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With this external quality assessment (EQA) scheme, we aim to investigate the diagnostic performance of the currently available methods for the detection of ALK alterations in non-small cell lung cancer on a national scale, namely, in situ hybridization (ISH), immunohistochemistry (IHC), and RNA/DNA sequencing (NGS). The EQA scheme cohort consisted of ten specimens, including four ALK positive and six ALK negative samples, which were thoroughly pretested using IHC, ISH, and RNA/DNA NGS. Unstained tumor sections were provided to the 57 participants, and the results were retrieved via an online questionnaire. ISH was used by 29, IHC by 38, and RNA/DNA sequencing by 19 participants. Twenty-eight institutions (97%) passed the ring trial using ISH, 33 (87%) by using IHC, and 18 (95%) by using NGS. The highest sensitivity and interrater agreement (Fleiss ' kappa) was observed for RNA/DNA sequencing (99%, 0.975), followed by ISH (94%, 0.898) and IHC (92%, 0.888). However, the proportion of samples that were not evaluable due to bad tissue quality was also higher for RNA/DNA sequencing (4%) compared with ISH (0.7%) and IHC (0.5%). While all three methods produced reliable results between the different institutions, the highest sensitivity and concordance were observed for RNA/DNA sequencing. These findings encourage the broad implementation of this method in routine diagnostic, although the application might be limited by technical capacity, economical restrictions, and tissue quality of formalin-fixed samples., (© 2021. The Author(s).)

Background and Objectives: Overall survival (OS) in patients with early-stage malignant melanoma differs. To date, there are no established prognostic markers. We aimed to contribute to a better understanding of potential prognostic immunohistochemical markers for risk stratification., Patients and Methods: 161 surgically resected early-stage malignant melanomas (stage pT1 and pT2) were analyzed for expression of 20 different proteins using immunohistochemistry. The results were correlated with OS. The cohort was randomly split into a discovery and a validation cohort., Results: High Bcl-2 expression, high nuclear S100A4 expression as well as a Ki67 proliferation index of ≥ 20 % were associated with shorter OS. Strong MITF immunoreactivity was a predictor for favorable prognosis. A combination of these four markers resulted in a multi-marker score with significant prognostic value in multivariate survival analysis (HR: 3.704; 95 % CI 1.484 to 9.246; p = 0.005). Furthermore, the score was able to differentiate a low-risk group with excellent OS rates (five-year survival rate: 100 %), an intermediate-risk group (five-year survival rate: 81.8 %) and a high-risk group (five-year survival rate: 52.6 %). The prognostic value was confirmed within the validation cohort., Conclusions: Combined immunohistochemical analysis of Bcl-2, nuclear S100A4, Ki67 and MITF could contribute to better risk stratification of early-stage malignant melanoma patients., (© 2019 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.)

Aims: Programmed death ligand 1 (PD-L1) immunohistochemistry has become a mandatory diagnostic test in the treatment of lung cancer. Several research initiatives have started to harmonise the five PD-L1 immunohistochemistry assays that have been used in clinical trials. Here, we report data on interlaboratory and interassay concordance for commercial assays ('assays') and laboratory-developed tests (LDTs) at 10 German testing sites., Methods and Results: To assess interlaboratory concordance, a tissue microarray containing 21 pulmonary carcinoma specimens was centrally prepared. Pre-cut sections were stained at 10 sites by the use of assays 28-8, 22C3, SP263, and SP142, as well as 11 LDTs. Assay performance was evaluated with a second tissue microarray containing 11 cell lines with defined PD-L1 expression. Quality control was centrally performed by manual and digital analyses. The assays yielded reproducible IHC staining patterns at all sites. In agreement with previous studies, 22C3, 28-8 and SP263 showed similar staining patterns, whereas SP142 was distinct. Among the LDTs, six of 11 protocols showed staining patterns similar to those of assays 22C3 and 28-8. Interlaboratory concordance of tumour cell scoring by use of a six-step system was moderate (Light's κ = 0.43-0.69), whereas the clinically approved cut-offs of ≥1% and ≥50% showed substantial concordance (κ = 0.73-0.89). Immune cell scoring by the use of SP142 yielded moderate concordance (κ = 0.42)., Conclusions: The data confirm the previously described staining patterns of the assays, and show that they can be reproducibly employed at different sites. LDTs with staining results similar to those of the assays are implementable, but have to be carefully validated., (© 2017 John Wiley & Sons Ltd.)

