Immunohistochemical Validation Research Articles

Induction of Proteinuria by Cannabinoid Receptors 1 Signaling Activation in CB1 Transgenic Mice

Proteinuria is not only a sign of kidney damage but is also involved in the progression of renal disease as an independent pathologic factor. Although patients with mutated type 1 cannabinoid receptors (CB1) polymorphism are associated with renal microvascular damage, the biologic role of CB1 signaling in proteinuria remains uncharacterized till now. Herein, we investigate whether CB1 participates in glomerular proteinuria in CB1 transgenic mice and treatment with CB1 agonist WIN55212-2 rat, neither of which are diabetic models. The CB1 transgenic mice and rats treated with CB1 agonist WiN55212-2 had higher kidney weight and urinary protein concentrations but not blood glucose levels compared with the wild-type group. A combination of laser-capture microsdissection, quantitative reverse transcription-polymerase chain reaction, immunoblotting and immunohistochemical validation revealed that CB1 transgenic mice and rats treated with CB1 agonist WIN55212-2 had higher vascular endothelial growth factor (vEGF) expression in renal glomeruli than that of the wild-type group. Geneticorpharmacological activation of CB1 by transgenic CB1 mice or treatment with WIN55212-2 reduced nephrin expression in the renal glomeruli compared with that of the wild-type group in the glomerular mesanglium. Taken together, CB1 transgenic mice and rats treated with CB1 agonist WIN55212-2 induced proteinuria with upregulation of CB1 resulting in impaired nephrin expression, by inducing excess vEGF reaction in the renal glomeruli. Genetic and pharmacological manipulation of CB1 signaling revealed vEGF-dependent nephrin depression of glomerulopathy. Controlling CB1 activity can be used an alternative strategy for sustaining renal function in the presence of CB1 activation.

Detection of micro-metastases by serial sectioning and immuno-histochemical validation of sentinel nodes in patients with breast carcinoma

Background Examination of sentinel lymph node (SLN) biopsy can give accurate nodal staging for breast cancer and assist in the management of patients. Serial sectioning and immuno-histochemical (IHC) staining of SLN can improve detection of micro-metastases. Methods Twenty-five patients with clinically node-negative breast cancer were recruited. A combination of radiotracer and blue-dye were used for identification of SLN. The nodes were thinly sliced and embedded. Serial sectioning and immuno-histochemical staining against AE1/3 were done if initial H&E sections were negative. Results Three cases (12%) had no tumour at initial H&E sections but tumour was found at serial levels and with IHC validation. These were micro-metastases of less than 2 mm diameter. Four other cases with macro-metastases were found at initial H&E sections of the SLN. Three false negative cases had axillary nodal (ALN) metastases with no tumour in the SLN despite levelling and IHC staining. Discussion and conclusion Thin slicing of tissue and serial sectioning improved the detection of micro-metastases. However, immuno-histochemical validation did not additionally increase the sensitivity of detection. The presence of ALN metastases without SLN involvement could be due to discontinuous or skip metastases to non-sentinel nodes or poor technique resulting in missed detection of the SLN.

Novel prognostic markers revealed by a proteomic approach separating benign from malignant insulinomas

The prognosis of pancreatic neuroendocrine tumors is related to size, histology and proliferation rate. However, this stratification needs to be refined further. We conducted a proteome study on insulinomas, a well-defined pancreatic neuroendocrine tumor entity, in order to identify proteins that can be used as biomarkers for malignancy. Based on a long follow-up, insulinomas were divided into those with metastases (malignant) and those without (benign). Microdissected cells from six benign and six malignant insulinomas were subjected to a procedure combining fluorescence dye saturation labeling with high-resolution two-dimensional gel electrophoresis. Differentially expressed proteins were identified using nano liquid chromatography–electrospray ionization/multi-stage mass spectrometry and validated by immunohistochemistry on tissue microarrays containing 62 insulinomas. Sixteen differentially regulated proteins were identified among 3000 protein spots. Immunohistochemical validation revealed that aldehyde dehydrogenase 1A1 and voltage-dependent anion-selective channel protein 1 showed significantly stronger expression in malignant insulinomas than in benign insulinomas, whereas tumor protein D52 (TPD52) binding protein was expressed less strongly in malignant insulinomas than in benign insulinomas. Using multivariate analysis, low TPD52 expression was identified as a strong independent prognostic factor for both recurrence-free and overall disease-related survival.

