and biphasic tumors) as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Results CK18 was expressed in normal epithelial cells of most organs but absent in normal squamous epithelium. At least an occasional weak CK18 positivity was seen in 90 of 115 (78.3%) tumor types. Wide-spread CK18 positivity was seen in 37 (31.9%) of tumor entities, including adenocarcinomas of the lung, prostate, colon and pancreas as well as ovarian cancer. Tumor categories with variable CK18 immunostaining included cancer types arising from CK18 positive precursor cells but show CK18 downregulation in a fraction of cases, tumor types arising from CK18 negative precursor cells occasionally exhibiting CK18 neo-expression, tumors derived from normal tissues with variable CK18 expression, and tumors with a mixed differentiation. CK18 downregulation was for example seen in renal cell cancers and breast cancers, whereas CK18 neo-expression was found in squamous cell carcinomas of various origins. Down-regulation of CK18 in invasive breast carcinomas of no special type and clear cell renal cell carcinomas (ccRCC) was related to adverse tumor features in both tumors (p ≤ 0.0001) and poor patient prognosis in ccRCC (p = 0.0088). Up-regulation of CK18 in squamous cell carcinomas was linked to high grade and lymph node metastasis (p &lt; 0.05). In summary, CK18 is consistently expressed in various epithelial cancers, especially adenocarcinomas. Conclusions Down-regulation or loss of CK18 expression in cancers arising from CK18 positive tissues as well as CK18 neo-expression in cancers originating from CK18 negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.

Abstract Background Tumor protein 63 (p63) is a transcription factor of the p53 gene family involved in differentiation of several tissues including squamous epithelium. p63 immunohistochemistry is broadly used for tumor classification but published data on its expression in cancer is conflicting. Methods To comprehensively catalogue p63 expression, tissue microarrays (TMAs) containing 12,620 tissue samples from 115 tumor entities and 76 normal tissue types were analyzed. Results p63 expression was seen in various normal tissues including squamous epithelium and urothelium. At least occasional weak p63 positivity could be detected in 61 (53%) of 115 different tumor types. The frequencies of p63 positivity was highest in squamous cell carcinomas irrespective of their origin (96–100%), thymic tumors (100%), urothelial carcinomas (81–100%), basal type tumors such as basal cell carcinomas (100%), and various salivary gland neoplasias (81–100%). As a rule, p63 was mostly expressed in cancers derived from p63 positive normal tissues and mostly not detectable in tumors derived from p63 negative cancers. However, exceptions from this rule occurred. A positive p63 immunostaining in cancers derived from p63 negative tissues was unrelated to aggressive phenotype in 422 pancreatic cancers, 160 endometrium cancers and 374 ovarian cancers and might be caused by aberrant squamous differentiation or represent stem cell properties. In 355 gastric cancers, aberrant p63 expression occurred in 4% and was linked to lymph node metastasis ( p = 0.0208). Loss of p63 in urothelial carcinomas - derived from p63 positive urothelium - was significantly linked to advanced stage, high grade ( p &lt; 0.0001 each) and poor survival ( p &lt; 0.0001) and might reflect clinically relevant tumor dedifferentiation. Conclusion The high prevalence of p63 expression in specific tumor types makes p63 immunohistochemistry a suitable diagnostic tool. Loss of p63 expression might constitute a feature of aggressive cancers.

Abstract Background The E-Cadherin gene ( CDH1, Cadherin 1 ), located at 16q22.1 encodes for a calcium-dependent membranous glycoprotein with an important role in cellular adhesion and polarity maintenance. Methods To systematically determine E-Cadherin protein expression in normal and cancerous tissues, 14,637 tumor samples from 112 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Results E-Cadherin was strongly expressed in normal epithelial cells of most organs. From 77 tumor entities derived from cell types normally positive for E-Cadherin, 35 (45.5%) retained at least a weak E-Cadherin immunostaining in ≥99% of cases and 61 (79.2%) in ≥90% of cases. Tumors with the highest rates of E-Cadherin loss included Merkel cell carcinoma, anaplastic thyroid carcinoma, lobular carcinoma of the breast, and sarcomatoid and small cell neuroendocrine carcinomas of the urinary bladder. Reduced E-Cadherin expression was linked to higher grade ( p = 0.0009), triple negative receptor status ( p = 0.0336), and poor prognosis ( p = 0.0466) in invasive breast carcinoma of no special type, triple negative receptor status in lobular carcinoma of the breast ( p = 0.0454), advanced pT stage ( p = 0.0047) and lymph node metastasis in colorectal cancer ( p &lt; 0.0001), and was more common in recurrent than in primary prostate cancer ( p &lt; 0.0001). Of 29 tumor entities derived from E-Cadherin negative normal tissues, a weak to strong E-Cadherin staining could be detected in at least 10% of cases in 15 different tumor entities (51.7%). Tumors with the highest frequency of E-Cadherin upregulation included various subtypes of testicular germ cell tumors and renal cell carcinomas (RCC). E-Cadherin upregulation was more commonly seen in malignant than in benign soft tissue tumors ( p = 0.0104) and was associated with advanced tumor stage ( p = 0.0276) and higher grade ( p = 0.0035) in clear cell RCC, and linked to advanced tumor stage ( p = 0.0424) and poor prognosis in papillary RCC ( p ≤ 0.05). Conclusion E-Cadherin is consistently expressed in various epithelial cancers. Down-regulation or loss of E-Cadherin expression in cancers arising from E-Cadherin positive tissues as well as E-Cadherin neo-expression in cancers arising from E-Cadherin negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.

Expansion of CD8+ cytotoxic Tlymphocytes is a prerequisite for anti-cancer immune activity and has gained interest in the era of immune checkpoint therapy.To understand the CD8+ T cell dynamics in the tumor microenvironment, we used multiplex fluorescence immunohistochemistry to quantitate CD8+ proliferation (Ki67 co-expression) in tissue microarrays from 1107 colorectal, 642 renal cell, 1066 breast, 375 ovarian, 451 pancreatic and 347 gastric cancer samples.The density and the percentage of proliferating (Ki67+) CD8+ T cells were both highly variable between tumor types as well as between patients with the same tumor type. Elevated density and percentage of proliferating CD8+ cytotoxic T cells were significantly associated with favorable tumor parameters such as low tumor stage, negative nodal stage (p ≤ 0.0041 each), prolonged overall survival (p ≤ 0.0028 each) and an inflamed immune phenotype (p = 0.0025) in colorectal cancer and, in contrast, linked to high tumor stage, advanced ISUP/Fuhrman/Thoenes grading (each p ≤ 0.003), shorter overall survival (p ≤ 0.0330 each) and an immune inflamed phenotype (p = 0.0094) in renal cell cancer. In breast, ovarian, pancreatic and gastric cancer the role of (Ki67+)CD8+ Tcells was not linked to clinicopathological data.Our data demonstrate a tumor type dependent prognostic impact of proliferating (Ki67+)CD8+ Tcells and an inverse impact in colorectal and renal cell cancer.

Trophoblast cell surface antigen 2 (TROP2) is the target of sacituzumab govitecan, an antibody-drug conjugate approved for treatment of triple negative breast cancer and urothelial carcinoma.A tissue microarray containing 18,563 samples from 150 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by TROP2 immunohistochemistry.TROP2 positivity was found in 109 tumor categories, including squamous cell carcinomas of various origins, urothelial, breast, prostate, pancreatic, and ovarian cancers (>95% positive). High TROP2 expression was linked to advanced stage (p = 0.0069) and nodal metastasis (p < 0.0001) in colorectal cancer as well as to nodal metastasis in gastric adenocarcinoma (p = 0.0246) and papillary thyroid cancer (p = 0.0013). Low TROP2 expression was linked to advanced stage in urothelial carcinoma (p < 0.0001), high pT (p = 0.0024), and high grade (p < 0.0001) in breast cancer, as well as with high Fuhrmann grade (p < 0.0001) and pT stage (p = 0.0009) in papillary renal cell carcinomas.TROP2 is expressed in many epithelial neoplasms. TROP2 deregulation can be associated with cancer progression in a tumor-type dependent manner. Since anti-TROP2 cancer drugs have demonstrated efficiency, they may be applicable to a broad range of tumor entities in the future.