Aims: Determining the origin of metastases is an important task of pathologists to allow for the initiation of a tumor-specific therapy. Recently, homeobox protein Hox-B13 (HOXB13) has been suggested as a new marker for the detection of prostatic origin. The aim of this study was to evaluate the diagnostic sensitivity of HOXB13 in comparison to commonly used immunohistochemical markers for prostate cancer., Materials and Methods: Histologically confirmed prostate cancer lymph node metastases from 64 cases were used to test the diagnostic value of immunohistochemical markers: prostate specific antigen (PSA), Prostatic acid phosphatase (PSAP), prostate specific membrane antigen (PSMA), homeobox gene NKX3.1 , prostein, androgen receptor (AR), HOXB13, and ETS-related gene (ERG). All markers were evaluated semi-quantitatively using Remmele's immune reactive score., Results: The detection rate of prostate origin of metastasis for single markers was 100% for NKX3.1, 98.1% for AR, 84.3% for PSMA, 80.8% for PSA, 66% for PSAP, 60.4% for HOXB13, 59.6% for prostein, and 50.0% for ERG., Conclusions: Our data suggest that HOXB13 on its own lacks sensitivity for the detection of prostatic origin. Therefore, this marker should be only used in conjunction with other markers, preferably the highly specific PSA. The combination of PSA with NKX3.1 shows a higher sensitivity and thus appears preferable in this setting., Competing Interests: The authors declare no conflict of interest.

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

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

The EML4-ALK pathway plays an important role in a significant subset of non-small cell lung cancer patients. Treatment options such as ALK tyrosine kinase inhibitors lead to improved progression free survival and overall survival. These therapeutic options are chosen on the basis of the identification of the underlying genetic signature of the EML-ALK translocation. Efficient and easily accessible testing tools are required to identify eligible patients in a timely fashion. While FISH techniques are commonly used to detect this translocation, the broad implementation of this type of ALK testing into routine diagnostics is not optimal due to technical, structural and financial reasons. Immunohistochemical techniques to screen for EML4-ALK translocations may therefore play an important role in the near future. This consensus paper provides recommendations for the test algorithm and quality of the respective test approaches, which are discussed in the light of the current literature., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Introduction: The goal of personalized medicine is to treat patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or toxic therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK-positive non-small-cell lung cancer (NSCLC) tumors, but to date the only approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate a new automated standardized ALK IHC assay., Methods: Archival NSCLC tumor specimens (n =103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc., Tucson, AZ). Specimens were scored binarily as positive if strong granular cytoplasmic brown staining was present in tumor cells. IHC results were compared with the FISH results and interevaluator comparisons made., Results: Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%), and accurate relative (93%) to the ALK FISH results. Similar results were observed using a majority score. IHC negativity was scored by seven of seven and six of seven evaluators on three and two FISH-positive cases, respectively. IHC positivity was scored on two FISH-negative cases by seven of seven readers. There was agreement among seven of seven and six of seven readers on 88% and 96% of the cases before review, respectively, and after review there was agreement among seven of seven and six of seven on 95% and 97% of the cases, respectively., Conclusions: On the basis of expert evaluation the ALK IHC test is sensitive, specific, and accurate, and a majority score of multiple readers does not improve these results over an individual reader's score. Excellent inter-reader agreement was observed. These data support the algorithmic use of ALK IHC in the evaluation of NSCLC.

