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1.  α-Methylacyl-CoA racemase (AMACR) serves as a prognostic biomarker for the early recurrence/metastasis of HCC 
Journal of Clinical Pathology  2014;67(11):974-979.
Aims
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, and it is still lacking effective prognostic biomarkers so far. Previous results of the iTRAQ-based quantitative proteomics study (iTRAQ-2DLC-MS/MS) have shown that α-methylacyl-CoA racemase (AMACR) might be a promising prognostic biomarker for the early recurrence/metastasis of hepatocellular carcinoma (HCC). Here a large-scale cohort clinical study was performed to evaluate its prognostic potential.
Methods
HCC samples from patients (n=158) were used for the construction of tissue microarray. The expression level of AMACR was determined by immunohistochemical staining. A large-scale cohort clinical study between the expression of AMACR and some major clinical parameter has been performed to assess the prognostic potential of AMACR for the early recurrence/metastasis of HCC.
Results
Some important clinical parameters such as α-fetoprotein, tumour numbers, dissemination to regional lymph nodes, tumour capsule and portal vein tumour thrombosis are significantly associated with the low expression of AMACR. The expression of AMACR was an independent factor for the survival of patients with HCC. The median survival time was 17 months in the low-expression group compared with 45 months in the high-expression group.
Conclusions
This study reveals that the AMACR might be a potential prognostic marker for predicting early recurrence/metastasis of HCC after hepatectomy.
doi:10.1136/jclinpath-2014-202378
PMCID: PMC4215266  PMID: 25092674
2.  Lamivudine/telbivudine-associated neuromyopathy: neurogenic damage, mitochondrial dysfunction and mitochondrial DNA depletion 
Journal of Clinical Pathology  2014;67(11):999-1005.
Aims
Myopathy or neuropathy has been associated with lamivudine/telbivudine therapy in hepatitis B patients. We aim to describe the pathological changes of lamivudine/telbivudine-associated neuromyopathy.
Methods
We retrospectively recruited six patients who were diagnosed with nucleotide analogues-associated myopathy or neuropathy. Muscle and nerve biopsy were performed, and the specimens were prepared for the light microscopy and electron microscopy. Genomic DNA was extracted from frozen muscle specimens, and the mitochondrial DNA (mtDNA) content was quantified by real-time PCR.
Results
Recovery of the myopathy can be achieved after the discontinuation or changing the drugs to entecavir. Muscle and nerve biopsy revealed similar changes under either the light or electronic microscopy in all the subjects. Quantitative real-time PCR revealed decrease of mtDNA content in the affected muscle.
Conclusions
MtDNA depletion results in mitochondrial dysfunction in the lamivudine/telbivudine-associated neuromyopathy. Myopathy was characterised by mitochondrial dysfunction accompanied with neurogenic damage due to axonal neuropathy. Ultrastructure changes of mitochondria included vacuolisation, simplification of the cristae and homogenised matrix.
doi:10.1136/jclinpath-2013-202069
PMCID: PMC4215273  PMID: 25190818
neuromuscular; NEUROPATHIES; NEUROPATHOLOGY
3.  Next-generation sequencing of adrenocortical carcinoma reveals new routes to targeted therapies 
Journal of Clinical Pathology  2014;67(11):968-973.
Aims
Adrenocortical carcinoma (ACC) carries a poor prognosis and current systemic cytotoxic therapies result in only modest improvement in overall survival. In this retrospective study, we performed a comprehensive genomic profiling of 29 consecutive ACC samples to identify potential targets of therapy not currently searched for in routine clinical practice.
Methods
DNA from 29 ACC was sequenced to high, uniform coverage (Illumina HiSeq) and analysed for genomic alterations (GAs).
Results
At least one GA was found in 22 (76%) ACC (mean 2.6 alterations per ACC). The most frequent GAs were in TP53 (34%), NF1 (14%), CDKN2A (14%), MEN1 (14%), CTNNB1 (10%) and ATM (10%). APC, CCND2, CDK4, DAXX, DNMT3A, KDM5C, LRP1B, MSH2 and RB1 were each altered in two cases (7%) and EGFR, ERBB4, KRAS, MDM2, NRAS, PDGFRB, PIK3CA, PTEN and PTCH1 were each altered in a single case (3%). In 17 (59%) of ACC, at least one GA was associated with an available therapeutic or a mechanism-based clinical trial.
Conclusions
Next-generation sequencing can discover targets of therapy for relapsed and metastatic ACC and shows promise to improve outcomes for this aggressive form of cancer.
doi:10.1136/jclinpath-2014-202514
PMCID: PMC4215283  PMID: 25078331
Cancer Genetics; Endocrine Pathology; Molecular Pathology; Oncology; Gene Amplification
4.  Guidance for laboratories performing molecular pathology for cancer patients 
Journal of Clinical Pathology  2014;67(11):923-931.
Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here.
doi:10.1136/jclinpath-2014-202404
PMCID: PMC4215286  PMID: 25012948
Molecular Pathology; Neoplasms; Quality Control; Laboratory Tests; Molecular Oncology
5.  Immune activation in HIV/HCV-infected patients is associated with low-level expression of liver expressed antimicrobial peptide-2 (LEAP-2) 
Journal of clinical pathology  2013;66(11):967-975.
Background
Immune activation is one of the main features of HIV/Hepatitis C virus (HCV) infections and has been linked to the disturbance of the gut-associated lymphoid tissue (GALT). In chronic HIV infection, loss of GALT integrity results in translocation of microbial products and chronic immune activation. We explored the relationship between bacterial translocation and specific colonic proteins, including liver expressed antimicrobial peptide (LEAP 2) which may play a role in modulating the bacterial translocation process.
Methods
A total of 40 subjects (10 HIV/HCV, 10 HIV, 10 HCV-infected patients and 10 controls) were enrolled and underwent serum and colonic tissue sampling. The levels of immune activation were evaluated by measuring plasma sCD27, and the levels of selected proinflammatory, Th2 and regulatory cytokines in both the plasma and supernatant of CD3-stimulated intraepithelial lymphocytes. We also evaluated LEAP-2 expression in the colon biopsies using Affymetrix Human Gene 1.0 ST (HuGene) and fluorescent immunohistochemistry.
Results
Increased levels of sCD27 were observed in HIV/HCV coinfected (p=0.03) and HIV monoinfected (p=0.04) patients compared with controls consistent with the presence of immune activation. The chip array identified LEAP-2 expression as a key marker associated with immune activation. LEAP-2 expression in HIV, HCV and HIV/HCV-infected patients was significantly lower compared with controls, and was significantly negatively correlated (p=0.03, r=−0.44) with sCD27.
Conclusions
Our data suggests that HCV and HIV infections are associated with decreased expression of LEAP-2 in colonic tissue. This may represent a key mechanism for enhanced microbial translocation and immune activation in HIV/HCV-infected patients.
doi:10.1136/jclinpath-2013-201581
PMCID: PMC3987848  PMID: 23940131
6.  High Antiphospholipid Antibody Levels are Associated with Statin Use and May Reflect Chronic Endothelial Damage in Non-Autoimmune Thrombosis: cross-sectional study 
Journal of clinical pathology  2012;65(6):551-556.
Objective
Persistently elevated antiphospholipid antibodies (aPL Ab) and positive lupus anticoagulant (LAC) are associated with an increased risk of thrombosis. Our objective was to explore whether aPL Ab and/or LAC positivity were associated with the traditional risk factors for thrombosis or with medication use in patients without autoimmune diseases hospitalized with arterial or venous thrombosis.
Design
Cross-sectional study
Setting
Montefiore Medical Center, a large urban tertiary care center
Patients
Two hundred and seventy patients (93 with deep vein thrombosis (DVT) or pulmonary embolism (PE), and 177 with non-hemorrhagic stroke (CVA)) admitted between January 2006 and December 2010 with a discharge diagnosis of either DVT, PE or CVA, who had LAC and aPL Abs measured within six months from their index admission. We excluded patients with lupus or antiphospholipid syndrome.
Main Outcome Measures
The main dependant variable was aPL Ab ≥ 40 units (aPL Ab+) and/or LAC+. Independent variables: traditional thrombosis risk factors, statin use, aspirin use, and warfarin use.
Results
Thirty one (11%) patients were LAC+ and/or aPL Ab+ (aPL/LAC+). None of the traditional risk factors at the time of DVT/PE/CVA was associated with aPL/LAC+. Current statin use was associated with an OR of 3.2 (95% CI 1.3, 7.9, p = 0.01) of aPL/LAC+, adjusted for age, ethnicity and gender. Aspirin or warfarin use was not associated with aPL Ab levels.
Conclusion
If statin therapy reflects the history of prior hyperlipidemia, high levels of aPL Abs may be a marker for prior endothelial damage caused by hyperlipidemia.
doi:10.1136/jclinpath-2011-200613
PMCID: PMC3760969  PMID: 22389514
Antiphospholipid antibodies; statins; endothelial damage; thrombosis
7.  Utility of multispectral imaging in automated quantitative scoring of immunohistochemistry 
Journal of clinical pathology  2012;65(6):496-502.