The Ki-67 labeling index (Ki-67 LI) is a strong prognostic marker in prostate cancer, although its analysis requires cumbersome manual quantification of Ki-67 immunostaining in 200-500 tumor cells. To enable automated Ki-67 LI assessment in routine clinical practice, a framework for automated Ki-67 LI quantification, which comprises three different artificial intelligence analysis steps and an algorithm for cell-distance analysis of multiplex fluorescence immunohistochemistry (mfIHC) staining, was developed and validated in a cohort of 12,475 prostate cancers. The prognostic impact of the Ki-67 LI was tested on a tissue microarray (TMA) containing one 0.6 mm sample per patient. A 'heterogeneity TMA' containing three to six samples from different tumor areas in each patient was used to model Ki-67 analysis of multiple different biopsies, and 30 prostate biopsies were analyzed to compare a 'classical' bright field-based Ki-67 analysis with the mfIHC-based framework. The Ki-67 LI provided strong and independent prognostic information in 11,845 analyzed prostate cancers (p < 0.001 each), and excellent agreement was found between the framework for automated Ki-67 LI assessment and the manual quantification in prostate biopsies from routine clinical practice (intraclass correlation coefficient: 0.94 [95% confidence interval: 0.87-0.97]). The analysis of the heterogeneity TMA revealed that the Ki-67 LI of the sample with the highest Gleason score (area under the curve [AUC]: 0.68) was as prognostic as the mean Ki-67 LI of all six foci (AUC: 0.71 [p = 0.24]). The combined analysis of the Ki-67 LI and Gleason score obtained on identical tissue spots showed that the Ki-67 LI added significant additional prognostic information in case of classical International Society of Urological Pathology grades (AUC: 0.82 [p = 0.002]) and quantitative Gleason score (AUC: 0.83 [p = 0.018]). The Ki-67 LI is a powerful prognostic parameter in prostate cancer that is now applicable in routine clinical practice. In the case of multiple cancer-positive biopsies, the sole automated analysis of the worst biopsy was sufficient. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract Background Despite modern imaging modalities, lymph-node staging before radical prostatectomy (RP) remains challenging in patients with prostate cancer (PCa). The visibility of lymph-node metastases (LNMs) is critically influenced by their size. Objective This study aims to describe the distribution of maximal tumor diameters (i.e., size) in LNMs of pN1-PCa at RP and its consequences on visibility in preoperative imaging and oncological outcomes. Design, setting, and participants A total of 2705 consecutive patients with pN1-PCa at RP, harboring a cumulative 7510 LNMs, were analyzed. Descriptive and multivariable analyses addressed the risk of micrometastases (MM)-only disease and the visibility of LNMs. Kaplan–Meier curves and Cox analyses were used for biochemical recurrence-free survival (BCRFS) stratified for MM-only disease. Results The median LNM size was 4.5mm (interquartile range (IQR): 2.0–9.0 mm). Of 7510 LNMs, 1966 (26%) were MM (≤ 2mm). On preoperative imaging, 526 patients (19%) showed suspicious findings (PSMA-PET/CT: 169/344, 49%). In multivariable analysis, prostate-specific antigen (PSA) (OR 0.98), age (OR 1.01), a Gleason score greater than 7 at biopsy (OR 0.73), percentage of positive cores at biopsy (OR 0.36), and neoadjuvant treatment (OR 0.51) emerged as independent predictors for less MM-only disease (p &lt; 0.05). Patients with MM-only disease compared to those harboring larger LNMs had a longer BCRFS (median 60 versus 29 months, p &lt; 0.0001). Conclusion Overall, 26% of LNMs were MM (≤ 2mm). Adverse clinical parameters were inversely associated with MM at RP. Consequently, PSMA-PET/CT did not detect a substantial proportion of LNMs. LNM size and count are relevant for prognosis.

Inter‐alpha‐trypsin inhibitor heavy chain 5 ( ITIH5 ) has been identified as a metastasis suppressor gene in pancreatic cancer. Here, we analyzed ITIH5 promoter methylation and protein expression in The Cancer Genome Atlas (TCGA) dataset and three tissue microarray cohorts ( n = 618), respectively. Cellular effects, including cell migration, focal adhesion formation and protein tyrosine kinase activity, induced by forced ITIH5 expression in pancreatic cancer cell lines were studied in stable transfectants. ITIH5 promoter hypermethylation was associated with unfavorable prognosis, while immunohistochemistry demonstrated loss of ITIH5 in the metastatic setting and worsened overall survival. Gain‐of‐function models showed a significant reduction in migration capacity, but no alteration in proliferation. Focal adhesions in cells re‐expressing ITIH5 exhibited a smaller and more rounded phenotype, typical for slow‐moving cells. An impressive increase of acetylated alpha‐tubulin was observed in ITIH5‐positive cells, indicating more stable microtubules. In addition, we found significantly decreased activities of kinases related to focal adhesion. Our results indicate that loss of ITIH5 in pancreatic cancer profoundly affects its molecular profile: ITIH5 potentially interferes with a variety of oncogenic signaling pathways, including the PI3K/AKT pathway. This may lead to altered cell migration and focal adhesion formation. These cellular alterations may contribute to the metastasis‐inhibiting properties of ITIH5 in pancreatic cancer.

To assess the reliability of the histological diagnosis of bladder cancer by assessing the interobserver variability of staging and grading in pTa/pT1 tumours and evaluating the clinical significance of discrepancies.All sections from 301 superficial bladder carcinomas were reviewed by one pathologist. The prognostic relevance of grade and stage from both the initial and review diagnosis were determined in 128 patients for whom there was long-term follow-up information.There were significant interobserver differences in both the grading and staging of tumours. From a total of 235 tumours that were initially considered pT1, the reviewer classified 35% as pTa, 56% as pT1, 6% as pT1- (at least pT1), and 3% as pT2-4. In 39% of all biopsies there were interobserver differences in tumour grade. The prognostic significance of grade and stage differed between the initial pathology report and the reviewer's diagnosis. The reviewer's staging allowed a better estimate of the risk of subsequent tumour progression than the initial staging. Progression was significantly more common in 49 tumours in which the reviewer agreed with stage pT1 than in 29 tumours that were down-staged from pT1 to pTa (P = 0.0116). However, the initial tumour grade (P = 0.0386) but not the reviewer's grade (P = 0.2645) was significantly linked to progression.These results show that grading and staging by different pathologists have varying prognostic implications. If possible, biopsies from bladder tumours should be independently evaluated by two different pathologists before radical therapy is administered.

Abstract Purpose: To comprehensively evaluate ephrin receptor B2 (EphB2) expression in normal and neoplastic tissues. EphB2 is a tyrosine kinase recently implicated in the deregulation of cell-to-cell communication in many tumors. Experimental Design: EphB2 protein expression was analyzed by immunohistochemistry on tissue microarrays that included 76 different normal tissues, &amp;gt;4,000 samples from 138 different cancer types, and 1,476 samples of colon cancer with clinical follow-up data. Results: We found most prominent EphB2 expression in the intestinal epithelium (colonic crypts) with cancer of the colorectum displaying the highest EphB2 positivity of all tumors. Positivity was found in 100% of 118 colon adenomas but in 33.3% of 45 colon carcinomas. EphB2 expression was also observed in 75 tumor categories, including serous carcinoma of the endometrium (34.8%), adenocarcinoma of the esophagus (33.3%), intestinal adenocarcinoma of the stomach (30.2%), and adenocarcinoma of the small intestine (70%). The occasional finding of strong EphB2 positivity in tumors without EphB2 positivity in the corresponding normal cells [adenocarcinoma of the lung (4%) and pancreas (2.2%)] suggests that deregulation of EphB2 signaling may involve up-regulation of the protein expression. In colon carcinoma, loss of EphB2 expression was associated with advanced stage (P &amp;lt; 0.0001) and was an indicator of poor overall survival (P = 0.0098). Conclusions: Our results provide an overview on the EphB2 protein expression in normal and neoplastic tissues. Deregulated EphB2 expression may play a role in several cancer types with loss of EphB2 expression serving as an indicator of the possible pathogenetic role of EphB2 signaling in the maintenance of tissue architecture of colon epithelium.

L1 is a cell adhesion molecule expressed at the invasive front of colorectal tumors with an important role in metastasis. The aim of the present study was to determine L1 protein expression in a large cohort of colorectal cancer patients and its impact on early metastatic spread and survival. A total of 375 patients that underwent surgical treatment for colorectal cancer were chosen retrospectively. A tissue microarray was constructed of 576 tissue samples from these patients and analyzed by immunohistochemistry with a monoclonal antibody against human L1 (UJ127). Lymph node and bone marrow micrometastasis were assessed with monoclonal antibodies Ber-EP4 and pancytokeratin A45-B/B3, respectively. Associations between L1 expression and lymph node, bone marrow micrometastasis and survival were investigated with Fisher's, log-rank test and Cox multivariate analysis. All statistical tests were two-sided. L1 was detected in a subset of 48 (13%) of 375 patients examined. Analysis of L1 expression and survival revealed a significantly worse outcome for L1-positive patients by log-rank test (P<0.05). Multivariate Cox regression analysis showed the strongest independent prognostic impact of L1 expression (P<0.05). Fisher's test revealed a significant association of L1 expression and presence of disseminated tumor cells in lymph nodes and bone marrow (P<0.05). L1 is a powerful prognostic marker for patients that undergo complete surgical resection. It may have a role in early metastatic spread, as L1 is associated with micrometastases to both the lymph nodes and bone marrow. Thus, L1 should be explored further as a target for adjuvant therapy for micrometastatic disease.