Background: Automated image analysis methods are becoming more and more important to extract and quantify image features in microscopy-based biomedical studies and several commercial or open-source tools are available. However, most of the approaches rely on pixel-wise operations, a concept that has limitations when high-level object features and relationships between objects are studied and if user-interactivity on the object-level is desired., Results: In this paper we present an open-source software that facilitates the analysis of content features and object relationships by using objects as basic processing unit instead of individual pixels. Our approach enables also users without programming knowledge to compose "analysis pipelines" that exploit the object-level approach. We demonstrate the design and use of example pipelines for the immunohistochemistry-based cell proliferation quantification in breast cancer and two-photon fluorescence microscopy data about bone-osteoclast interaction, which underline the advantages of the object-based concept., Conclusions: We introduce an open source software system that offers object-based image analysis. The object-based concept allows for a straight-forward development of object-related interactive or fully automated image analysis solutions. The presented software may therefore serve as a basis for various applications in the field of digital image analysis.

Trastuzumab-based therapy has been shown to confer overall survival benefit in HER2-positive patients with advanced gastric cancer in a large multicentric trial (ToGA study). Subgroup analysis identified adenocarcinomas of the stomach and gastroesophageal (GE) junction with overexpression of HER2 according to immunohistochemistry (IHC) as potential responders. Due to recent approval of trastuzumab for HER2 positive metastatic gastric and GE-junction cancer in Europe (EMEA) HER2 diagnostics is now mandatory with IHC being the primary test followed by fluorescence in situ hybridization (FISH) in IHC2+ cases. However, in order to not miss patients potentially responding to targeted therapy determination of a HER2-positive status for gastric cancer required modification of scoring as had been proposed in a pre-ToGA study. To validate this new HER2 status testing procedure in terms of inter-laboratory and inter-observer consensus for IHC scoring a series of 547 gastric cancer tissue samples on a tissue microarray (TMA) was used. In the first step, 30 representative cores were used to identify specific IHC HER2 scoring issues among eight French and German laboratories, while in the second step the full set of 547 cores was used to determine IHC HER2 intensity and area score concordance between six German pathologists. Specific issues relating to discordance were identified and recommendations formulated which proved to be effective to reliably determine HER2 status in a prospective test series of 447 diagnostic gastric cancer specimens.

For the clinical diagnosis and appropriate therapy of patients with cancer of unknown primary (CUP), biopsies of the tumor metastases are generally examined histopathologically and by immunohistochemistry (IHC). Gene expression profiling (GEP) is a new diagnostic technique that might further contribute to tumor specification. Biopsies of 43 CUP cases underwent a retrospective central histopathological and immunohistochemical review and centrally performed GEP using CupPrint(TM). Two samples could not be evaluated by IHC due to small biopsy size or loss of immunoreactivity. One of these and 17 other samples were not suited for GEP due to RNA degradation. In 13 out of the remaining 24 cases (54%), the same primary was proposed by IHC and GEP and was supported by the clinical findings, furthermore leading to the doubtless identification of the primary in four out of these. In seven cases, there was discordance between IHC and GEP, with the clinical picture being more in line with IHC in three and with GEP in four cases. Four cases had to remain undecided because the primary tumors suggested by IHC and GEP were not supported. In conclusion, overlap between IHC and GEP results and the clinical presentation was noted in the majority of those true CUP cases that were evaluable with both techniques. Therefore, GEP can be a complementary diagnostic technique assisting immunohistochemical profiling of cancer biopsies with unknown primary.

Five percent of cancer cases present as metastases. If the primary tumor cannot be identified after diagnostic workup, the disease is referred to as cancer of unknown primary (CUP) and is classified as C80.9 according to ICD-10. In Germany, CUP is among the ten most common causes of tumor-related death, with mortality similar to mortality in gastric or pancreatic cancer. Biopsies of the tumor manifestation are generally examined histopathologically and by immunohistochemistry (IHC). Gene expression profiling (GEP) is a new diagnostic technique that might further contribute to tumor specification.In a retrospective study, 43 CUP cases underwent central immunohistochemical review and centrally performed GEP using a classifier based on 495 genes. There was concordance between IHC, GEP and clinical picture in 54% of cases. In four cases, the combination of methods led to an unequivocal identification of the primary tumor.In conclusion, we regard detailed IHC workup and complementary GEP advisable for the purposes of targeted therapy, as well as to identify or exclude specific tumors in a CUP situation.