Downregulation of S100 Calcium Binding Protein A9 in Esophageal Squamous Cell Carcinoma.

The development of esophageal squamous cell carcinoma (ESCC) is poorly understood and the major regulatory molecules involved in the process of tumorigenesis have not yet been identified. We had previously employed a quantitative proteomic approach to identify differentially expressed proteins in ESCC tumors. A total of 238 differentially expressed proteins were identified in that study including S100 calcium binding protein A9 (S100A9) as one of the major downregulated proteins. In the present study, we carried out immunohistochemical validation of S100A9 in a large cohort of ESCC patients to determine the expression and subcellular localization of S100A9 in tumors and adjacent normal esophageal epithelia. Downregulation of S100A9 was observed in 67% (n = 192) of 288 different ESCC tumors, with the most dramatic downregulation observed in the poorly differentiated tumors (99/111). Expression of S100A9 was restricted to the prickle and functional layers of normal esophageal mucosa and localized predominantly in the cytoplasm and nucleus whereas virtually no expression was observed in the tumor and stromal cells. This suggests the important role that S100A9 plays in maintaining the differentiated state of epithelium and suggests that its downregulation may be associated with increased susceptibility to tumor formation.

Immunohistochemical validation and expression profiling of NKG2D ligands in a wide spectrum of human epithelial neoplasms.

The MHC class I-chain-related proteins (MICs) and the UL16-binding proteins (ULBPs) are inducible stress response molecules that work as activators of a specific receptor, NKG2D, which is expressed on effector cells, such as NK cells and subsets of T cells. In this study, we sought to explore the biological significance of NKG2D ligands in human neoplasms by comprehensively examining the immunohistochemical expression profile of NKG2D ligands in a variety of human epithelial neoplasms. Following careful validation of the immunohistochemical specificity and availability of anti-human ULBP antibodies for formalin-fixed paraffin-embedded (FFPE) materials, the expression of NKG2D ligands was analyzed in FFPE tissue microarrays comprising 22 types of epithelial neoplastic tissue with their non-neoplastic counterpart from various organs. Hierarchical cluster analysis demonstrated a positive relationship among ULBP2/6, ULBP3, ULBP1, and ULBP5, whose expression patterns were similar across all of the neoplastic tissues examined. In contrast, MICA/B, as well as ULBP4, did not appear to be related to any other ligand. These expression profiles of NKG2D ligands in human neoplasms based on well-validated specific antibodies, followed by hierarchical cluster analysis, should help to clarify some functional aspects of these molecules in cancer biology, and also provide a path to the development of novel tumor-type-specific treatment strategies.

Epithelial‐myoepithelial carcinoma arising in the palate: a rare case report and review of the literature

AbstractThis report describes our recent experience with a case of epithelial‐myoepithelial carcinoma of the left palate and reviews previously reported cases of this rare malignancy. A 68‐year‐old woman presented with painless nodular swelling of the left hard palate. Histopathological and immunohistochemical analyses confirmed a diagnosis of epithelial‐myoepithelial carcinoma. We performed a partial maxillectomy through a lateral rhinotomy incision under general anaesthesia. During the surgery, the entire tumour mass was removed with clear surgical margins. The patient continued to undergo clinical follow‐up and radiological assessments for a period of 2.5 years with no evidence of recurrent or metastatic disease. Epithelial‐myoepithelial carcinoma is a rare malignancy that requires immunohistochemical validation of diagnosis and may be successfully treated with surgery alone.