Background: Friend leukaemia integration-1 (FLI-1) antibody is a useful marker for Ewing's sarcoma/ primitive neuroectodermal tumour (EWS/PNET) and vascular tumours.However, it is also expressed in subsets of lymphoblastic lymphoma, Merkel cell carcinoma (MCC) and desmoplastic small round cell tumour (DSRCT).Aim: To determine expression of FLI-1 in various benign and malignant neoplasms, by immunohistochemical analysis on 4323 tumours using multiple tumour microarrays, as well as on whole sections.Results: FLI-1 was expressed in 46/62 EWS/PNETs, 2/3 olfactory neuroblastomas, 7/102 small cell carcinomas of the lung, 10/34 MCCs, 1/14 rhabdomyosarcoma, 19/132 non-Hodgkin's lymphomas, 2/3 DSRCTs, and in 53/74 benign and malignant vascular tumours.In addition, 27/508 squamous cell carcinomas, 19/837 adenocarcinomas, 10/400 urothelial bladder cancers, 1/40 basal cell carcinomas, 3/ 29 liposarcomas, 1/40 glioblastoma multiforme and 9/29 medullar carcinomas of the breast expressed FLI-1.The sensitivity and specificity of FLI-1 to distinguish EWS/PNET from all types of malignancies were 74.2% and 96.0%, respectively.Finally, the sensitivity and specificity of FLI-1 to distinguish EWS/PNET from other small round cell tumours (SRCTs) were 74.2% and 91.6%, respectively.Conclusion: This study was the first to show that FLI-1 can be seen in a variety of solid tumours, some of which had never been explored before.This finding should be kept in mind, especially when using FLI-1 as a marker for finding the primary origin of poorly differentiated metastatic tumour.Finally, despite the expression of FLI-1 in numerous malignancies, it is still considered to be highly sensitive and specific in distinguishing EWS/ PNET from other tumour types in general and from other SRCTs in particular.

Immunohistochemistry (IHC) is the gold standard methodology for in-situ protein expression analysis in tissue samples. The combination of IHC and tissue microarray (TMA) technology allows for the simultaneous analysis of hundreds of tissue samples with an unprecedented degree of experimental standardization. The same immunostaining protocols used for conventional large sections can be used for TMAs, including antigen retrieval procedures for staining of routinely archived formalin-fixed tissue samples. The development of optimal IHC protocols is highly important for TMA studies because minor protocol variations often have a marked impact on the outcome of the staining. Preabsorption and isotype-specific control experiments should be included as the last step in protocol development to proof target protein-specific binding. Such controls are particularly important for new antibodies with unknown staining patterns.

Multiple different biologically and clinically relevant genes are often amplified in invasive breast cancer, including HER2, ESR1, CCND1, and MYC. So far, little is known about their role in tumor progression. To investigate their significance for tumor invasion, we compared pure ductal carcinoma in situ (DCIS) and DCIS associated with invasive cancer with regard to the amplification of these genes. Fluorescence in situ hybridization (FISH) was performed on a tissue microarray containing samples from 130 pure DCIS and 159 DCIS associated with invasive breast cancer. Of the latter patients, we analyzed the intraductal and invasive components separately. In addition, lymph node metastases of 23 patients with invasive carcinoma were included. Amplification rates of pure DCIS and DCIS associated with invasive cancer did not differ significantly (pure DCIS vs. DCIS associated with invasive cancer: HER2 22.7 vs. 24.2%, ESR1 19.0 vs. 24.1%, CCND1 10.0 vs. 14.8%, MYC 11.8 vs. 6.5%; P > 0.05). Furthermore, we observed a high concordance of the amplification status for all genes if in situ and invasive carcinoma of individual patients were compared. This applied also to the corresponding lymph node metastases. Our results indicate no significant differences between the gene amplification status of DCIS and invasive breast cancer concerning HER2, ESR1, CCND1, and MYC. Therefore, our data suggest an early role of all analyzed gene amplifications in breast cancer development but not in the initiation of invasive tumor growth.

Abstract MMSET (WHSC1/NSD2) is a SET domain–containing histone lysine methyltransferase the expression of which is deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation associated with poor prognosis. Recent studies have shown that MMSET mRNA levels are increased in other tumor types as well. We have carried out immunohistochemical staining of tissue microarrays and found that MMSET protein is frequently and highly expressed in neuroblastoma (MMSET positive in 75% of neuroblastomas, n = 164). The expression level of MMSET in neuroblastomas was significantly associated with poor survival, negative prognostic factors, and metastatic disease. Moreover, a subset of neuroblastomas for which pre- and postchemotherapy biopsies were available displayed a strong decrease in MMSET protein levels after chemotherapy. In agreement with neuroblastomas becoming more differentiated after treatment, we show that retinoic acid–induced differentiation of human neuroblastoma cells in vitro also leads to a strong decrease in MMSET levels. Furthermore, we show that the high levels of MMSET in normal neural progenitor cells are strongly downregulated during differentiation. Importantly, we show that MMSET is required for proliferation of neuroblastoma cells and brain-derived neural stem cells. Taken together, our results suggest that MMSET is implicated in neuroblastomagenesis possibly by supporting proliferation of progenitor cells and negatively regulating their differentiation. In this respect, MMSET might be a strong candidate therapeutic target in a subset of neuroblastomas with unfavorable prognosis. Cancer Res; 71(12); 4226–35. ©2011 AACR.