Reliable assessment of in situ pathology analyses like immunohistochemistry, RNA in situ hybridization, and FISH are critically important in diagnosis and treatment decisions. We describe the development of small tissue microarrays (TMA) for the internal control and quantitation of these techniques, the iCon-TMA. In its simple variant, these iCon-TMA (abbreviated from internal control TMA) consist of 2 tissue cylinders, one being clearly positive and the other clearly negative for a specific marker. In its more elaborate format, the iCon-TMAs carry additional tissue spots providing a scale for quantitatively assessable markers, e.g. representing scores of negative, weak, moderate, or strong positivity. The specimen with the yet unknown reactivity is then applied to the iCon-TMA section, and both are simultaneously processed and analyzed, thus providing a convenient measure for in situ quality control and quantitation.

Uroplakins are specific differentiation products of terminally differentiated superficial urothelial cells. We tested the value of a new commercially available monoclonal antibody against uroplakin III (clone AU 1) as a paraffin-reactive immunohistochemical marker for primary and metastatic urothelial carcinomas. The study cases included 67 urothelial carcinomas of the urinary tract (35 primary tumors, 32 metastases) and 318 nonurothelial carcinomas, as well as 5 benign Brenner tumors and 2 transitional cell carcinomas of the ovaries. Uroplakin III was detected in 21 (60%) of the primary urothelial carcinomas and 17 (53%) of the metastases, resulting in an overall sensitivity of 0.57. The studied Brenner tumors also were immunoreactive for uroplakin III. All other studied carcinomas were consistently uroplakin III-negative (specificity 1.00). We found the new monoclonal antibody AU 1 against uroplakin III to be a highly specific paraffin-reactive immunohistochemical marker for urothelial tumors with a moderate sensitivity for the identification of primary and metastatic urothelial carcinomas.

The immunohistochemical detection of estrogen receptors (ERs) and progesterone receptors (PRs) is useful for the differentiation of cytostatically treatable breast and ovarian carcinomas from other metastatic adenocarcinomas of unknown primary sites. We retrospectively studied metastases of 68 breast, 24 ovarian, 15 bronchogenic, 35 gastric, 22 pancreatic, 23 colonic, 27 renal cell, and 26 primary bronchogenic carcinomas by using a panel of 3 antibodies against ERs and 4 antibodies against PRs and applying both a conventional immunohistochemical detection method and the highly sensitive tyramide signal amplification (TSA) technique. Antibody 6F11 against ERs was slightly more sensitive than clone 1D5 with both detection methods, staining 5% more breast carcinomas and 4% more ovarian carcinomas with TSA, compared with conventional immunohistochemical analysis. Furthermore, clone 6F11 detected ERs in 4 of 26 primary bronchogenic carcinomas and 1 of 35 metastases of gastric carcinomas with conventional immunohistochemical techniques; an additional 9 primary and 6 metastatic bronchogenic carcinomas and 2 metastatic gastric carcinomas were positive with TSA. The resulting specificities for breast and ovarian carcinomas versus all other carcinomas were 0.97 with conventional immunohistochemical techniques and 0.85 with TSA. Antibody 1A6 against PRs was slightly more sensitive but least specific for breast and ovarian carcinomas (specificity was 0.96 with conventional immunohistochemical methods and 0.91 with TSA), compared with antibody hPRa3 and a polyclonal antibody (specificity was 1.00 with both detection methods). Antibodies TE111 against ERs and 10A9 against PRs were significantly less sensitive than the other antibodies. We conclude that TSA cannot be recommended for the detection of ERs and PRs to differentiate breast and ovarian carcinomas from other metastatic adenocarcinomas of unknown primary sites.