Quantitative proteome profiling of lymph node-positivevs. -negative colorectal carcinomas pinpoints MX1 as a marker for lymph node metastasis

We used high-resolution mass spectrometry to measure the abundance of more than 9,000 proteins in 19 individually dissected colorectal tumors representing lymph node metastatic (n = 10) and nonmetastatic (n = 9) phenotypes. Statistical analysis identified MX1 and several other proteins as overexpressed in lymph node-positive tumors. MX1, IGF1-R and IRF2BP1 showed significantly different expression in immunohistochemical validation (Wilcoxon test p = 0.007 for IGF1-R, p = 0.04 for IRF2BP1 and p = 0.02 for MX1 at the invasion front) in the validation cohort. Knockout of MX1 by siRNA in cell cultures and wound healing assays provided additional evidence for the involvement of this protein in tumor invasion. The collection of identified and quantified proteins to our knowledge is the largest tumor proteome dataset available at the present. The identified proteins can give insights into the mechanisms of lymphatic metastasis in colorectal carcinoma and may act as prognostic markers and therapeutic targets after further prospective validation.

Keratin 17 in premalignant and malignant squamous lesions of the cervix: proteomic discovery and immunohistochemical validation as a diagnostic and prognostic biomarker

Most previously described immunohistochemical markers of cervical high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma may help to improve diagnostic accuracy but have a minimal prognostic value. The goals of the current study were to identify and validate novel candidate biomarkers that could potentially improve diagnostic and prognostic accuracy for cervical HSIL and squamous cell carcinoma. Microdissected tissue sections from formalin-fixed paraffin-embedded normal ectocervical squamous mucosa, low-grade squamous intraepithelial lesion (LSIL), HSIL and squamous cell carcinoma sections were analyzed by mass spectrometry-based shotgun proteomics for biomarker discovery. The diagnostic specificity of candidate biomarkers was subsequently evaluated by immunohistochemical analysis of tissue microarrays. Among 1750 proteins identified by proteomic analyses, keratin 4 (KRT4) and keratin 17 (KRT17) showed reciprocal patterns of expression in the spectrum of cases ranging from normal ectocervical squamous mucosa to squamous cell carcinoma. Immunohistochemical studies confirmed that KRT4 expression was significantly decreased in squamous cell carcinoma compared with the other diagnostic categories. By contrast, KRT17 expression was significantly increased in HSIL and squamous cell carcinoma compared with normal ectocervical squamous mucosa and LSIL. KRT17 was also highly expressed in immature squamous metaplasia and in endocervical reserve cells but was generally not detected in mature squamous metaplasia. Furthermore, high levels of KRT17 expression were significantly associated with poor survival of squamous cell carcinoma patients (Hazard ratio=14.76, P=0.01). In summary, both KRT4 and KRT17 expressions are related to the histopathology of the cervical squamous mucosa; KRT17 is highly overexpressed in immature squamous metaplasia, in HSIL, and in squamous cell carcinoma and the level of KRT17 in squamous cell carcinoma may help to identify patients who are at greatest risk for cervical cancer mortality.