Reelin is a secreted, signaling protein associated with neuronal cell positioning and migration. Recently, reelin was found to be epigenetically silenced in gastric and pancreatic cancers in which down-regulation was associated with increased migratory ability and reduced survival. Here we analyzed reelin expression by immunohistochemistry in 17 normal breast tissue samples from reduction mammoplasties and in two independent tissue microarrays of 136 and more than 2000 breast cancer biopsy samples, respectively. Results were analyzed with regard to clinical parameters, including BRE (Bloom, Richardson, Elston) grade, nodal status, estrogen receptor and HER2 status, and overall survival. Reelin was expressed in the luminal epithelium and myoepithelium of the normal human breast but not in cancerous breasts. Loss of reelin protein expression correlated significantly with decreased survival (P = 0.01) and positive lymph node status (P < 0.001). By measuring reelin expression and promoter methylation status in 39 primary breast tumors, as well as in breast cancer-derived cell lines before and after decitabine treatment, we established that reelin expression levels correlated inversely with promoter methylation status, whereas demethylation increased reelin mRNA expression in vitro. Reelin overexpression in MDA-MB231 cells, as well as incubation with recombinant reelin, suppressed cell migration, invadopodia formation, and invasiveness in vitro. We conclude that reelin may play an important role in controlling invasiveness and metastatic potential of breast cancer cells and that its expression is controlled by promoter methylation. Reelin is a secreted, signaling protein associated with neuronal cell positioning and migration. Recently, reelin was found to be epigenetically silenced in gastric and pancreatic cancers in which down-regulation was associated with increased migratory ability and reduced survival. Here we analyzed reelin expression by immunohistochemistry in 17 normal breast tissue samples from reduction mammoplasties and in two independent tissue microarrays of 136 and more than 2000 breast cancer biopsy samples, respectively. Results were analyzed with regard to clinical parameters, including BRE (Bloom, Richardson, Elston) grade, nodal status, estrogen receptor and HER2 status, and overall survival. Reelin was expressed in the luminal epithelium and myoepithelium of the normal human breast but not in cancerous breasts. Loss of reelin protein expression correlated significantly with decreased survival (P = 0.01) and positive lymph node status (P < 0.001). By measuring reelin expression and promoter methylation status in 39 primary breast tumors, as well as in breast cancer-derived cell lines before and after decitabine treatment, we established that reelin expression levels correlated inversely with promoter methylation status, whereas demethylation increased reelin mRNA expression in vitro. Reelin overexpression in MDA-MB231 cells, as well as incubation with recombinant reelin, suppressed cell migration, invadopodia formation, and invasiveness in vitro. We conclude that reelin may play an important role in controlling invasiveness and metastatic potential of breast cancer cells and that its expression is controlled by promoter methylation. Reelin is a secreted signaling protein and an important regulator of neuronal cell positioning during embryonic brain development (for review, see refs. 1Tissir F Goffinet AM Reelin and brain development.Nat Rev Neurosci. 2003; 4: 496-505Crossref PubMed Scopus (623) Google Scholar, 2Jossin Y Neuronal migration and the role of reelin during early development of the cerebral cortex.Mol Neurobiol. 2004; 30: 225-251Crossref PubMed Scopus (51) Google Scholar). It was first described as a regulator of neuronal cell migration of the neocortex during the development of the early cortical plate.3D'Arcangelo G Miao GG Chen SC Soares HD Morgan JI Curran T A protein related to extracellular matrix proteins deleted in the mouse mutant reeler.Nature. 1995; 374: 719-723Crossref PubMed Scopus (1487) Google Scholar In mice mutated in reelin (reeler) or in members of its downstream signaling pathway (reeler-like mice) cortical cell layering is severely disturbed.3D'Arcangelo G Miao GG Chen SC Soares HD Morgan JI Curran T A protein related to extracellular matrix proteins deleted in the mouse mutant reeler.Nature. 1995; 374: 719-723Crossref PubMed Scopus (1487) Google Scholar, 4Goldowitz D Cushing RC Laywell E D'Arcangelo G Sheldon M Sweet HO Davisson M Steindler D Curran T Cerebellar disorganization characteristic of reeler in scrambler mutant mice despite presence of reelin.J Neurosci. 1997; 17: 8767-8777PubMed Google Scholar, 5Howell BW Hawkes R Soriano P Cooper JA Neuronal position in the developing brain is regulated by mouse disabled-1.Nature. 1997; 389: 733-737Crossref PubMed Scopus (619) Google Scholar, 6Sheldon M Rice DS D'Arcangelo G Yoneshima H Nakajima K Mikoshiba K Howell BW Cooper JA Goldowitz D Curran T Scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice.Nature. 1997; 389: 730-733Crossref PubMed Scopus (556) Google Scholar, 7Trommsdorff M Gotthardt M Hiesberger T Shelton J Stockinger W Nimpf J Hammer RE Richardson JA Herz J Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2.Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1087) Google Scholar, 8Falconer DS Two new mutations, trembler and reeler, with neurological actions in the house mouse.J Genet. 1951; 50: 192-201Crossref PubMed Scopus (422) Google Scholar In the brain reelin acts mainly through binding to the low-density lipoprotein receptors VLDLR and ApoER2.9D'Arcangelo G Homayouni R Keshvara L Rice DS Sheldon M Curran T Reelin is a ligand for lipoprotein receptors.Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar On reelin binding the receptors cluster and signal down to the adaptor protein Dab1, which becomes phosphorylated10Hiesberger T Trommsdorff M Howell BW Goffinet A Mumby MC Cooper JA Herz J Direct binding of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation.Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar in an Src family kinase-dependent process.11Arnaud L Ballif BA Forster E Cooper JA Fyn tyrosine kinase is a critical regulator of disabled-1 during brain development.Curr Biol. 2003; 13: 9-17Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 12Keshvara L Benhayon D Magdaleno S Curran T Identification of reelin-induced sites of tyrosyl phosphorylation on disabled 1.J Biol Chem. 2001; 276: 16008-16014Crossref PubMed Scopus (120) Google Scholar pDab1 can activate the phosphatidylinositol 3-kinase/protein kinase B pathway, leading to glycogen synthase kinase-3β inhibition and actin remodeling.13Ballif BA Arnaud L Cooper JA Tyrosine phosphorylation of Disabled-1 is essential for Reelin-stimulated activation of Akt and Src family kinases.Brain Res Mol Brain Res. 2003; 117: 152-159Crossref PubMed Scopus (78) Google Scholar, 14Beffert U Morfini G Bock HH Reyna H Brady ST Herz J Reelin-mediated signaling locally regulates protein kinase B/Akt and glycogen synthase kinase 3β.J Biol Chem. 2002; 277: 49958-49964Crossref PubMed Scopus (248) Google Scholar In another process, pDab1 can interact with the Lis1 complex, leading to changes in microtubular formation and nucleokinesis.15Assadi AH Zhang G Beffert U McNeil RS Renfro AL Niu S Quattrocchi CC Antalffy BA Sheldon M Armstrong DD Wynshaw-Boris A Herz J D'Arcangelo G Clark GD Interaction of reelin signaling and Lis1 in brain development.Nat Genet. 2003; 35: 270-276Crossref PubMed Scopus (185) Google Scholar The reelin promoter contains large CpG islands, and its expression in neurons is strongly controlled by promoter methylation and acetylation.16Chen Y Sharma RP Costa RH Costa E Grayson DR On the epigenetic regulation of the human reelin promoter.Nucleic Acids Res. 2002; 30: 2930-2939Crossref PubMed Scopus (234) Google Scholar Demethylation of the promoter or treatment with histone deacetylase inhibitors increases reelin mRNA expression in neuronal NT2 cells in vitro.16Chen Y Sharma RP Costa RH Costa E Grayson DR On the epigenetic regulation of the human reelin promoter.Nucleic Acids Res. 2002; 30: 2930-2939Crossref PubMed Scopus (234) Google Scholar In addition, hypermethylation of the reelin promoter can be found in brain regions of patients with schizophrenia.16Chen Y Sharma RP Costa RH Costa E Grayson DR On the epigenetic regulation of the human reelin promoter.Nucleic Acids Res. 2002; 30: 2930-2939Crossref PubMed Scopus (234) Google Scholar The physiological function of reelin has so far only been described in the brain. Although it has been shown to be present in other organs of the adult mouse including spinal cord, liver, kidney, testis, and ovary17Ikeda Y Terashima T Expression of reelin, the gene responsible for the reeler mutation, in embryonic development and adulthood in the mouse.Dev Dyn. 1997; 210: 157-172Crossref PubMed Scopus (126) Google Scholar as well as in serum,18Smalheiser NR Costa E Guidotti A Impagnatiello F Auta J Lacor P Kriho V Pappas GD Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells.Proc Natl Acad Sci USA. 2000; 97: 1281-1286Crossref PubMed Scopus (131) Google Scholar no biological function has so far been attributed to reelin outside the brain. However, recent publications established a role for the reelin signaling pathway in different cancers. Whereas strong reelin expression was associated with high-grade prostate cancer,19Perrone G Vincenzi B Zagami M Santini D Panteri R Flammia G Verzi A Lepanto D Morini S Russo A Bazan V Tomasino RM Morello V Tonini G Rabitti C Reelin expression in human prostate cancer: a marker of tumor aggressiveness based on correlation with grade.Mod Pathol. 2007; 20: 344-351Crossref PubMed Scopus (33) Google Scholar transcriptional silencing of reelin in gastric and pancreatic cancers through epigenetic control mechanisms was associated with a poor outcome.20Sato N Fukushima N Chang R Matsubayashi H Goggins M Differential and epigenetic gene expression profiling identifies frequent disruption of the RELN pathway in pancreatic cancers.Gastroenterology. 2006; 130: 548-565Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 21Dohi O, Takada H, Wakabayashi N, Yasui K, Sakakura C, Mitsufuji S, Naito Y, Taniwaki M, Yoshikawa T: Epigenetic silencing of RELN in gastric cancer. Int J Oncol 36:85–92Google Scholar Knockdown of reelin, of its receptors VLDLR and ApoER2, or of Dab1 in a pancreatic cancer cell line increased cell migration and colony formation, whereas demethylation and treatment with trichostatin A (TSA) increased reelin expression.20Sato N Fukushima N Chang R Matsubayashi H Goggins M Differential and epigenetic gene expression profiling identifies frequent disruption of the RELN pathway in pancreatic cancers.Gastroenterology. 2006; 130: 548-565Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar In the light of these somewhat contradictory results in three different cancers, we aimed to establish whether reelin was expressed in the human breast and whether its expression played a role in breast cancer. Here we describe, for the first time, that reelin is expressed in the normal breast epithelium and that reelin is down-regulated in breast cancer. Overexpression of reelin in a breast cancer cell line strongly reduces invadopodia formation and migration, as well as invasiveness in a collagen I matrix, whereas loss of reelin expression in breast cancers is significantly associated with positive lymph node status and decreased overall survival. We show that regulation of reelin expression is strongly linked to promoter methylation in breast cancer cell lines and in primary breast cancers. Cell lines MDA-MB 231 (HTB-26), MDA-MB361 (HTB-27), MDA-MB435S (HTB-129D), MDA-MB 436 (HTB-130), MDA-MB453 (HTB-131), MDA-MB468 (HTB-132), MCF-7 (HTB-22), BT-20 (HTB-19), BT-474 (HTB-20), BT-549 (HTB-122), and ZR-75-30 (CRL-1504) were purchased from the American Type Culture Collection (LGC Standards, Teddington, UK) and grown according to the conditions set out by the American Type Culture Collection. Reelin-overexpressing HEK293 cells were a generous gift from Drs. Foerster and Frotscher (Freiburg, Germany) and have been described previously.22Forster E Tielsch A Saum B Weiss KH Johanssen C Graus-Porta D Muller U Frotscher M Reelin, Disabled 1, and β1 integrins are required for the formation of the radial glial scaffold in the hippocampus.Proc Natl Acad Sci USA. 2002; 99: 13178-13183Crossref PubMed Scopus (224) Google Scholar These were grown in Dulbecco's modified Eagle's medium (DMEM)/10% fetal calf serum in the presence of 1 mg/ml G418 (Invitrogen, Paisley, UK). Cells were treated with 1 μmol/L 5-aza-2′-deoxycytidine (Decitabine) and 300 nmol/L TSA (Sigma-Aldrich, Dorset, UK) alone or in combination. Cells were treated with 5-aza-2′-deoxycytidine for 7 days with fresh inhibitor added every day. No obvious signs of cell death were observed during this period. TSA was added before the last day of 5-aza-2′-deoxycytidine treatment. Doxorubicin (200 ng/ml, Sigma-Aldrich) was used to treat cells for 24 hours. MDA-MB231 cells were transfected with a reelin expression vector (pCrl; generous gift from T. Curran, The Children's Hospital of Philadelphia, Philadelphia, PA). This vector expresses full-length reelin under control of a cytomegalovirus promoter in a pcDNA3 backbone (neomycin-resistant) and was first cloned by D'Arcangelo et al.23D'Arcangelo G Nakajima K Miyata T Ogawa M Mikoshiba K Curran T Reelin is a secreted glycoprotein recognized by the CR-50 monoclonal antibody.J Neurosci. 1997; 17: 23-31Crossref PubMed Google Scholar Cells were transfected at 80% confluence with 2 μg of unlinearized plasmid DNA, using Lipofectamine 2000 (Invitrogen) for 24 hours. Stable transfectants were selected by growing cells in 500 μg/ml G418. After serial dilution in a 96-well plate, 10 clones were cultured, and reelin protein levels were analyzed by Western blotting (data not shown). Finally, one clone expressing the full-length reelin and one clone expressing a truncated fragment of reelin were selected for further investigation. The procedure was repeated using linearized pCrl and two further clones were selected that showed either full-length or truncated reelin expression. RNA was isolated using a Mini-RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA (500 ng) was used for cDNA synthesis with Roche Transcriptor reverse transcriptase, and 1 μl of cDNA was used per PCR reaction. The PCR was performed using a Roche Light-Cycler with probe 72 (Roche Probe Library, Roche Diagnostics Ltd., Burgess Hill, UK) and the following primers: forward 5′-TGCTGGAATACACTAAGGATGC-3′ and reverse 5′-GAAGGCACTGGGTCTGTACG-3′. All reactions were run in triplicate, and the results were normalized to an internal control (β-actin, forward 5′-ATTGGCAATGAGCGGTTC-3′ and reverse 5′-GGATGCCACAGGACTCCAT-3′; probe 11). Genomic DNA was isolated using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions, and 200 ng of genomic DNA was modified by sodium bisulfate using a Zymo EZ DNA Methylation Kit (Zymo Research, Orange, CA). Approximately 20 ng of modified DNA was used in each PCR reaction. Primers used were as follows: unmethylated forward primer 5′-GGGTGTTTTTTTAGGTTTGGTTG-3′; unmethylated reverse primer 5′-CAACTCCCAAAATTACTTTAAACCAC-3′; methylated forward primer 5′-CGTTTTTTTAGGTTTGGTCGA-3′; and methylated reverse primer 5′-GACTCCCAAAATTACTTTAAACCG-3′. Cycling conditions were as follows: an initial 8 cycles of 95°C for 2 minutes, 60°C for 30 seconds, and 72°C for 30 seconds followed by 32 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes. Products were separated on 2% agarose gels and visualized by ethidium bromide staining. Unmethylated control DNA was extracted from normal peripheral blood; chemically modified methylated control DNA (CpGenome Universal Methylated DNA) was purchased from Chemicon Europe (Cambridge Bioscience, Cambridge, UK). DNA (500 ng) was modified using the Zymo EZ DNA Methylation Kit (Zymo Research) according to the manufacturer's instructions. Primers for bisulfite sequencing were designed using the MethPrimer protocol.24Li LC Dahiya R MethPrimer: designing primers for methylation PCRs.Bioinformatics. 2002; 18: 1427-1431Crossref PubMed Scopus (1980) Google Scholar Modified DNA (25 ng) was amplified using Thermo-Start Master Mix (Abgene Ltd., Epsom, UK) containing 500 nmol/L of each primer (forward 5′-GTAATATGTAGGGAAATGAGTATTT-3′ and reverse 5′-CCCAAAAAAAACAAAACACACTAA-3′) using an amplification program consisting of an initial incubation for 15 minutes at 95°C, followed by 40 cycles of 30 seconds at 95°C, 30 seconds annealing at 50°C, and 30 seconds extension at 72°C and a final 5-minute incubation at 72°C. Products were eluted from agarose gels using Zymoclean Gel DNA Recovery Kits (Zymo Research) and cloned into TA vectors (Invitrogen) according to the manufacturer's instructions. DNA from at least 12 individual clones was isolated using TempliPhi amplification (GE Healthcare UK Limited, Bucks, UK) and analyzed by Sanger sequencing. After bisulfite treatment and PCR, the degree of methylation at each CpG position in a sequence was determined from the ratio of T and C. PCR primers (forward 5′-BIOT-GGGTTTTAAGAAGGTGTG-3′ and reverse 5′-CTCCCAAAATTACTTTAAAC-3′) were designed to amplify a 158-bp fragment across the CpG islands and optimized for software dedicated to methylation analysis. PCR conditions were as follows: one initial 95°C step for 8 minutes followed by 40 cycles of 95°C for 30 seconds, 53°C for 30 seconds, 72°C for 40 seconds, and a final extension for 7 minutes at 72°C. The PCR products were separated on a 2% agarose gel and visualized using a transilluminator. The PCR products were finally analyzed by pyrosequencing using a Biotage Sample Prep kit (Biotage, Uppsala, Sweden) and the specific sequencing primer (PCR F). After the pyrosequencing run, analysis of the methylation percentage of each CpG site was performed using PyroMark ID software (Biotage). Placental DNA was used as a negative control of methylation (0% average methylation) and a commercial methylated DNA (Millipore, Watford, UK) was used as a positive control (98% average methylation). Reelin-specific antibody clone 142 (Millipore) was used for all immunocytochemical assays (dilution 1:800), using the IMPRESS kit (Vector Laboratories UK, Peterborough, UK) and antigen retrieval with EDTA/Tris buffer (pH 8). In brief, after dewaxing in xylene, tissues were treated with 0.3% H2O2 before antigen retrieval and overnight antibody treatment; 2.5% horse serum was used as a blocking agent. Tissue sections were counterstained with Hematoxylin Z (Cellpath Ltd., Newtown, UK). Because no blocking peptides were available and the epitope of antibody 142 is not known, antibody specificity was tested by a 30-minute preincubation with 10 μl of reelin protein solution derived from stably transfected HEK293 cells.22Forster E Tielsch A Saum B Weiss KH Johanssen C Graus-Porta D Muller U Frotscher M Reelin, Disabled 1, and β1 integrins are required for the formation of the radial glial scaffold in the hippocampus.Proc Natl Acad Sci USA. 2002; 99: 13178-13183Crossref PubMed Scopus (224) Google Scholar Reelin-expressing cells and green fluorescent protein-transfected HEK293 control cells were grown to near confluence, washed twice with PBS, and incubated for 48 hours in serum-free DMEM. Ten milliliters of supernatant was concentrated ∼50 times using Amicon Ultra-15 filter (Millipore) devices with a 30-kDa cutoff and washed once with 10 ml of PBS to obtain 200 μl of concentrated solution. The material used in the pilot study was taken from a cohort of patients who were enrolled into the Ludwig Trial V Ludwig Breast Cancer Study Group.25Ludwig Breast Cancer Study Group Combination adjuvant chemotherapy for node-positive breast cancer. Inadequacy of a single perioperative cycle.N Engl J Med. 1988; 319: 677-683Crossref PubMed Scopus (145) Google Scholar The cancers used were representative of the trial cohort. From 1981 to 1985, the International Breast Cancer Study Group (formerly the Ludwig Breast Cancer Study Group) conducted a randomized clinical study to assess the effect of early commencement of adjuvant cyclophosphamide, methotrexate, and 5-fluorouracil chemotherapy in patients with lymph node-negative and lymph node-positive breast carcinoma. The larger set of tissue arrays consisted of 2197 cases of primary breast cancer collected from 1985 to 1999 at the University Hospital Basel, Institute of Clinical Pathology and Institute of Pathology and Triemli Hospital Zürich26Ruiz C Seibt S Al Kuraya K Siraj AK Mirlacher M Schraml P Maurer R Spichtin H Torhorst J Popovska S Simon R Sauter G Tissue microarrays for comparing molecular features with proliferation activity in breast cancer.Int J Cancer. 2006; 118: 2190-2194Crossref PubMed Scopus (95) Google Scholar and have been used previously in our laboratory.27Stein T Price KN Morris JS Heath VJ Ferrier RK Bell AK Pringle MA Villadsen R Petersen OW Sauter G Bryson G Mallon EA Gusterson BA Annexin A8 is up-regulated during mouse mammary gland involution and predicts poor survival in breast cancer.Clin Cancer Res. 2005; 11: 6872-6879Crossref PubMed Scopus (43) Google Scholar The median age of these patients was 62 years (range, 26 to 101 years). The mean follow-up time was 68 months (range, 1 to 176 months). The tissue microarrays were reviewed by two pathologists to define histological grade according to the Elston and Ellis BRE scoring system28Elston CW Ellis IO Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up.Histopathology. 1991; 19: 403-410Crossref PubMed Scopus (4786) Google Scholar and histological tumor type. In addition, pathological stage, tumor diameter, and nodal status were known from the primary pathology reports. HER2 status by fluorescence in situ hybridization analysis and estrogen receptor (ER) status by immunohistochemistry were analyzed in Al-Kuraya et al29Al-Kuraya K Schraml P Sheikh S Amr S Torhorst J Tapia C Novotny H Spichtin H Maurer R Mirlacher M Simon R Sauter G Predominance of high-grade pathway in breast cancer development of Middle East women.Mod Pathol. 2005; 18: 891-897Crossref PubMed Scopus (58) Google Scholar and Ruiz et al.26Ruiz C Seibt S Al Kuraya K Siraj AK Mirlacher M Schraml P Maurer R Spichtin H Torhorst J Popovska S Simon R Sauter G Tissue microarrays for comparing molecular features with proliferation activity in breast cancer.Int J Cancer. 2006; 118: 2190-2194Crossref PubMed Scopus (95) Google Scholar Reelin staining of tumor cells was scored for intensity (0, no; 1, weak; 2, moderate; and 3, strong staining) and percentage of positive cells (1, 0 to 4%; 2, 5 to 19%; 3, 20 to 39%; 4, 40 to 59%; 5, 60 to 79%; 6, 80 to 100%). These values were multiplied to obtain an H-score (0 to 18) and grouped into no (H-score = 0), low (H-score = 1–6), and high reelin staining (H-score > 6). Coverslips were precleaned with 20% nitric acid for 20 minutes followed by an extensive wash with water. Ethanol-sterilized coverslips were then coated with 50 μg/ml poly-l-lysine (Sigma-Aldrich) for 20 minutes at room temperature, followed by fixation with 0.5% glutaraldehyde for 15 minutes. The coverslips were washed three times with PBS, inverted on a 50-μl drop of Oregon Green 488 gelatin matrix (Invitrogen), and incubated for 20 minutes at room temperature. After washing with PBS, the residual reactive groups in the gelatin matrix were quenched with 5 mg/ml sodium borohydride for 15 minutes followed by extensive washing with PBS. The coverslips were then ethanol-sterilized for 5 minutes followed by quenching in DMEM for 1 hour. To assess the ability of cells to form invadopodia and gelatin degradation, 5 × 104 cells were seeded on the coverslips coated with 488 gelatin and incubated at 37°C for 5 hours. Foci of degraded matrix were visible as dark areas that lacked fluorescence. The total pixels of the degradation area corresponding to the cells (n = 60 for each cell line; in triplicate) were analyzed and quantified by the Gelatin Degradation plug-in of ImageJ. Cells on coverslips were fixed with 4% paraformaldehyde for 10 minutes followed by permeabilization with 0.1% Triton X-100 for 10 minutes. Actin filaments were visualized with rhodamine phalloidin (Molecular Probes, Eugene, OR). Confocal images were collected using an Olympus laser scanning microscope (FluoView FV1000) with ×63/1.4 NA oil objectives. Images were processed with Volocity. Monolayers of confluent cells in a six-well plate were wounded by scraping with a P200 plastic pipette tip and rinsed three times with growth medium to remove floating cells. Images were periodically collected every 10 minutes over the next 36 hours with a ×10 objective on an inverted phase microscope (Nikon, Tokyo, Japan) equipped with a chamber to maintain the atmosphere of 5% CO2 and 37°C. Cell motility was evaluated by the time the cells took to close the same distance of wound. MDA-MB231 cells (1 × 105) were plated in serum-free medium in the presence or absence of reelin. Reelin protein was harvested as described above; 20 μl of concentrated protein or control supernatant was used per well, and cells were incubated for 20 hours at 5% CO2 and 37°C. Cells were stained with calcein AM (Invitrogen) and visualized with an inverted fluorescent microscope (Olympus XI51). Cells that had not migrated through the pores were removed from the top chamber before image capture at ×100 magnification. Cells from three randomly selected areas were counted from each well. Inverted invasion assays were performed as described previously.30Hennigan RF Hawker KL Ozanne BW Fos-transformation activates genes associated with invasion.Oncogene. 1994; 9: 3591-3600PubMed Google Scholar In brief, collagen I (PureCol, concentrate to 11 mg/ml and mix 3:1 with 10× DMEM, 2 mmol/L glutamine, and 170 mmol/L NaHCO3) was allowed to polymerize in transwell inserts (Costar, Cambridge, MA) for 1 hour at 37°C. Inserts were then inverted, and 4 × 104 cells were seeded directly onto the opposite face of the filters. After a 4-hour incubation at 37°C, the inserts were reversed. Transwell inserts were finally placed in serum-free DMEM, and DMEM supplemented with 10% fetal calf serum, 25 ng/ml epidermal growth factor (MDA-MB-231), or 10% FCS, 25 ng/ml epidermal growth factor plus 100 μg/ml G418 (reelin stable clones) was placed on top of the matrix, providing a chemotactic gradient. At 120 hours after seeding, invading cells were stained with calcein AM and visualized by Leica confocal microscopy. Serial optical sections were captured at 15-μm intervals. Statistical analyses were performed using JMP (SAS Institute Inc., Cary, NC). Association of reelin expression with clinicopathologic parameters and HER2 and ER status were evaluated using a χ2 test. The Kaplan-Meier method was used to visualize association of reelin expression with cancer-specific survival, and the log-rank test was used to test the significance between stratified groups. Cox proportional multivariate analysis was used to test the statistical independence and significance of reelin expression with other potential prognostic factors. All statistical tests were two-sided, and P < 0.05 was considered as significant. To see whether reelin was expressed in the breast epithelium we stained 17 samples of morphologically normal human breast tissue from reduction mammoplasties for reelin protein using immunocytochemistry. All sections showed a distinct immunoreactivity in the cytoplasm of the myoepithelium. This could be found in the major ducts, terminal ducts, and lobuloalveolar units. The luminal epithelia showed varying degrees of reelin-positive staining (Figure 1, A–F). This staining was blocked on preincubation with reelin protein derived from reelin-overexpressing HEK293 cells, but not when the antibody was preincubated with a control medium from green fluorescent protein-expressing HEK293 cells (Supplemental Figure S1, see http://ajp.amjpathol.org). Other immunoreactive cells were endothelial cells, mast cells, myofibroblasts in reactive stroma, and smooth muscle cells in vessel walls and peripheral nerves. To study a possible change in reelin expression in breast cancer, we performed a pilot study using a small, well characterized group of 39 breast cancers of varying grades and compared reelin-staining intensity between the tumor tissue and adjacent normal epithelium. Of these, 27 showed complete loss of staining for the protein in the tumor tissue (Figure 1, B–D), whereas 3 showed reduced expression relative to the normal breast epithelium. Nine samples were positive for reelin in the normal as well as the tumor tissue (Figure 1, E and F). To establish whether down-regulation of reelin expression in breast cancers had any clinical relevance with regard to outcome, two tissue array studies were performed. First, a pilot study was performed on 136 breast cancers from patients who were randomized to the International Breast Cancer Study Group Trial V. These were representative of the trial cohort. Staining intensity for reelin immunoreactivity was scored on a scale of 0 (negative) to 3 (strong). In this s