To test the possibility of immunohistochemical differentiation of cytostatically treatable metastatic breast carcinomas from other metastatic adenocarcinomas of unknown primary site, we studied a total of 328 metastatic adenocarcinomas including 35 bronchogenic, 26 pancreatic, 25 colonic, 39 gastric, 45 renal, 29 ovarian and 129 breast carcinomas with a panel of 13 commercially available monoclonal antibodies. The expression of gross cystic disease fluid protein 15 and/or oestrogen or progesterone receptors had a sensitivity of 0.83, a specificity of 0.93 and a predictive accuracy of 0.92 for carcinomas of the breast against all other carcinomas. Excluding ovarian carcinomas, this combination had a sensitivity, specificity and predictive accuracy for mammary carcinomas of 0.83, 0.98 and 0.98, respectively. Carcinoembryonic antigen and/or cytokeratin 20 identified bronchogenic, gastric, pancreatic and colorectal carcinomas versus breast carcinomas lacking gross cystic disease fluid protein 15 and oestrogen or progesterone receptors with a sensitivity, specificity and predictive accuracy of 0.82, 0.99 and 0.95, respectively. Vimentin differentiates renal cell carcinomas from gross cystic disease fluid protein 15 and oestrogene or progesterone receptor negative breast carcinomas with a sensitivity, specificity and predictive accuracy of 0.93, 0.82 and 0.84. Thus, it should be possible to differentiate most metastatic mammary carcinomas from metastatic adenocarcinomas of other common primary sites, even if the former lack expression of gross cystic disease fluid protein 15 and oestrogen or progesterone receptors.

The increasing importance of targeting drugs in the treatment of several tumor entities (breast, colon, lung, malignant melanoma (MM), lymphoma, and so on) and the necessity of a companion diagnostic (human epidermal growth factor receptor 2, Kirsten rat sarcoma viral oncogene, epidermal growth factor receptor (EGFR), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), and so on) is leading to new challenges for surgical pathology. As all the biomarkers to be specifically detected are tissue based, a precise and reliable diagnostic is absolutely crucial. To meet this challenge, surgical pathology has adapted a number of molecular methods (semi-quantitative immunohistochemistry, fluorescence in situ hybridization), PCR and its multiple variants, (pyro/Sanger) sequencing, next-generation sequencing, DNA-arrays, methylation analyses, and so on) to be applicable for formalin-fixed paraffin-embedded (FFPE) tissue. To read a patients' tissue as 'deeply' as possible and to obtain information on morphological, genetic, proteomic as well as epigenetic background is the actual task of pathologists and molecular biologists in order to provide the clinicians with information relevant for individualized medicine. The intensified cooperation of clinicians and pathologists will provide the basis of improved clinical drug selection as well as guide development of new cancer gene therapies and molecularly targeted drugs by research units and the pharmaceutical industry. This review will give some information on (1) biomarker detection methods adapted to FFPE tissue, (2) the potency of predictive pathology in tumor detection and treatment and (3) the implications of pathology on the development of new drugs in molecularly targeted and gene therapies. [ABSTRACT FROM AUTHOR]

Background: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases.Methods: Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed.Results: Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model.Conclusion: We conclude that in breast cancer, Mb is co-expressed with ERalpha and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours. [ABSTRACT FROM AUTHOR]

GOLPH2 is coding the 73-kDa type II Golgi membrane antigen GOLPH2/GP73. Upregulation of GOLPH2 mRNA has been recently reported in expression array analyses of prostate cancer. As GOLPH2 protein expression in prostate tissues is currently unknown, this study aimed at a comprehensive analysis of GOLPH2 protein in benign and malignant prostate lesions. Immunohistochemically detected GOLPH2 protein expression was compared with the basal cell marker p63 and the prostate cancer marker alpha-methylacyl-CoA racemase (AMACR) in 614 radical prostatectomy specimens. GOLPH2 exhibited a perinuclear Golgi-type staining pattern and was preferentially seen in prostatic gland epithelia. Using a semiquantitative staining intensity score, GOLPH2 expression was significantly higher in prostate cancer glands compared with normal glands (P<0.001). GOLPH2 protein was upregulated in 567 of 614 tumours (92.3%) and AMACR in 583 of 614 tumours (95%) (correlation coefficient 0.113, P = 0.005). Importantly, GOLPH2 immunohistochemistry exhibited a lower level of intratumoral heterogeneity (25 vs 45%). Further, GOLPH2 upregulation was detected in 26 of 31 (84%) AMACR-negative prostate cancer cases. These data clearly suggest GOLPH2 as an additional ancillary positive marker for tissue-based diagnosis of prostate cancer. [ABSTRACT FROM AUTHOR]