Background
Automated scanning devices and image analysis software provide a means to overcome the limitations of manual semiquantitative scoring of immunohistochemistry. Common drawbacks to automated imaging systems include an inability to classify tissue type and an inability to segregate cytoplasmic and nuclear staining.
Methods
Immunohistochemistry for the membranous marker α-catenin, the cytoplasmic marker stathmin and the nuclear marker Ki-67 was performed on tissue microarrays (TMA) of archival formalin-fixed paraffin-embedded tissue comprising 471 (α-catenin and stathmin) and 511 (Ki-67) cases of prostate adenocarcinoma. These TMA were quantitatively analysed using two commercially available automated image analysers, the Ariol SL-50 system and the Nuance system from CRi. Both systems use brightfield microscopy for automated, unbiased and standardised quantification of immunohistochemistry, while the Nuance system has spectral deconvolution capabilities.
Results
Overall concordance between scores from both systems was excellent (r=0.90; 0.83–0.95). The software associated with the multispectral imager allowed accurate automated classification of tissue type into epithelial glandular structures and stroma, and a single-step segmentation of staining into cytoplasmic or nuclear compartments allowing independent evaluation of these areas. The Nuance system, however, was not able to distinguish reliably between tumour and non-tumour tissue. In addition, variance in the labour and time required for analysis between the two systems was also noted.
Conclusion
Despite limitations, this study suggests some beneficial role for the use of a multispectral imaging system in automated analysis of immunohistochemistry.
doi:10.1136/jclinpath-2012-200734
PMCID: PMC3437674  PMID: 22447914
9.  ER-α36, a novel isoform of ER-α66, is commonly over-expressed in apocrine and adenoid cystic carcinomas of the breast 
Journal of Clinical Pathology  2010;64(1):54-57.
Background
ER-α36 is a novel 36 kDa isoform of the full-length oestrogen receptor alpha (ER-α66). ER-α36 primarily localises to the cytoplasm and the plasma membrane, and responds to membrane-initiated oestrogen and antioestrogen signalling pathways.
Aim
To examine the expression of ER-α36 in apocrine and adenoid cystic carcinoma of the breast, both of which are consistently ER-α66 negative and currently lack effective targeted therapeutic options.
Methods
19 pure apocrine carcinomas (17 invasive and two in-situ carcinomas) and 11 adenoid cystic carcinomas of the breast were evaluated for ER-α36 expression, along with expressions of ER-α66, progesterone receptor (PR) and androgen receptor (AR) using immunohistochemical methods.
Results
All pure apocrine carcinomas showed a characteristic steroid receptor expression profile (ER-α66 and PR negative, AR strongly positive). ER-α36 expression was detected in 18/19 pure apocrine carcinomas (94.7%, 95% CI 75.1 to 98.7) in predominantly membranous and cytoplasmic distribution. When positive, pure apocrine carcinomas uniformly (100% of cells) expressed ER-α36. All adenoid cystic carcinomas were uniformly negative for all three classic steroid receptors, but ER-α36 was detected in 8/11 cases (72.7%, 95% CI 42.8 to 90) with the similar sub-cellular pattern of expression as in the pure apocrine carcinomas. When positive, adenoid cystic carcinomas expressed ER-α36 in the majority of cells (average 76%).
Conclusion
ER-α36, a novel isoform of ER-α66, is frequently over-expressed in apocrine and adenoid cystic carcinomas of the breast. These results indicate a potential for a novel targeted treatment in these cancers.
doi:10.1136/jcp.2010.082776
PMCID: PMC3380072  PMID: 21045236
10.  Comparison of the RNA-based EndoPredict multigene test between core biopsies and corresponding surgical breast cancer sections 
Journal of Clinical Pathology  2012;65(7):660-662.
Aim
This study compared the perfomance of the RNA-based EndoPredict multigene test on core biopsies and surgical breast cancer specimens and analysed the influence of biopsy-induced tissue injuries on the test result.
Methods
80 formalin-fixed paraffin-embedded samples comprising paired biopsies and surgical specimens from 40 ER-positive, HER2-negative patients were evaluated. Total RNA was extracted and the EndoPredict score was determined.
Results
RNA yield was considerably lower in core biopsies, but sufficient to measure the assay in all samples. The EndoPredict score was highly correlated between paired samples (Pearson r=0.92), with an excellent concordance of classification into a low or high risk of metastasis (overall agreement 95%).
Conclusions
The measurements are comparable between core biopsies and surgical sections, which suggest that the EndoPredict assay can be performed on core biopsy tissue. Inflammatory changes induced by presurgical biopsies had no significant effect on the RNA-based risk assessment in surgical specimens.
doi:10.1136/jclinpath-2012-200716
PMCID: PMC3426896  PMID: 22447922
Breast; breast cancer; breast pathology; cancer; cancer genetics; cancer research; EGFR; endocrine pathology; gynaecological pathology; molecular oncology; molecular pathology; oncology; ovary; statistics; tumour markers
11.  Quality standards and samples in genetic testing 
Journal of Clinical Pathology  2012;65(5):389-393.