Steroid hormone receptor expression in breast cancer stroma

Around two-thirds of all breast carcinomas respond to adjuvant endocrine therapy with the presence of ERa in tumour epithelial cells used to guide therapy. Other members of the steroid receptor superfamily have also been detected in breast cancer epithelial cells, notably PR, AR, whose presence and potential prognostic role in breast cancer is gaining momentum [1, 2], and there is ongoing interest in the potential role of ERb in breast carcinogenesis [3]. However, scientists are now looking beyond the epithelial landscape with the recognition that interaction of tumour epithelial cells with its surrounding microenvironment, particularly the stroma plays a role in promoting tumour growth and progression. While recognising that the stroma is multicellular, much of this attention has focused on fibroblasts residing in tumour stroma, termed carcinoma-associated fibroblasts (CAFs) as there is growing appreciation that CAFs may provide a favourable environment to augment tumour growth [4]. A recent report in Breast Cancer Research and Treatment by Knower et al. [5] used low density Taqman arrays followed by qPCR and immunohistochemical validation to examine the presence of a panel of 48 nuclear receptors in CAFs, derived from ERa-positive breast carcinomas and in normal breast adipose fibroblasts (NAFs). Receptor expression was grouped as absent, low, moderate or high. Intriguingly, of the four nuclear hormone receptors previously associated with breast carcinogenesis listed above, none were identified as being highly expressed in breast stroma; ERb was absent, ERa and PR showed low expression, while moderate expression of AR was seen. As a group with a major interest in the role of ERs in breast carcinogenesis, we have routinely monitored nonepithelial expression of ERs in clinical (ERa) and research settings (ERb) over the years. In contrast to Knower et al. [5], we readily detect ERa in cultured breast fibroblasts from adjacent normal and cancer-associated breast tissue by qPCR. This is carried forward in observations by our group during the routine histopathological reporting of breast carcinoma, where the presence of ERa in stroma is seen in 60 % of all cases, although this can be very focal. Aware of reported issues with some ERa antibodies [6], we evaluated this further using three different antibodies directed against ERa, 1D5 (Dako), 6F11 (Novocastra) and SP1 (Ventana). Using all three antibodies four fifths of tumours expressed ERa in fibroblasts in concordant areas suggesting the signal is genuine (Fig. 1). The lack of ERa expression observed by Knower et al. [5] is thus surprising. We do agree that ERb gene expression is low, although it is usually detectable by qPCR. However, it is commonly found in breast fibroblasts by immunohistochemistry both total ERb [7] and specific isoforms (ERb1, -2 and -5) [8]. ERb is also subject to complex regulation [9], hence nonconcordance of gene and protein is anticipated. The nuclear receptors whose expression was modified in CAFs included RAR-related orphan receptor-a (ROR-a); thyroid hormone receptor-b (TR-b); vitamin D receptor (VDR); and peroxisome proliferator-activated receptor-c (PPAR-c) [5], which one may not immediately associate This letter to the editor refers to the article at 10.1007/s10549-0132716-6 and a rebuttal letter to this letter to the editor is available at 10. 1007/s10549-014-2837-6

Oligonucleotide Microarray Identifies Genes Differentially Expressed during Tumorigenesis of DMBA-Induced Pancreatic Cancer in Rats

The extremely dismal prognosis of pancreatic cancer (PC) is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA)-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR) and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes) were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

Regorafenib Effects on Human Colon Carcinoma Xenografts Monitored by Dynamic Contrast-Enhanced Computed Tomography with Immunohistochemical Validation

ObjectiveTo investigate dynamic contrast-enhanced computed tomography for monitoring the effects of regorafenib on experimental colon carcinomas in rats by quantitative assessments of tumor microcirculation parameters with immunohistochemical validation.Materials and MethodsColon carcinoma xenografts (HT-29) implanted subcutaneously in female athymic rats (n = 15) were imaged at baseline and after a one-week treatment with regorafenib by dynamic contrast-enhanced computed tomography (128-slice dual-source computed tomography). The therapy group (n = 7) received regorafenib daily (10 mg/kg bodyweight). Quantitative parameters of tumor microcirculation (plasma flow, mL/100 mL/min), endothelial permeability (PS, mL/100 mL/min), and tumor vascularity (plasma volume, %) were calculated using a 2-compartment uptake model. Dynamic contrast-enhanced computed tomography parameters were validated with immunohistochemical assessments of tumor microvascular density (CD-31), tumor cell apoptosis (TUNEL), and proliferation (Ki-67).ResultsRegorafenib suppressed tumor vascularity (15.7±5.3 to 5.5±3.5%; p<0.05) and tumor perfusion (12.8±2.3 to 8.8±2.9 mL/100 mL/min; p = 0.063). Significantly lower microvascular density was observed in the therapy group (CD-31; 48±10 vs. 113±25, p<0.05). In regorafenib-treated tumors, significantly more apoptotic cells (TUNEL; 11844±2927 vs. 5097±3463, p<0.05) were observed. Dynamic contrast-enhanced computed tomography tumor perfusion and tumor vascularity correlated significantly (p<0.05) with microvascular density (CD-31; r = 0.84 and 0.66) and inversely with apoptosis (TUNEL; r = −0.66 and −0.71).ConclusionsRegorafenib significantly suppressed tumor vascularity (plasma volume) quantified by dynamic contrast-enhanced computed tomography in experimental colon carcinomas in rats with good-to-moderate correlations to an immunohistochemical gold standard. Tumor response biomarkers assessed by dynamic contrast-enhanced computed tomography may be a promising future approach to a more personalized and targeted cancer therapy.