High activity of histone deacetylases (HDACs) causes epigenetic alterations associated with malignant cell behaviour. Consequently, HDAC inhibitors have entered late-phase clinical trials as new antineoplastic drugs. However, little is known about expression and function of specific HDAC isoforms in human tumours including prostate cancer. We investigated the expression of class I HDACs in 192 prostate carcinomas by immunohistochemistry and correlated our findings to clinicopathological parameters including follow-up data. Class I HDAC isoforms were strongly expressed in the majority of the cases (HDAC1: 69.8%, HDAC2: 74%, HDAC3: 94.8%). High rates of HDAC1 and HDAC2 expression were significantly associated with tumour dedifferentiation. Strong expression of all HDACs was accompanied by enhanced tumour cell proliferation. In addition, HDAC2 was an independent prognostic marker in our prostate cancer cohort. In conclusion, we showed that the known effects of HDACs on differentiation and proliferation of cancer cells observed in vitro can also be confirmed in vivo. The class I HDAC isoforms 1, 2 and 3 are differentially expressed in prostate cancer, which might be important for upcoming studies on HDAC inhibitors in this tumour entity. Also, the highly significant prognostic value of HDAC2 clearly deserves further study. [ABSTRACT FROM AUTHOR]

Aims: Archived tissue blocks preserve the antigenicity of samples for a long time under normal storage conditions, whereas tissue sections may show a diminished immunoreactivity over time. Little is known about the processes responsible for antigenicity loss and how tissue sections should be conserved for extended storage. Oxidation and drying are presumed mechanisms of antigenicity loss. To prove this, degradation of immunoreactivity was provoked by chemical oxidation, photo-oxidation and artificial drying. Methods: First, paraffin sections of an oestrogen receptor (ER) positive breast carcinoma were subjected for variable time periods to H202, ultraviolet A (UVA) irradiation and dry heat (56°C) prior to ER immunohistochemistry. Second, using heat and UVA irradiation several other antigens (ER, PR, HER-2neu, p53, p63, p16, PSA, CK5/6, CK7, CK20, SMA, Fli-1, c-kit, CD20 and EGFR) were tested, using internal control tissue microarray sections. Results: While H202 had no effect on the ER-staining intensity, extended drying showed a detectable decrease in staining after 10 days, and UVA irradiation induced a decrease after 3 days. Entire antigenicity loss was not observed. Except for HER-2neu, PSA and Fli-1, all antigens showed some diminution in antigenicity, but no entire antigenicity loss, after heating and UVA irradiation. Conclusions: This study confirms that photo-oxidation and drying have an influence on immunohistochemistry outcome, and protocols for testing the stability of specific antigens are provided. [ABSTRACT FROM AUTHOR]

Activation of nuclear factor-κB (NF-κB) signaling was observed in pancreatic adenocarcinoma cell lines and tumours. However, information on the expression of RelA/p65, the major transcription activating NF-κB subunit, in these carcinomas and possible correlations thereof with NF-κB activation and patient survival is not available. To provide this missing translational link, we analysed expression of RelA/p65 in 82 pancreatic adenocarcinomas by immunohistochemistry. Moreover, we measured activation of the NF-κB pathway in 11 tumours by quantitative PCR for NF-κB target genes. We observed strong cytoplasmic or nuclear expression of RelA/p65 in 42 and 37 carcinomas, respectively. High cytoplasmic and nuclear expression of RelA/p65 had negative prognostic impact with 2-year survival rates for patients without cytoplasmic or nuclear RelA/p65 positivity of 41 and 40% and rates for patients with strong cytoplasmic or nuclear RelA/p65 expression of 22 and 20%, respectively. High RelA/p65 expression was correlated to increased expression of NF-κB target genes. The observation that high expression of RelA/p65 is correlated to an activation of the NF-κB pathway and indicates poor patient survival identifies a patient subgroup that might particularly benefit from NF-κB-inhibiting agents in the treatment of pancreatic cancer. Based on our findings, this subgroup could be identified by applying simple immunohistochemical techniques.British Journal of Cancer (2007) 97, 523–530. doi:10.1038/sj.bjc.6603878 www.bjcancer.com Published online 10 July 2007 [ABSTRACT FROM AUTHOR]