The most critical performance indicator for medical laboratories is the delivery of accurate test results. In any laboratory, there is always the possibility that random or systematic errors may occur and place human health and welfare at risk. Laboratory quality assurance programmes continue to drive improvements in analytical accuracy. The most rigorously scrutinised data on laboratory errors, which come from transfusion medicine, reveal that the incidence of analytical errors has fallen to levels where most of the residual risk is now found in preanalytical links in the chain from patient to result, particularly activities associated with ordering of tests and sample collection. This insight is important for genetic testing because, like pretransfusion testing of patients with unknown blood groups, a substantial proportion of genotyping results cannot be immediately verified. An increasing number of clinical decisions, associated personal and social choices, and legal outcomes are now influenced by genetic test results in the absence of other confirmatory data. An incorrect test result may lead to unnecessary and irreversible interventions, which may in themselves have associated risks for the patient, inaccurate risk assessment regarding the disease, missed opportunities for disease prevention or even wrongful conviction in a court of law. Unfortunately, there is limited information available about the risk of preanalytical errors associated with, and few published guidelines regarding, sample collection for genetic testing. The growing number and range of important decisions made on the basis of genetic findings warrant a reappraisal of current standards to minimise risks in genetic testing.
doi:10.1136/jclinpath-2011-200519
PMCID: PMC3337464  PMID: 22259179
Genetic testing; medical errors; risk management; standards; quality, genetics; DNA; general; ethics; cancer genetics
12.  POSTMORTEM CANDIDEMIA: MARKER OF DISSEMINATED DISEASE 
Journal of clinical pathology  2009;63(4):337-340.
Aim
The significance of finding Candida species in heart blood cultures obtained at postmortem examination has never been studied. Therefore, we describe the findings of autopsy patients with postmortem candidemia and compare them to autopsy patients with antemortem candidemia.
Method
Twenty-three patients with Candida species isolated from heart blood at autopsy were identified over a ten-year period. These patients were compared to 10 autopsy patients found during the same time period with antemortem blood cultures isolating Candida species, but not positive postmortem heart blood cultures. Ante- and postmortem records were reviewed.
Results
All 23 patients with Candida species isolated from postmortem blood culture had one or more antemortem risk factors for disseminated candidiasis such as positive antemortem blood cultures, isolation of Candida from sterile internal sites, neutropenia, recent abdominal surgery, broadspectrum antibiotic administration or the use of central venous catheters or other invasive devices. Eight patients had histologic proof of invasive candidiasis in addition to the positive heart blood cultures. This group did not differ with respect to risk factors from 10 autopsy patients with disseminated candidiasis and antemortem blood cultures with Candida species. However, all the patients with antemortem candidemia had histologic evidence of disseminated candidiasis at autopsy.
Conclusion
Candidemia, when documented by heart blood culture performed at autopsy or by antemortem blood culture, is an insensitive, but highly specific indicator of disseminated candidiasis.
doi:10.1136/jcp.2009.070607
PMCID: PMC3086197  PMID: 19939858
Candida; disseminated candidiasis; candidemia; postmortem; autopsy
13.  Differential expression of microRNA-675, microRNA-139-3p and microRNA-335 in benign and malignant adrenocortical tumours 
Journal of Clinical Pathology  2011;64(6):529-535.
Background
For the clinical management of adrenocortical neoplasms it is crucial to correctly distinguish between benign and malignant tumours. Even histomorphologically based scoring systems do not allow precise separation in single lesions, thus novel parameters are desired which offer a more accurate differentiation. The tremendous potential of microRNAs (miRNAs) as diagnostic biomarkers in surgical pathology has recently been shown in a broad variety of tumours.
Methods
In order to elucidate the diagnostic impact of miRNA expression in adrenocortical neoplasms, a cohort of 20 adrenocortical specimens including normal adrenal tissue (n=4), adrenocortical adenomas (ACAs) (n=9), adrenocortical carcinomas (ACCs) (n=4) and metastases (n=3) was analysed using TaqMan low density arrays to identify specific miRNA profiles in order to distinguish between benign and malignant adrenocortical lesions. Results were validated in a validation cohort (n=16).