Immunohistochemical validation of INI1/SMARCB1 in a spectrum of musculoskeletal tumors: An experience at a Tertiary Cancer Referral Centre

The purpose of this study was to evaluate and validate immunohistochemical (IHC) expression of INI1/SMARCB1 in various musculoskeletal tumors in the light of the established literature.Twenty-seven cases of epithelioid sarcoma (ES); 4 of extrarenal rhabdoid tumor (ERRT) of soft tissue and 97 other tumors, including 16 cases of synovial sarcoma (SS), were evaluated for IHC expression of INI1 on formalin-fixed, paraffin-embedded tissue sections of various biopsies.Out of 128 tumors, INI1/SMARCB1 staining was completely lacking in cases of ES (23/27) (85.1%), ERRTs (4/4) (100%), myoepithelial tumors (4/14) (28.5%) and in (1/16) (6.2%) cases of SS. Fourteen out of 15 SSs displayed a reduced staining pattern. Other 67 studied tumors were INI1-positive. Sensitivity for complete INI1 negativity in ES was 85.1%, and specificity with respect to its differentials, excluding ERRTs, was 94.8%.Complete lack of INI1 immunostaining in most ESs indicates its value as a diagnostic marker for ESs, including those occurring at rare sites; in ERRTs and in some myoepithelial tumors, within an appropriate clinicopathological context, in all kinds of biopsies. ES, at least in some cases, is immunohistochemically the most closely related tumor to an ERRT. A unique pattern of reduced INI1 expression in a SS is useful during triage of some cases for molecular testing. Its expression should be interpreted in the tumor cells, rather than intermixed stromal cells and or inflammatory cells that retain INI1 expression.

Introduction and First Clinical Application of a Simplified Immunohistochemical Validation System Confirms Prognostic Impact of KI-67 and CK20 for Stage T1 Urothelial Bladder Carcinoma: Single-Center Analysis of Eight Biomarkers in a Series of Three Hundred Six Patients

BackgroundBiomarkers could help to estimate the prognosis of solid tumors. One of the reasons that many immunohistochemical (IHC) markers are not used routinely is the high interobserver variability and various cutoff values. In the present study, we used a simplified IHC method with a group of 8 biomarkers in stage pT1 urothelial bladder carcinoma (UBC). Patients and MethodsIHC expression of CK20, KI-67, STK15, MUC7, periostin, fibronectin, survivin, and CXCR4 was assessed independently by 2 reviewers in a series of 306 stage pT1 UBC specimens from a single center in 10% steps from < 10% up to > 90%. A general center < 10% vs. ≥ 10% was set for further analysis for all markers. All patients initially underwent a bladder-sparing approach. Kaplan-Meier analyses and multivariate Cox regression analyses of recurrence-free survival (RFS), progression-free survival (PFS), and cancer-specific survival (CSS) were performed. ResultsA cutoff point ≥ 10% was shown to be valid and reliable for marker expression, with 96% interobserver agreement. Of the studied marker expressions, ≥ 10% for Ki-67 showed a statistically significant worse RFS (54% vs. 64%; P = .004), PFS (66% vs. 73%; P = .001), and CSS (71% vs. 77%; P = .015); ≥ 10% for CK20 showed a worse RFS (57% vs. 58%; P = .009). Multivariate Cox regression analysis revealed CK20 to be an independent prognostic factor for recurrence (hazard ratio [HR], 2.08; confidence interval [95% CI]; 1.21-3.57; P = .008) and Ki-67 for progression (HR, 2.11; CI, 1.02-4.37; P = .045). ConclusionWe proposed and applied a simplified IHC evaluation that increases interobserver agreement and confirms the prognostic role of Ki-67 and CK20 for stage T1 UBC.