Determination of oestrogen receptor alpha (ER) represents at present the most important predictive factor in breast cancers. Data of ours and of other authors suggest that promising predictive/prognostic factors may also include pS2, metallothionein (MT) and CD24. Present study aimed at determining prognostic and predictive value of immunohistochemical determination of ER, pS2, MT, and CD24 expression in sections originating from 104 patients with breast cancer. An univariate and multivariate analysis was performed. Both univariate and multivariate analyses demonstrated that cytoplasmic-membranous expression of CD24 (CD24c-m) represents a strong unfavourable prognostic factor in the entire group and in most of the subgroups of patients. In several subgroups of the patients also a prognostic value was demonstrated of elevated expression of pS2 and of membranous expression of CD24. Our studies demonstrated that all patients with good prognostic factors (higher ER and pS2 expressions, lower MT expression, CD24c-m negativity) survived total period of observation (103 months). The study documented that cytoplasmic-membranous expression of CD24 represented an extremely strong unfavourable prognostic factor in breast cancer. Examination of the entire panel of the studied proteins permitted to select a group of patients of an exceptionally good prognosis. [ABSTRACT FROM AUTHOR]

Human kallikrein 14 (KLK14) is a steroid hormone-regulated member of the tissue kallikrein family of serine proteases, for which a prognostic and diagnostic value in breast cancer has been suggested. To further characterise the value of KLK14 as a breast tumour marker, we have carefully analysed KLK14 expression in normal breast tissue and breast cancer both on the RNA level by real-time RT-PCR (n=39), and on the protein level (n=127) using a KLK14-specific antibody for immunohistochemistry. We correlated KLK14 protein expression data with available clinico-pathological parameters (mean follow-up time was 55 months) including patient prognosis. KLK14 RNA expression as quantified by real-time RT-PCR was significantly more abundant in breast tumours compared to normal breast tissue (P=0.027), an issue that had not been clarified recently. Concordantly with the RNA data, cytoplasmic KLK14 protein expression was significantly higher in invasive breast carcinomas compared to normal breast tissues (P=0.003). Furthermore, KLK14 protein expression was associated with higher tumour grade (P=0.041) and positive nodal status (P=0.045) but was not significantly associated with shortened disease-free or overall patient survival time in univariate analyses. We conclude that KLK14 is clearly overexpressed in breast cancer in comparison to normal breast tissues and is positively associated with conventional parameters of tumour aggressiveness, but due to a missing association with survival times, the use of KLK14 immunohistochemistry as a prognostic marker in breast cancer is questionable.British Journal of Cancer (2006) 94, 540–547. doi:10.1038/sj.bjc.6602956 www.bjcancer.com Published online 24 January 2006 [ABSTRACT FROM AUTHOR]