To identify molecular features associated with clinico‐pathological parameters and TMPRSS2‐ERG fusion status in prostate cancer, we employed MALDI mass spectrometric imaging (MSI) to a prostate cancer tissue microarray (TMA) containing formalin‐fixed, paraffin‐embedded tissues samples from 1,044 patients for which clinical follow‐up data were available. MSI analysis revealed 15 distinct mass per charge ( m / z )‐signals associated to epithelial structures. A comparison of these signals with clinico‐pathological features revealed statistical association with favorable tumor phenotype such as low Gleason grade, early pT stage or low Ki67 labeling Index (LI) for four signals ( m / z 700, m / z 1,502, m / z 1,199 and m / z 3,577), a link between high Ki67LI for one signal ( m / z 1,013) and a relationship with prolonged time to PSA recurrence for one signal ( m / z 1,502; p = 0.0145). Multiple signals were associated with the ERG‐fusion status of our cancers. Two of 15 epithelium‐associated signals including m / z 1,013 and m / z 1,502 were associated with detectable ERG expression and five signals ( m / z 644, 678, 1,044, 3,086 and 3,577) were associated with ERG negativity. These observations are in line with substantial molecular differences between fusion‐type and non‐fusion type prostate cancer. The signals observed in this study may characterize molecules that play a role in the development of TMPRSS2‐ERG fusions, or alternatively reflect pathways that are activated as a consequence of ERG‐activation. The combination of MSI and large‐scale TMAs reflects a powerful approach enabling immediate prioritization of MSI signals based on associations with clinico‐pathological and molecular data.