CD30-positive peripheral T-cell lymphomas share molecular and phenotypic features

Peripheral T-cell lymphoma, not otherwise specified is a heterogeneous group of aggressive neoplasms with indistinct borders. By gene expression profiling we previously reported unsupervised clusters of peripheral T-cell lymphomas, not otherwise specified correlating with CD30 expression. In this work we extended the analysis of peripheral T-cell lymphoma molecular profiles to prototypical CD30(+) peripheral T-cell lymphomas (anaplastic large cell lymphomas), and validated mRNA expression profiles at the protein level. Existing transcriptomic datasets from peripheral T-cell lymphomas, not otherwise specified and anaplastic large cell lymphomas were reanalyzed. Twenty-one markers were selected for immunohistochemical validation on 80 peripheral T-cell lymphoma samples (not otherwise specified, CD30(+) and CD30(-); anaplastic large cell lymphomas, ALK(+) and ALK(-)), and differences between subgroups were assessed. Clinical follow-up was recorded. Compared to CD30(-) tumors, CD30(+) peripheral T-cell lymphomas, not otherwise specified were significantly enriched in ALK(-) anaplastic large cell lymphoma-related genes. By immunohistochemistry, CD30(+) peripheral T-cell lymphomas, not otherwise specified differed significantly from CD30(-) samples [down-regulated expression of T-cell receptor-associated proximal tyrosine kinases (Lck, Fyn, Itk) and of proteins involved in T-cell differentiation/activation (CD69, ICOS, CD52, NFATc2); upregulation of JunB and MUM1], while overlapping with anaplastic large cell lymphomas. CD30(-) peripheral T-cell lymphomas, not otherwise specified tended to have an inferior clinical outcome compared to the CD30(+) subgroups. In conclusion, we show molecular and phenotypic features common to CD30(+) peripheral T-cell lymphomas, and significant differences between CD30(-) and CD30(+) peripheral T-cell lymphomas, not otherwise specified, suggesting that CD30 expression might delineate two biologically distinct subgroups.

190 Immunohistochemical Validation of [18f]-Fluorodeoxyglucose as a Novel Biomarker of Inflamed Vulnerable Carotid Plaque: A Sub-Study of the Canadian Atherosclerosis Imaging Network (CAIN)

Atherosclerosis is a leading cause of morbidity and mortality within Canada. ‘Vulnerable' rupture prone plaque is associated with pronounced inflammatory activity and increased density of macrophage cells. Macrophages have increased metabolic rates and require high-levels of energy for phagocytic activity. Therefore, the uptake of radiolabelled glucose or [18F]-fluorodeoxyglucose (18FDG) imaged with hybrid positron emission tomography (PET) and computed tomography (CT) may serve as a surrogate marker of inflammatory activity within plaque.

Automated Core–Penumbra Quantification in Neonatal Ischemic Brain Injury

Neonatal hypoxic-ischemic brain injury (HII) and arterial ischemic stroke (AIS) result in irreversibly injured (core) and salvageable (penumbral) tissue regions. Identification and reliable quantification of salvageable tissue is pivotal to any effective and safe intervention. Magnetic resonance imaging (MRI) is the current standard to distinguish core from penumbra using diffusion-perfusion mismatch (DPM). However, subtle MR signal variations between core-penumbral regions make their visual delineation difficult. We hypothesized that computational analysis of MRI data provides a more accurate assessment of core and penumbral tissue evolution in HII/AIS. We used two neonatal rat-pup models of HII/AIS (unilateral and global hypoxic-ischemia) and clinical data sets from neonates with AIS to test our noninvasive, automated computational approach, Hierarchical Region Splitting (HRS), to detect and quantify ischemic core-penumbra using only a single MRI modality (T2- or diffusion-weighted imaging, T2WI/DWI). We also validated our approach by comparing core-penumbral images (from HRS) to DPM with immunohistochemical validation of HII tissues. Our translational and clinical data results showed that HRS could accurately and reliably distinguish the ischemic core from penumbra and their spatiotemporal evolution, which may aid in the vetting and execution of effective therapeutic interventions as well as patient selection.