Results
Concerning the differential diagnosis of ACAs and ACCs, 159 out of 667 miRNAs were up- and 89 were down-regulated in ACAs. Using real-time PCR analysis of three of the most significantly expressed single key miRNAs allowed separation of ACAs from ACCs. ACCs exhibited significantly lower levels of miR-139-3p (up to 8.49-fold, p<0.001), miR-675 (up to 23.25-fold, p<0.001) and miR-335 (up to 5.25-fold, p<0.001). A validation cohort of 16 specimen with known Weiss score showed up-regulation of miR-335 and miR-675 in the majority of cases with probable malignant course, although overlapping values exist.
Conclusion
miRNA profiling of miR-675 and miR-335 helps in discriminating ACCs from ACAs. miRNA analysis may indicate malignant behaviour in cases with indeterminate malignant potential.
doi:10.1136/jcp.2010.085621
PMCID: PMC3099361  PMID: 21471143
miRNA; adrenal gland; adrenocortical cancer; miRNA array; micro array; molecular pathology
14.  EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis 
Journal of Clinical Pathology  2011;64(5):415-420.
Aims
Epithelial cell adhesion molecule (EpCAM) is a cell surface protein with oncogenic features that is expressed on healthy human epithelia and corresponding malignant tumours. EpCAM expression frequently correlates with more aggressive tumour behaviour and new EpCAM-specific therapeutic agents have recently been approved for clinical use in patients with cancer. However, no consensus exists on how and when to evaluate EpCAM expression in patients with cancer.
Material and methods
EpCAM expression was assessed by a well-established immunohistochemical staining protocol in 2291 primary tumour tissues and in 108 metastases using the EpCAM-specific antibody clone VU1D9. A total immunostaining score was calculated as the product of a proportion score and an intensity score. Four expression subgroups (no, weak, moderate and intense) were defined. As described previously, the term ‘EpCAM overexpression’ was reserved for tissues showing a total immunostaining score >4.
Results
EpCAM was highly expressed in most tumours of gastrointestinal origin and in some carcinomas of the genitourinary tract. However, hepatocellular carcinomas, clear cell renal cell cancer, urothelial cancer and squamous cell cancers were frequently EpCAM negative. EpCAM expression in breast cancer depended on the histological subtype; lobular histology usually showed no or weak expression. Most metastases were EpCAM positive and they frequently reflected the expression phenotype of the primary tumour.
Conclusion
EpCAM expression was detected on adenocarcinomas of various primary sites. If EpCAM-specific antibodies are intended to be used in patients with cancer, we recommend prior immunohistochemical evaluation of EpCAM expression, particularly in patients with renal cell cancer, hepatocellular carcinoma, urothelial carcinoma, breast cancer and squamous cell carcinomas.
doi:10.1136/jcp.2011.090274
PMCID: PMC3088404  PMID: 21415054
Antibodies; immunohistochemistry
15.  Correction 
doi:10.1136/jcp.2010.078253corr1
PMCID: PMC3252589
16.  Preliminary study on the correlation between grading and histology of solitary pulmonary nodules and contrast enhancement and [18F]fluorodeoxyglucose standardised uptake value after evaluation by dynamic multiphase CT and PET/CT 
Journal of Clinical Pathology  2010;64(2):114-119.
Aim
To evaluate whether the histology and grading of solitary pulmonary nodules (SPNs) correlated with the results of dynamic multiphase multidetector CT (MDCT) and the [18F]fluorodeoxyglucose standardised uptake value (SUV) in 30 patients.
Methods
Chest x-rays of 270 patients with incidentally detected SPNs were retrospectively evaluated. Thirty patients with histologically proven SPNs were enrolled. On MDCT and positron emission tomography (PET)/CT images, two experts measured the density of nodules in all perfusion phases and the SUV. Net enhancement (NE) was calculated by subtracting peak pre-contrast density from peak post-contrast density. The Pearson test was used to correlate nodule NE, SUV, grading, histology and diameter.
Results
Of the 30 malignant SPNs, six were classified as G1 (median NE, 31.5 Hounsfield units (HU); median SUV, 4.8 units), 15 were classified as G2 (median NE, 49 HU; median SUV, 6 units), and nine were classified as G3 (median NE, 32 HU; median SUV, 4.5 units). A highly negative correlation was found in G3 SPNs between NE and the corresponding diameters (r=−0.834; p=0.00524). NE increased with the increase in diameter (r=0.982; p=0.284). SUV increased as the SPN diameter increased (r=0.789; p=0.421). NE and SUV were higher in G2 than G1 SPNs, and lower in G2 than G3 SPNs (r=0.97; p=0.137).