The Coxsackie- and Adenovirus Receptor (CAR) has been assigned two crucial attributes in carcinomas: (a) involvement in the regulation of growth and dissemination and (b) binding for potentially therapeutic adenoviruses. However, data on CAR expression in cancer types are conflicting and several entities have not been analysed to date.The expression of CAR was assessed by immunohistochemical staining of tissue microarrays (TMA) containing 3714 specimens derived from 100 malignancies and from 273 normal control tissues.The expression of CAR was detected in all normal organs, except in the brain. Expression levels, however, displayed a broad range from being barely detectable (for example, in the thymus) to high abundance expression (for example, in the liver and gastric mucosa). In malignancies, a high degree of variability was notable also, ranging from significantly elevated CAR expression (for example, in early stages of malignant transformation and several tumours of the female reproductive system) to decreased CAR expression (for example, in colon and prostate cancer types).Our results provide a comprehensive insight into CAR expression in neoplasms and indicate that CAR may offer a valuable target for adenovirus-based therapy in a subset of carcinomas. Furthermore, these data suggest that CAR may contribute to carcinogenesis in an entity-dependent manner.

Based on next-generation sequencing of early-onset prostate cancer (PCa), we earlier demonstrated that PCa in young patients is prone to rearrangements involving androgen-regulated genes-such as transmembrane protease, serine 2 (TMPRSS2)-v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) fusion-and provided data suggesting that this situation might be caused by increased androgen signaling in younger men. In the same study, an accumulation of chromosomal deletions was found in cancers of elderly patients. To determine how age-dependent molecular features relate to cancer phenotype, an existing data set of 11,152 PCas was expanded by additional fluorescence in situ hybridization analyses of phosphatase and tensin homolog (PTEN), 6q15 and 5q21. The results demonstrate that the decrease in TMPRSS2-ERG fusions with increasing patient age is limited to low-grade cancers (Gleason ≤3+4) and that the significant increase in the deletion frequency with age was strictly limited to ERG-negative cancers for 6q15 and 5q21 but to ERG-positive cancers for PTEN. These data suggest that the accumulation of non-androgen-linked genomic alterations with advanced patient age may require an appropriate microenvironment, such as a positive or negative ERG status. The strong link of ERG activation to young patient age and low-grade cancers may help to explain a slight predominance of low-grade cancers in young patients.