High MCM3 expression is an independent biomarker of poor prognosis and correlates with reduced RBM3 expression in a prospective cohort of malignant melanoma

BackgroundMalignant melanoma is the most lethal form of skin cancer with a variable clinical course even in patients with thin melanomas and localized disease. Despite increasing insights into melanoma biology, no prognostic biomarkers have yet been incorporated into clinical protocols. Reduced expression of the RNA binding motif protein 3 (RBM3) has been shown to correlate with tumour progression and poor prognosis in melanoma and several other cancer forms. In ovarian cancer, an inverse association was found between expression of RBM3 and the minichromosome maintenance 3 (MCM3) gene and protein. In melanoma, gene expression analysis and immunohistochemical validation has uncovered MCM3 as a putative prognostic biomarker. The aim of the present study was to examine the associations of MCM3 expression with clinical outcome and RBM3 expression in a prospective, population-based cohort of melanoma.MethodsImmunohistochemical MCM3 expression was examined in 224 incident cases of primary melanoma from the Malmö Diet and Cancer Study, previously analysed for RBM3 expression. Spearman´s Rho and Chi-Square tests were used to explore correlations between MCM3 expression, clinicopathological factors, and expression of RBM3 and Ki67. Kaplan Meier analysis, the log rank test, and univariable and multivariable Cox proportional hazards modelling were used to assess the impact of MCM3 expression on disease-free survival (DFS) and melanoma-specific survival (MSS).ResultsHigh MCM3 expression was significantly associated with unfavourable clinicopathological features and high Ki67 expression. A significant inverse correlation was seen between expression of MCM3 and RBM3 (p = 0.025). High MCM3 expression was associated with a reduced DFS (HR = 5.62) and MSS (HR = 6.03), and these associations remained significant in multivariable analysis, adjusted for all other factors (HR = 5.01 for DFS and HR = 4.96 for MSS). RBM3 expression remained an independent prognostic factor for MSS but not DFS in the multivariable model.ConclusionsThese findings provide validation of the utility of MCM3 expression as an independent biomarker for prognostication of patients with primary melanoma. Moreover, the inverse association and prognostic impact of MCM3 and RBM3 expression indicate a possible interaction of these proteins in melanoma progression, the functional basis for which merits further study.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1814908129755401

Use of exon-based transcriptome profiling to identify novel signaling pathways and survival-associated genes in diffuse large B-cell lymphoma.

8074 Background: Identification of biological prognostic factors that could be used to define poor risk diffuse large B-cell lymphoma (DLBCL) patients is a main concern. Methods: Study population consisted of 38 de novo high risk DLBCL patients less than 65 years old. The patients were treated in the Nordic phase II protocol with six courses of R-CHOEP14 followed by systemic central nervous system prophylaxis with one course of high dose methotrexate and one course of high dose cytarabine. Exon array-based profiling was used to screen signaling pathways and differentially expressed genes between the clinically high risk patients, who had relapsed or remained in remission in response to dose dense chemoimmunotherapy. At the time of the analysis, median follow up was 34 months, progression free survival (PFS) 78% and overall survival (OS) 78%. Results: The screen between relapsed patients and the patients in remission using criteria of p ≤ 0.05 and fold change ≥ 1.6 revealed 566 differentially expressed genes (131 protein coding), of which 24 were likely to be involved in conventional signaling pathways, including those regulating antigen processing and presentation (CIITA, HLA-DQA2, HLA-DQB1, RFXAP), Jak-STAT signaling (SOCS3), Notch signaling (NOTCH1) and Toll-like receptor signalling (IRF5). In cox univariate analysis, 12 of 24 genes were found to associate with PFS (p&lt;0.05). Of these, high expression of CIITA, DLL4, HLA-DQA2, HLA-DQB1, IRF5, NOTCH1, PER1, RFXAP, SEMA4D and ZFP36 had a favorable impact on PFS, whereas high levels of ENPP3 and PRKAR2B were associated with adverse outcome. Differential expression of four genes was confirmed by quantitative PCR, and prognostic value of six genes validated using Lymphoma/Leukemia Molecular Profiling Project microarray data set. Immunohistochemical validation of the findings in a larger patient cohort is ongoing. Germinal centre B-cell signature did not predict survival in this cohort. Conclusions: The results provide evidence that exon-based transcriptome profiling can identify biologically relevant signaling pathways and genes that discriminate the outcome of homogenously treated young high risk DLBCL patients.