Conclusions
The significant correlation in dedifferentiated (G3) SPNs between NE and diameter (r=−0.834; p=0.00524) supports the theory that stroma and neoangiogenesis are fundamental in SPN growth. The highly negative correlation between NE and diameter demonstrates a net decrease in perfusion despite an increase in dimension. The multidisciplinary approach used herein may result in a more precise prognosis and consequently a better therapeutic outcome, particularly in patients with undifferentiated lung cancer.
doi:10.1136/jcp.2010.076562
PMCID: PMC3030774  PMID: 21169276
Angiogenesis; diagnostics; histopathology; lung cancer; tumour angiogenesis
17.  Expression of intestinal MUC17 membrane-bound mucin in inflammatory and neoplastic diseases of the colon 
Journal of clinical pathology  2010;63(8):702-707.
Aim
To determine the cellular location and expression of MUC17 mucin in specimens of normal, inflamed and neoplastic colon.
Methods
Immunohistochemical analysis of human surgical resection specimens (n=106) was performed with a specific antibody to the MUC17 apomucin protein. A semi-quantitative scoring system was used to measure MUC17 expression. In various colon cancer cell lines, the MUC17 expression was examined by immunoblot analysis and normal RT-PCR.
Results
MUC17 was highly expressed on the surface epithelium and crypts of colonic mucosa. In contrast, the expression of MUC17 was significantly decreased in colonic mucosa of chronic ulcerative colitis (p<0.0001) and ischaemic colitis (p=0.003). Similarly, MUC17 expression was decreased in hyperplastic polyps (p=0.0003), tubular and tubulovillous adenomas (p<0.0001) and colon cancers (p<0.0001). Furthermore, of eight different colon cancer cell lines, MUC17 expression was only detected in LS174T and LS180 cells.
Conclusion
Results indicate that the potential protective effects of this membrane-bound mucin are primarily or secondarily diminished in inflammatory and neoplastic conditions. Further research is needed to determine the specific role of MUC17 in the pathogenesis of these conditions.
doi:10.1136/jcp.2010.078717
PMCID: PMC2997570  PMID: 20702471
18.  High-density tissue microarrays from prostate needle biopsies 
Journal of Clinical Pathology  2010;64(1):88-90.
Background
Formalin-fixed prostate biopsies are frequently the only tissue collected at the time of prostate cancer diagnosis. There is therefore a requirement for techniques that allow the use of these prostate biopsy specimens in a high-throughput analysis of immunohistochemical and fluorescence-in-situ-hybridisation-detected biomarkers.
Methods
The authors have previously described methods that allow tissue microarray (TMA) construction from prostate biopsies. Here, we describe significant technical innovations that provide an easier and more robust system of biopsy–TMA construction.
Results and discussion
The TMAs produced are of a high density (up to 104 cores each, 8×13) and allow a multiplex analysis of biomarkers in the context of clinical trials.
doi:10.1136/jcp.2010.082339
PMCID: PMC3002837  PMID: 21081515
Prostate cancer; tissue microarrays; biomarkers; needle biopsy; cancer; histopathology; molecular pathology; prostate
19.  Paxillin expression and amplification in early lung lesions of high-risk patients, lung adenocarcinoma and metastatic disease 
Journal of Clinical Pathology  2010;64(1):16-24.
Background
Paxillin is a modular protein that localises to cell adhesion sites where it facilitates bidirectional communication between the intracellular actin cytoskeleton and the extracellular matrix. These complex and dynamic interactions are essential for cell adhesion, cell migration and cell survival. The authors have previously demonstrated that paxillin is overexpressed in lung cancer tissues and identified somatic paxillin mutations in 9% of lung cancers. A murine in vivo xenograft model of the most common paxillin mutation (A127T) showed increased cell proliferation and invasive tumour growth, establishing an important role for paxillin in the development of lung cancer.
Methods
The authors analysed 279 bronchoscopy-aided biopsy specimens from 92 high-risk patients. Adenocarcinoma with bronchioloalveolar features and pure bronchioloalveolar carcinoma (BAC) were analysed with fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC).
Results
Paxillin is overexpressed in premalignant areas of hyperplasia, squamous metaplasia and goblet cell metaplasia, as well as dysplastic lesions and carcinoma in high-risk patients. Concordance between increased paxillin gene copy number and paxillin overexpression was observed in cases of adenocarcinoma eusomic for chromosome 12.
Conclusions
Paxillin overexpression occurs during the earliest stages of lung cancer development. FISH and IHC analysis of lung adenocarcinoma suggests that relatively small-scale genomic rearrangements of chromosome 12 are associated with paxillin overexpression in lung adenocarcinoma.
doi:10.1136/jcp.2010.075853
PMCID: PMC3002839  PMID: 21045234
Lung cancer; paxillin; high-risk patient; premalignant lesion; preinvasive lesion; cytogenetics; adenocarcinoma; bronchioloalveolar carcinoma; cancer; cell adhesion molecules
20.  Ocular adnexal marginal zone B cell lymphoma infiltrated by IgG4-positive plasma cells 
Journal of Clinical Pathology  2010;63(12):1059-1065.