Mitochondria are suggested to be important organelles for cancer initiation and promotion. This study was designed to evaluate the prognostic value of MTC02, a marker for mitochondrial content, in prostate cancer.Immunohistochemistry of using an antibody against MTC02 was performed on a tissue microarray (TMA) containing 11,152 prostate cancer specimens. Results were compared to histological phenotype, biochemical recurrence, ERG status and other genomic deletions by using our TMA attached molecular information.Tumor cells showed stronger MTC02 expression than normal prostate epithelium. MTC02 immunostaining was found in 96.5% of 8,412 analyzable prostate cancers, including 15.4% tumors with weak, 34.6% with moderate, and 46.5% with strong expression. MTC02 expression was associated with advanced pathological tumor stage, high Gleason score, nodal metastases (p < 0.0001 each), positive surgical margins (p = 0.0005), and early PSA recurrence (p < 0.0001) if all cancers were jointly analyzed. Tumors harboring ERG fusion showed higher expression levels than those without (p < 0.0001). In ERG negative prostate cancers, strong MTC02 immunostaining was linked to deletions of PTEN, 6q15, 5q21, and early biochemical recurrence (p < 0.0001 each). Moreover, multiple scenarios of multivariate analyses suggested an independent association of MTC02 with prognosis in preoperative settings.Our study demonstrates high-level MTC02 expression in ERG negative prostate cancers harboring deletions of PTEN, 6q15, and 5q21. Additionally, increased MTC02 expression is a strong predictor of poor clinical outcome in ERG negative cancers, highlighting a potentially important role of elevated mitochondrial content for prostate cancer cell biology.

The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients.

DNA mismatch repair (MMR) is integral to the maintenance of genetic stability. We aimed to evaluate the clinical impact of MMR gene expression in prostate cancer. The MMR genes MSH6, MLH1 and PMS2 were analyzed by immunohistochemistry on a tissue microarray containing 11152 prostate cancer specimens. Results were compared with ETS-related gene status and deletions of PTEN, 3p13, 5q21 and 6q15. MSH6, MLH1 and PMS2 expression was detectable in 89.5%, 85.4% and 85.0% of cancers and was particularly strong in cancers with advanced pathological tumor stage (P < 0.0001 each), high Gleason grade (P < 0.0001 each), nodal metastasis (P ≤ 0.0083) and early biochemical recurrence (P < 0.0001). High levels of MMR gene expression paralleled features of genetic instability, such as the number of genomic deletions per cancer; strong expression of all three MMR genes was found in 24%, 29%, 30%, 33% and 42% of cancers with no, one, two, three or four to five deletions (P < 0.0001). The prognostic value of the analyzed MMR genes was largely driven by the subset of cancers lacking ERG fusion (P < 0.0001), while the prognostic impact of MMR gene overexpression was only marginal in ERG-positive cancers. Multivariate analyses suggested an independent prognostic relevance of MMR genes in ERG-negative prostate cancers when compared with prognostic parameters available at the time of initial biopsy. In conclusion, MMR overexpression is common in prostate cancer and is linked to poor outcome as well as features indicating genetic instability. ERG fusion should be analyzed along with MMR gene expression in potential clinical tests.

IMP3 is an RNA binding protein required for ribosomal RNA processing, which has been suggested to be a prognostic marker in a large variety of human types of cancer. However, available data on the prevalence of IMP3 expression are largely discrepant. To systematically investigate the epidemiology and clinical relevance of IMP3 expression in human cancers we employed a two-step tissue microarrays (TMAs) approach. First, a normal tissue TMA and a multi-tumor TMA were analyzed for immunohistochemically detectable expression of IMP3 in 76 different normal tissue types and 3889 cancer samples from 95 different tumor categories. In a second step, we searched for associations between IMP3 expression and tumor phenotype and patient prognosis in TMAs containing 697 urinary bladder cancers, 1711 colon cancers, 343 esophageal adenocarcinomas, 251 esophageal squamous cell cancers, 673 lung cancers), 275 pancreatic cancers and 230 stomach cancers. In normal tissues, unequivocal IMP3 expression was found in placenta, lymphocytes and some types of glandular epithelial cells. In cancers, at least one case with weak expression could be found in 76 out of 95 (80%) different tumor types and 64 entities (67%) had at least one tumor with strong positivity. IMP3 expression was most frequently found in testicular cancer (including 71% seminomas and 96% non-seminomas), neuroblastoma (88%), and squamous cell cancer of various origins. Significant associations were found between IMP3 and adverse tumor features in esophageal adenocarcinomas and cancers of the urinary bladder, lung, stomach, and pancreas. In summary, IMP3 was frequently expressed in many different tumor types, and was typically associated with aggressive tumor features.

Evidence suggests that class III β-tubulin (βIII-tubulin) may represent a prognostic and predictive molecular marker in prostate cancer. βIII-Tubulin expression was determined by IHC in 8179 prostate cancer specimens in a TMA format. Results were compared with tumor phenotype, biochemical recurrence, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) status, and deletions on PTEN, 3p13, 5q21, and 6q15. βIII-Tubulin expression was detectable in 25.6% of 8179 interpretable cancers. High βIII-tubulin expression was strongly associated with both TMPRSS2:ERG rearrangement and ERG expression (P < 0.0001 each). High βIII-tubulin expression was tightly linked to high Gleason grade, advanced pT stage, and early prostate-specific antigen (PSA) recurrence in all cancers (P < 0.0001 each), but also in the subgroups of ERG-negative and ERG-positive cancers. When all tumors were analyzed, the prognostic role of βIII-tubulin expression was independent of Gleason grade, pT stage, pN stage, surgical margin status, and preoperative PSA. Independent prognostic value became even more evident if the analysis was limited to preoperatively available features, such as biopsy specimen Gleason grade, preoperative PSA, cT stage, and βIII-tubulin expression (P < 0.0001 each). βIII-Tubulin expression was associated with PTEN (P < 0.0001) when all tumors were analyzed, but also in the subgroups of ERG-negative and ERG-positive cancers. βIII-Tubulin expression is an independent prognostic parameter. The significant associations with key genomic alterations of prostate cancer, such as TMPRSS2:ERG fusions and PTEN deletions, suggest interactions with several pivotal pathways involved in prostate cancer.

BackgroundThe optimal regimen for adjuvant breast cancer chemotherapy is undefined. We compared sequential to concurrent combination of doxorubicin and cyclophosphamide with docetaxel chemotherapy in women with node-positive non-metastatic breast cancer. We report the final, 10-year analysis of disease-free survival (DFS), overall survival (OS), and long-term safety.Patients and methodsA total of 3298 women with HER2 nonamplified breast cancer were randomized to doxorubicin and cyclophosphamide every 3 weeks for four cycles followed by docetaxel (AC → T) every 3 weeks for four cycles or docetaxel, doxorubicin, and cyclophosphamide (TAC) every 3 weeks for six cycles. The patients received standard radiotherapy and endocrine therapy and were followed up for 10 years with annual clinical evaluation and mammography.ResultsThe 10-year DFS rates were 66.5% in the AC → T arm and 66.3% in the TAC arm (P = 0.749). OS was 79.9% in the AC → T arm and 78.9% in the TAC arm (P = 0.506). TAC was associated with higher rates of febrile neutropenia, although G-CSF primary prophylaxis greatly reduced this risk. AC → T was associated with a higher rate of myalgia, hand-foot syndrome, fluid retention, and sensory neuropathy.ConclusionThis 10-year analysis of the BCIRG-005 trial confirmed that the efficacy of TAC was not superior to AC → T in women with node-positive early breast cancer. The toxicity profiles differ between arms and were consistent with previous reports. The TAC regimen with G-CSF support provides shorter adjuvant treatment duration with less toxicity.Trial RegistrationClinicalTrials.gov Identifier NCT00312208.

Enhancer of zeste homolog 2 (EZH2) plays an important role in tumor development and progression by interacting with histone and nonhistone proteins. In the current study, we analyzed prevalence and prognostic impact of EZH2 in prostate cancer. EZH2 expression was analyzed by immunohistochemistry on a tissue microarray containing more than 12400 prostate cancer specimens. Results were compared to tumor phenotype, biochemical recurrence and molecular subtypes defined by ERG status as well as genomic deletions of 3p, 5q, 6q and PTEN. EZH2 immunostaining was detectable in 56.6% of 10168 interpretable cancers and considered strong in 1.1%, moderate in 12.2% and weak in 43.3% of cases. High EZH2 expression was strongly associated with high Gleason grade (P < 0.0001), advanced pathological tumor stage (P < 0.0001), positive nodal status (P < 0.0001), elevated preoperative PSA level (P = 0.0066), early PSA recurrence (P < 0.0001) and increased cell proliferation P < 0.0001). High-level EZH2 staining was also associated with TMPRSS2:ERG rearrangement and ERG expression in prostate cancers (P < 0.0001) and was linked to deletions of PTEN, 6q15, 5q21 and 3p13 (P < 0.0001 each) particularly in ERG-negative cancers. The prognostic impact of EZH2 was independent of established pre- and postoperatively assessed clinicopathological parameters. EZH2 has strong prognostic impact in prostate cancer and might contribute to the development of a fraction of genetically instable and particularly aggressive prostate cancers. EZH2 analysis might therefore be of clinical value for risk stratification of prostate cancer.