Downregulation of cornulin in esophageal squamous cell carcinoma

Early events in the development of esophageal squamous cell carcinoma (ESCC) are poorly understood and many of the key molecules involved have not yet been identified. We previously used isobaric tags for a relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach to identify differentially expressed proteins in ESCC tissue as compared to the adjacent normal mucosa. Cornulin was identified as one of the major downregulated molecules in ESCC. Cornulin is a member of the S100 fused-type protein family, which has an EF-hand calcium binding motif and multiple tandem repeats of specific peptide motifs. Cornulin was 5-fold downregulated in ESCC as compared to normal epithelium mirroring our previous findings in a gene expression study of ESCC. In the present study, we performed immunohistochemical validation of cornulin (CRNN) in a larger set of patients with ESCC. Downregulation of cornulin was observed in 89% (n=239) of 266 different ESCC tissues arrayed on tissue microarrays (TMAs). Expression of cornulin was observed in the prickle and functional cell layers of normal esophageal mucosa, localized predominantly in the cytoplasm and perinuclear region. The large majority of ESCC cases had little or no expression of cornulin in the carcinoma or stroma. These findings suggest that cornulin is an important molecule in normal esophageal pathology and is likely lost during the conversion of normal to neoplastic epithelium.

Integrated Analysis Reveals Critical Genomic Regions in Prostate Tumor Microenvironment Associated with Clinicopathologic Phenotypes

Recent studies suggest that tumor microenvironment (stroma) is important in carcinogenesis and progression. We sought to integrate global genomic structural and expressional alterations in prostate cancer epithelium and stroma and their association with clinicopathologic features. We conducted a genome-wide LOH/allelic imbalance (AI) scan of DNA from epithelium and stroma of 116 prostate cancers. LOH/AI hot or cold spots were defined as the markers with significantly higher or lower LOH/AI frequencies compared with the average frequency for markers along the same chromosome. These data were then integrated with publicly available transcriptome data sets and our experimentally derived data. Immunohistochemistry on an independent series was used for validation. Overall, we identified 43 LOH/AI hot/cold spots, 17 in epithelium and stroma (P < 0.001), 18 only in epithelium (P < 0.001), and eight only in stroma (P < 0.001). Hierarchical clustering of expression data supervised by genes within LOH/AI hot/cold spots in both epithelium and stroma accurately separated samples into normal epithelium, primary cancer, and metastatic cancer groups, which could not be achieved with data from only epithelium. Importantly, our experimental expression data of the genes within the LOH/AI hot/cold spots in stroma accurately clustered normal stroma from cancer stroma. We also identified 15 LOH/AI markers that were associated with Gleason score, which were validated functionally in each compartment by transcriptome data. Independent immunohistochemical validation of STIM2 within a stromal significant LOH marker (identified as associated with Gleason grade) confirmed its downregulation in the transition from moderate to high Gleason grade. Compartment-specific genomic and transcriptomic alterations accurately distinguish clinical and pathologic outcomes, suggesting new biomarkers for prognosis and targeted therapeutics.