Aims
To report the clinicopathological characteristics of patients with ocular adnexal marginal zone B cell lymphoma (MZBL) with IgG4-positive plasma cells.
Methods
114 biopsy samples of ocular adnexal MZBLs were analysed. MZBLs with IgG4-positive plasma cells were included when the IgG4:IgG ratio was >40% (IgG4-related group). The serum levels of each subclass of immunoglobulins and soluble interleukin-2 receptor in the IgG4-related group were compared with those in 61 consecutive patients having MZBL without IgG4-positive plasma cells (IgG4-unrelated group). They were also compared with those in 10 patients having ocular adnexal IgG4-related lymphoplasmacytic disorder (IgG4-related inflammatory group).
Results
Ten (9%) of the patients were diagnosed with MZBL with IgG4-positive plasma cells. The IgG4-related group had a significantly greater degree of sclerosis and reactive follicles in the MZBLs (p=0.0004 and p=0.01, respectively). The serum levels of IgG, IgG1, IgG4, IgE and soluble interleukin 2 receptor in the IgG4-related group were significantly higher than those in the IgG4-unrelated group (p=0.003, p=0.009, p<0.0001, p<0.0001 and p=0.0007, respectively). The serum levels did not differ significantly from those of the IgG4-related inflammatory group. The IgG4-related group also had reactive IgG4-positive lymphoplasmacytic infiltrations in the recurrent lesion and in the stomach.
Conclusions
IgG4-positive plasma cells had infiltrated into ocular adnexal MZBLs in 9% of cases. It is suggested that ocular adnexal MZBLs with IgG4-positive plasma cells have unique histological and serological characteristics that overlap those of ocular adnexal IgG4-related lymphoplasmacytic infiltrative disorder and systemic conditions.
doi:10.1136/jcp.2010.082156
PMCID: PMC2991078  PMID: 20980530
Immunopathology; lymphoma; marginal zone B cell lymphoma; ocular adnexa; ophthalmology
21.  Aberrant upregulation of MUC4 mucin expression in cutaneous condyloma acuminatum and squamous cell carcinoma suggests a potential role in the diagnosis and therapy of skin diseases 
Journal of clinical pathology  2010;63(7):579-584.
Aim
Mucins comprise a family of high-molecular-weight glycoproteins. MUC4, a large transmembrane mucin, has recently emerged as a novel marker for diagnosis, prognosis and therapy in several malignancies. However, its role in skin pathologies remains unknown. The aim of this study was to analyse the expression of MUC4 in cutaneous pathologies by immunohistochemistry for potential diagnostic, prognostic and therapeutic applications.
Methods
A total of 330 tissue spots representing the normal skin, and benign and malignant cutaneous diseases, were analysed after staining with the monoclonal antibody to human MUC4 (clone 8G7).
Results
While the normal epidermis showed a negative to weak-positive expression of MUC4, its expression was significantly upregulated in squamous cell carcinomas (SCCs) where the intensity of staining correlated negatively with tumour grade and positively with age. A moderately strong MUC4 expression was also noted in 2/20 cancer adjacent normal skin and 2/21 chronically inflamed skin tissues, while 10/19 cases of vulval condyloma acuminate, 3/12 of vulval hyperplasia and 2 cases of verruca vulgaris also showed strong MUC4 positivity. Malignant melanoma, basal cell carcinoma and cutaneous cysts were negative.
Conclusion
The results indicate that MUC4 expression is aberrantly upregulated in cutaneous SCCs, vulval condylomas and verruca vulgaris. Further, it appears that MUC4 expression in the skin may be modulated by chronic inflammation and the presence of an adjacent cutaneous malignancy in certain cases. These observations suggest a novel role for MUC4 mucin in the pathogenesis of cutaneous SCC and a possible application as a diagnostic and/or prognostic marker in cutaneous pathologies.
doi:10.1136/jcp.2010.076125
PMCID: PMC2920126  PMID: 20591909
22.  Keratocystoma of the parotid gland: case report and immunohistochemical investigation 
Journal of Clinical Pathology  2010;63(8):758-760.
doi:10.1136/jcp.2010.076521
PMCID: PMC2976056  PMID: 20702483
Histopathology; immunohistochemistry; neoplasms; salivary gland
23.  Programmed cell death 4 (PDCD4) expression during multistep Barrett's carcinogenesis 
Journal of Clinical Pathology  2010;63(8):692-696.
Aim
To test the contribution of programmed cell death 4 (PDCD4) tumour suppressor gene in Barrett's carcinogenesis.