Thymidylate synthase (TYMS) plays a role in DNA synthesis and is a target for 5-fluorouracil.In this study TYMS was analyzed by immunohistochemistry on a tissue microarray containing 11,152 prostate cancers.TYMS expression was higher in neoplastic than in normal prostate epithelium and was detectable in 72.9% of 10,223 interpretable cancers.It was considered strong in 21.9%, moderate in 33.4% and weak in 17.6% of tumors.TYMS overexpression was associated with deletions at 5q21 (p < 0.0001), 6q15 (p < 0.0001) and 3p13 (p = 0.0083) and gradually increased with the total number of these deletions present in the respective cancer sample (p < 0.0001).TYMS expression was unrelated to PTEN deletions (p = 0.9535) but tightly linked to high Gleason grade, advanced pathological tumor stage and early PSA recurrence (p < 0.0001).The prognostic value of TYMS was independent from the ERG status and deletions at 3p13, 5q21, and 6q15.In multivariate analyses the prognostic role of TYMS expression was independent of Gleason grade, pT stage, preoperative PSA, pN stage, or resection margins.TYMS expression analysis might result in clinically useful information in prostate cancer.The striking link to some but not all chromosomal aberrations might suggest a mechanistical link with specific types of DNA damage.

Sequestosome 1 (p62) is a multifunctional adapter protein accumulating in autophagy-defective cells.To evaluate the clinical impact and relationship with key genomic alterations in prostate cancer, p62 protein levels were analyzed by immunohistochemistry on a tissue microarray containing 12,427 prostate cancers. Data on ERG status and deletions of PTEN, 3p13, 5q21, and 6q15 were available from earlier studies.p62 immunostaining was absent in benign prostatic glands but present in 73% of 7,822 interpretable prostate cancers. Strong cytoplasmic p62 staining was tightly linked to high Gleason grade, advanced pathologic tumor (pT) stage, positive nodal status, positive resection margin, and early PSA recurrence (P < 0.0001 each). Increased levels of p62 were significantly linked to TMPRSS2-ERG fusions, both by FISH and immunohistochemical analysis (P < 0.0001 each). For example, moderate or strong p62 immunostaining was seen in 28.5% of cancers with TMPRSS2-ERG fusion detected by FISH and in 23.1% of cancers without such rearrangements (P < 0.0001). Strong p62 staining was significantly linked to the presence of all tested deletions, including PTEN (P < 0.0001), 6q15 (P < 0.0001), 5q21 (P = 0.0002), 3p13 (P = 0.0088), and 6q15 (P < 0.0001), suggesting a link between p62 accumulation and loss of genomic stability. The prognostic role of p62 protein accumulation was striking and independent of Gleason grade, pT stage, pN stage, surgical margin status, and preoperative PSA, regardless of whether preoperative or postoperative parameters were used for modeling.Our study identifies cytoplasmic accumulation of p62 as a strong predictor of an adverse prognostic behavior of prostate cancer independently from established clinicopathologic findings.

Presence of small (tertiary) Gleason 5 pattern is linked to a higher risk of biochemical recurrence in prostate cancer. It is unclear, however, how to integrate small Gleason 5 elements into clinically relevant Gleason grade groups. To analyze the prognostic impact of Gleason 5 patterns in prostate cancer and to develop a method for integrating tertiary Gleason 5 patterns into a quantitative Gleason grading system. Prostatectomy specimens from 13 261 consecutive patients and of 3295 matched preoperative biopsies were available. Percentages of Gleason 3, 4, and 5 had been recorded for each cancer. Outcome measurements and statistical analysis: Our data demonstrate that minimal Gleason 5 areas have strong prognostic impact in Gleason 7 carcinomas, while further expansion of the Gleason 5 pattern population has less impact. We thus defined an integrated quantitative Gleason score (IQ-Gleason) by adding a lump score of 10 to the percentage of unfavorable Gleason pattern (Gleason 4/5) if any Gleason 5 was present and by adding another 7.5 points in case of a Gleason 5 fraction >20%. There was a continuous increase of the risk of prostate-specific antigen recurrence with increasing IQ-Gleason. This was also true for subgroups with identical Cancer of the Prostate Risk Assessment Postsurgical scores (p < 0.0001) or Gleason grade groups (p < 0.0001). The IQ-Gleason represents a simple and efficient approach for combining both quantitative Gleason grading and tertiary Gleason grades in one highly prognostic numerical variable. Prostatectomy specimens (13 261) were analyzed to estimate the relevance of small Gleason 5 elements in prostate cancers. Even the smallest Gleason 5 areas markedly increased the risk of prostate-specific antigen recurrence after surgery. Larger fractions of Gleason 5 patterns had less further impact on prognosis. Based on this, a numerical Gleason score (integrated quantitative Gleason score) was defined by the percentages of Gleason 4 and 5 patterns, enabling a refined estimate of patient prognosis.

Overexpression of class III β‑tubulin (TUBB3), a factor that confers dynamic properties to microtubules, is a candidate biomarker for resistance to microtubule‑targeting chemotherapeutics in breast and other types of solid cancer. Discrepant results from previous studies, with respect to the association of TUBB3 expression levels with breast cancer phenotype and patient prognosis, prompted the present study to investigate TUBB3 expression in a large cohort of breast cancer cases, with available clinical follow‑up data. A preexisting breast cancer prognosis tissue microarray, containing a single 0.6 mm tissue core from each of 2,197 individual patients with breast cancer, was analyzed for TUBB3 expression by immunohistochemistry. The results of the present study revealed that TUBB3 expression was less frequent in lobular breast cancer cases (34%), compared with that of cancer cases of alternative histologies, including breast cancer of no special type (60%; P<0.0001). High TUBB3 positivity was associated with high tumor grade (P<0.0001), negativity for estrogen (P<0.0001) and progesterone receptors (P<0.004), as well as the presence of human epidermal growth factor 2 amplification (P<0.0001) and a triple‑negative phenotype (P<0.0001). TUBB3 overexpression was additionally associated with reduced patient survival if all breast cancer cases of any histology were jointly analyzed (P=0.0088); however this link was not evident in the subset of breast cancer cases of no special type, or in a multivariate analysis including the established prognostic factors of tumor stage, grade and nodal stage. In conclusion, the present study demonstrated that TUBB3 overexpression was associated with adverse features of breast cancer, and that TUBB3 may possess a distinct role in lobular breast cancer cases, compared with alternative histological subtypes. The results of the present study do not support a clinically relevant role for TUBB3 as a prognostic marker in breast cancer.

Abstract Background Positive surgical margins (PSMs) represent a poor prognostic factor at radical prostatectomy (RP). To investigate the impact of PSM, its length, the focality, and the PSM Gleason, on biochemical recurrence (BCR) in organ‐confined RP patients. Methods Within a high‐volume center database, we identified patients who harbored organ‐confined (pathologic stage T2 disease) prostate cancer (PCa) at RP (2010‐2016). Kaplan‐Meier analyses and multivariable Cox regression models were used to test the effect of the PSM on the BCR risk. Results Overall, 8770 patients were identified. Of those, 6.6% (n = 579) harbored PSM. BCR‐free survival at 72 months after RP was 77.7% vs 89.0% for patients with vs without PSM ( P &lt; .001). BCR‐free survival rates at 72 months were 77.4% vs 73.6% ( P = .1) for unifocal vs multifocal PSM, 77.2% vs 71.8% ( P = .03) for Gleason pattern 3 vs ≥4 at the margin and 88.4% vs 66.3% ( P &lt; .001) for &lt;3 vs ≥3 mm length of margin. In multivariable Cox models PSM was an independent predictor for BCR (hazard ratio [HR] = 2.40, P &lt; .001). However, in subgroups with PSM, only ≥3 mm PSM represented an independent predictor (HR = 1.93, P = .04), while focality and Gleason at the margin were no significant predictors. Conclusion PSM represents an independent predictor for BCR in organ‐confined PCa at RP. Moreover, Gleason ≥4 at the margin and ≥3 mm PSM length were associated with worse BCR‐free survival. Closer surveillance of patients with organ‐confined PCa at RP and PSM can help to identify those who qualify for early salvage radiotherapy.

Syndecan-1 (CD138) is a transmembrane proteoglycan known to be expressed in various normal and malignant tissues. It is of interest because of a possible prognostic role of differential expression in tumors and its role as a target for indatuximab, a monoclonal antibody coupled with a cytotoxic agent. To comprehensively analyze CD138 in normal and neoplastic tissues, we used tissue microarrays (TMAs) for analyzing immunohistochemically detectable CD138 expression in 2,518 tissue samples from 85 different tumor entities and 76 different normal tissue types. The data showed that CD138 expression is abundant in tumors. At least an occasional weak CD138 immunostaining could be detected in 71 of 82 (87%) different tumor types, and 58 entities (71%) had at least one tumor with a strong positivity. In normal tissues, a particularly strong expression was found in normal squamous epithelium of various organs, goblet and columnar cells of the gastrointestinal tract, and in hepatocytes. The highly standardized analysis of most human cancer types resulted in a ranking order of tumors according to the frequency and levels of CD138 expression. CD138 immunostaining was highest in squamous cell carcinomas such as from the esophagus (100%), cervix uteri (79.5%), lung (85.7%), vagina (89.7%) or vulva (73.3%), and in invasive urothelial cancer (76.2%). In adenocarcinomas, CD138 was also high in lung (82.9%) and colorectal cancer (85.3%) but often lower in pancreas (73.3%), stomach (54.2% in intestinal type), or prostate carcinomas (16.3%). CD138 expression was usually low or absent in germ cell tumors, sarcomas, endocrine tumors including thyroid cancer, and neuroendocrine tumors. In summary, the preferential expression in squamous cell carcinomas of various sites makes these cancers prime targets for anti-CD138 treatments once these might become available. Abundant expression in many different normal tissues might pose obstacles to exploiting CD138 as a therapeutic target, however.