Background NTRK1 , NTRK2 and NTRK3 fusions are present in a plethora of malignancies across different histologies. These fusions represent the most frequent mechanism of oncogenic activation of these receptor tyrosine kinases, and biomarkers for the use of TRK small molecule inhibitors. Given the varying frequency of NTRK1/2/3 fusions, crucial to the administration of NTRK inhibitors is the development of optimal approaches for the detection of human cancers harbouring activating NTRK1/2/3 fusion genes. Materials and methods Experts from several Institutions were recruited by the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) to review the available methods for the detection of NTRK gene fusions, their potential applications, and strategies for the implementation of a rational approach for the detection of NTRK1/2/3 fusion genes in human malignancies. A consensus on the most reasonable strategy to adopt when screening for NTRK fusions in oncologic patients was sought, and further reviewed and approved by the ESMO TR and PM WG and the ESMO leadership. Results The main techniques employed for NTRK fusion gene detection include immunohistochemistry, fluorescence in situ hybridization (FISH), RT-PCR, and both RNA-based and DNA-based next generation sequencing (NGS). Each technique has advantages and limitations, and the choice of assays for screening and final diagnosis should also take into account the resources and clinical context. Conclusion In tumours where NTRK fusions are highly recurrent, FISH, RT-PCR or RNA-based sequencing panels can be used as confirmatory techniques, whereas in the scenario of testing an unselected population where NTRK1/2/3 fusions are uncommon, either front-line sequencing (preferentially RNA-sequencing) or screening by immunohistochemistry followed by sequencing of positive cases should be pursued. [ABSTRACT FROM AUTHOR]

Strong stromal and cytoplasmic SPARC expression was associated with worse DFS and OS in the overall study population of CONKO-001. The negative prognostic impact was restricted to patients who received adjuvant treatment with gemcitabine. Our data implements the role of SPARC in a more general context: High SPARC expression is associated with a decreased DFS and OS in patients treated with gemcitabine (or chemotherapy in general?)Background Previous investigations in pancreatic cancer suggested a prognostic role for secreted protein acidic and rich in cysteine (SPARC) expression in the peritumoral stroma but not for cytoplasmic SPARC expression. The aim of this study was to evaluate the impact of SPARC expression in pancreatic cancer patients treated with gemcitabine compared with untreated patients. Patients and methods CONKO-001 was a prospective randomized phase III study investigating the role of adjuvant gemcitabine when compared with observation. Tissue samples of 160 patients were available for SPARC immunohistochemistry on tissue microarrays to evaluate its impact on patient outcome. Results Strong stromal SPARC expression was associated with worse disease-free survival (DFS) and overall survival (OS) in the overall study population (DFS: P = 0.005, OS: P = 0.033). Its negative prognostic impact was restricted to patients treated with gemcitabine (DFS: P = 0.007, OS: P = 0.006). High cytoplasmic SPARC expression also was associated with worse patient outcome (DFS: P = 0.041, OS: P = 0.011). Again the effect was restricted to patients treated with gemcitabine (DFS: P = 0.002, OS: P = 0.003). In multivariable analysis, SPARC expression was independently predictive of patient outcome. Conclusions Our data confirm the prognostic significance of SPARC expression after curatively intended resection. The negative prognostic impact was restricted to patients who received adjuvant treatment with gemcitabine, suggesting SPARC as a predictive marker for response to gemcitabine. [ABSTRACT FROM PUBLISHER]

Background Mucin-1 (MUC1) is a promising antigen for the development of tumor vaccines. We evaluated the frequency of MUC1 expression and its impact on therapy response and survival after neoadjuvant chemotherapy for breast cancer. Patients and methods Pre-treatment core biopsies of patients from the GeparTrio neoadjuvant trial (NCT 00544765) were evaluated for MUC1 by immunohistochemistry (IHC; N = 691) and quantitative RT-PCR (qRT-PCR; N = 286) from formalin-fixed paraffin-embedded (FFPE) samples. Results MUC1 protein and mRNA was detectable in the majority of cases and was associated with hormone-receptor-positive status (P < 0.001). High MUC1 protein and mRNA expression were associated with lower probability of pathologic complete response (P = 0.017 and P < 0.001) and with longer patient survival (P = 0.03 and P < 0.001). In multivariable analysis, MUC1 protein and mRNA expression were independently predictive (P = 0.001 and P < 0.001). MUC1 protein and mRNA expression were independently prognostic for overall survival (P = 0.029 and P = 0.015). Conclusions MUC1 is frequently expressed in breast cancer and detectable on mRNA and protein level from FFPE tissue. It provides independent predictive information for therapy response and survival after neoadjuvant chemotherapy. In clinical immunotherapy trials, MUC1 expression may serve as a predictive marker. [ABSTRACT FROM PUBLISHER]