Methods
PDCD4 immunohistochemical expression was assessed in 88 biopsy samples obtained from histologically proven long-segment Barrett's mucosa (BM; 25 non-intestinal columnar metaplasia, 25 intestinal metaplasia (IM), 16 low-grade intraepithelial neoplasia (LG-IEN), 12 high-grade IEN (HG-IEN) and 10 Barrett's adenocarcinoma (BAc)). As controls, 25 additional samples of native oesophageal mucosa (N) were obtained from patients with dyspepsia. To further support the data, the expression levels of miR-21, an important PDCD4 expression regulator, in 14 N, 5 HG-IEN and 11 BAc samples were determined by quantitative real-time PCR analysis.
Results
PDCD4 immunostaining decreased progressively and significantly with the progression of the phenotypic changes occurring during Barrett's carcinogenesis (p<0.001). Normal basal squamous epithelial layers featured strong PDCD4 nuclear immunoreaction (mostly coexisting with weak–moderate cytoplasmic staining). Non-intestinal columnar metaplasia and intestinal metaplasia preserved a strong nuclear immunostaining; conversely, a significant decrease in PDCD4 nuclear expression was seen in dysplastic (LG-IEN and HG-IEN) and neoplastic lesions. Weak–moderate cytoplasmic immunostaining was evident in cases of LG-IEN, while HG-IEN and BAc samples showed weak cytoplasmic or no protein expression. As expected, miR-21 expression was significantly upregulated in HG-IEN and BAc samples, consistently with PDCD4 dysregulation.
Conclusions
These data support a significant role for PDCD4 downregulation in the progression of BM to BAc, and confirm miR-21 as a negative regulator of PDCD4 in vivo. Further efforts are needed to validate PDCD4 as a potential prognostic marker in patients with Barrett's oesophagus.
doi:10.1136/jcp.2010.078253
PMCID: PMC2976066  PMID: 20702469
Barrett's oesophagus; immunohistochemistry; oesophageal cancer; PDCD4; tumour markers; tumour suppressor gene
24.  The poor man's cell block 
Journal of Clinical Pathology  2010;63(9):837-838.
The authors describe a simple method for making formalin or isopropyl alcohol vapour fixed cell blocks from fine needle aspiration cytology specimens that we refer to as ‘The Poor Man's Cell Block.’
doi:10.1136/jcp.2010.078410
PMCID: PMC2976618  PMID: 20671053
FNA; fine needle aspiration; cytology; cell block; immunohistochemistry; cytopathology; immunocytochemistry
25.  FISH assay development for the detection of p16/CDKN2A deletion in malignant pleural mesothelioma 
Journal of Clinical Pathology  2010;63(7):630-634.
Aims
To develop a fluorescence in-situ hybridisation (FISH) assay for detecting p16/CDKN2A deletion on paraffin tissue sections for use as an ancillary test to distinguish reactive from malignant mesothelial proliferations.
Method
Dual-colour FISH for p16/CDKN2A and chromosome 9 (CEP-9) was performed on 11 benign mesothelial proliferations and 54 malignant pleural mesothelioma (MPM) cases to establish cut-off values for p16/CDKN2A deletion. A third MYC probe was used to verify cases showing homozygous deletion. Eight equivocal biopsies were used for assay testing.
Results
Cut-off values for p16/CDKN2A deletion were calculated based on FISH signalling patterns obtained from the benign controls (mean percent nuclei plus three standard deviations). Hemizygous deletion was defined as >44% of nuclei showing the hemizygous (one p16/CDKN2A, two CEP-9 signals) or >15% of nuclei showing the monosomy (one p16/CDKN2A, one CEP-9 signal) deletion patterns. None of the benign cases showed a homozygous deletion pattern (no p16/CDKN2A, at least one CEP-9 signal). In the malignant cases, the percentage of nuclei showing homozygous deletion ranged from 1% to 87%. Therefore, the cut-off value for homozygous deletion was defined as >10%. P16/CDKN2A deletion was detected in 61% (33/54) of MPM cases. Among the equivocal biopsies, four showed homozygous and one showed hemizygous p16/CDKN2A deletion. Age over 60 years, asbestos exposure and p16/CDKN2A deletion were associated with a worse prognosis.
Conclusion
Distinction between benign and malignant mesothelial proliferations can be diagnostically challenging. FISH for p16/CDKN2A deletion is a useful test for confirming the diagnosis of MPM.
doi:10.1136/jcp.2010.076794
PMCID: PMC2989172  PMID: 20591913
Mesothelioma; p16; CDKN2A; gene deletion; fluorescence in situ hybridisation; FISH; malignant tumours; pleura

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