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Prostate cancer (PCa) progression relies on androgen receptor (AR) function, making AR a top candidate for PCa therapy. However, development of drug resistance is common, which eventually leads to development of castration‐resistant PCa. This warrants a better understanding of the pathophysiology of PCa that facilitates the aberrant activation of key signaling pathways including AR. MicroRNAs (miRNAs) function as regulators of cancer progression as they modulate various cellular processes. Here, we demonstrate a multidimensional function of miR‐30e through the regulation of genes involved in various signaling pathways. We noted loss of miR‐30e expression in prostate tumors, which, when restored, led to cell cycle arrest, induction of apoptosis, improved drug sensitivity of PCa cells and reduced tumor progression in xenograft models. We show that experimental upregulation of miR‐30e reduces expression of mRNAs including AR, FBXO45, SRSF7 and MYBL2 and a novel long noncoding RNA (lncRNA) HELLPAR, which are involved in cell cycle, apoptosis and ubiquitination, and the effects could be rescued by inhibition of miR‐30e expression. RNA immunoprecipitation analysis confirmed direct interactions between miR‐30e and its RNA targets. We noted a newly identified reciprocal relationship between miR‐30e and HELLPAR, as inhibition of HELLPAR improved stabilization of miR‐30e. Transcriptome profiling and quantitative real‐time PCR (qRT‐PCR) validation of miR‐30e‐expressing PCa cells showed differential expression of genes involved in cell cycle progression, apoptosis and ubiquitination, which supports our in vitro study. This study demonstrates an integrated function of miR‐30e on dysregulation of miRNA/lncRNA/mRNA axes that may have diagnostic and therapeutic significance in aggressive PCa.

Abstract The activation and growth of tumour-initiating cells with stem-like properties in distant organs characterize colorectal cancer (CRC) growth and metastasis. Thus, inhibition of colon cancer stem cell (CCSC) growth holds promise for CRC growth and metastasis prevention. We and others have shown that farnesyl dimethyl chromanol (FDMC) inhibits cancer cell growth and induces apoptosis in vitro and in vivo. We provide the first demonstration that FDMC inhibits CCSC viability, survival, self-renewal (spheroid formation), pluripotent transcription factors (Nanog, Oct4, and Sox2) expression, organoids formation, and Wnt/β-catenin signalling, as evidenced by comparisons with vehicle-treated controls. In addition, FDMC inhibits CCSC migration, invasion, inflammation (NF-kB), angiogenesis (vascular endothelial growth factor, VEGF), and metastasis (MMP9), which are critical tumour metastasis processes. Moreover, FDMC induced apoptosis (TUNEL, Annexin V, cleaved caspase 3, and cleaved PARP) in CCSCs and CCSC-derived spheroids and organoids. Finally, in an orthotopic (cecum-injected CCSCs) xenograft metastasis model, we show that FDMC significantly retards CCSC-derived tumour growth (Ki-67); inhibits inflammation (NF-kB), angiogenesis (VEGF and CD31), and β-catenin signalling; and induces apoptosis (cleaved PARP) in tumour tissues and inhibits liver metastasis. In summary, our results demonstrate that FDMC inhibits the CCSC metastatic phenotype and thereby supports investigating its ability to prevent CRC metastases.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with an overall median 5-year survival rate of 8%. This poor prognosis is because of the development of resistance to chemotherapy and radiation therapy and lack of effective targeted therapies. IκB kinase enhancer (IKBKE) overexpression was previously implicated in chemoresistance. Because IKBKE is frequently elevated in PDAC and IKBKE inhibitors are currently in clinical trials, we evaluated IKBKE as a therapeutic target in this disease. Depletion of IKBKE was found to significantly reduce PDAC cell survival, growth, cancer stem cell renewal, and cell migration and invasion. Notably, IKBKE inhibitor CYT387 and IKBKE knockdown dramatically activated the MAPK pathway. Phospho-RTK array analyses showed that IKBKE inhibition leads to rapid upregulation of ErbB3 and IGF-1R expression, which results in MAPK-ERK pathway activation—thereby limiting the efficacy of IKBKE inhibitors. Furthermore, IKBKE inhibition leads to stabilization of FOXO3a, which is required for RTK upregulation on IKBKE inhibition. Finally, we demonstrated that the IKBKE inhibitors synergize with the MEK inhibitor trametinib to significantly induce cell death and inhibit tumor growth and liver metastasis in an orthotopic PDAC mouse model.

Abstract Background Vitamin E δ-tocotrienol (VEDT), a vitamin E compound isolated from sources such as palm fruit and annatto beans, has been reported to have cancer chemopreventive and therapeutic effects. Methods We report a novel function of VEDT in augmenting tumor necrosis factor-related apoptosis-inducing ligand- (TRAIL-) induced apoptosis in pancreatic cancer cells. The effects of VEDT were shown by its ability to trigger caspase-8-dependent apoptosis in pancreatic cancer cells. Results When combined with TRAIL, VEDT significantly augmented TRAIL-induced apoptosis of pancreatic cancer cells. VEDT decreased cellular FLICE inhibitory protein (c-FLIP) levels without consistently modulating the expression of decoy death receptors 1, 2, 3 or death receptors 4 and 5. Enforced expression of c-FLIP substantially attenuated VEDT/TRAIL-induced apoptosis. Thus, c-FLIP reduction plays an important part in mediating VEDT/TRAIL-induced apoptosis. Moreover, VEDT increased c-FLIP ubiquitination and degradation but did not affect its transcription, suggesting that VEDT decreases c-FLIP levels through promoting its degradation. Of note, degradation of c-FLIP and enhanced TRAIL-induced apoptosis in pancreatic cancer cells were observed only with the anticancer bioactive vitamin E compounds δ-, γ-, and β-tocotrienol but not with the anticancer inactive vitamin E compounds α-tocotrienol and α-, β-, γ-, and δ-tocopherol. Conclusions c-FLIP degradation is a key event for death receptor-induced apoptosis by anticancer bioactive vitamin E compounds in pancreatic cancer cells. Moreover, VEDT augmented TRAIL inhibition of pancreatic tumor growth and induction of apoptosis in vivo. Combination therapy with TRAIL agonists and bioactive vitamin E compounds may offer a novel strategy for pancreatic cancer intervention.

Abstract Background FGD4 (Frabin) is an F-actin binding protein with GTP/GDP exchange activity specific for CDC42. It is involved in reorganization of the actin cytoskeleton, which requires both actin binding and CDC42 activating function of FGD4. Expression of FGD4 is altered in patients with heterogeneous hereditary motor and sensory neuropathies as a result of demyelination of peripheral nerves. Methods In this study, we examined the expression of FGD4 in prostate cancer specimens using immunohistochemistry and studied the function of FGD4 in maintaining cell phenotype, behavior and drug sensitivity using overexpression and siRNA-based silencing approaches. We used Mann-Whitney test for comparative analysis of FGD4 expression. Results Our results show that the expression of FGD4 is upregulated in cancerous prostates compared to the luminal cells in benign prostatic hyperplasia, although the basal cells showed high staining intensities. We noted a gradual increase in the staining intensity of FGD4 with increasing aggressiveness of the disease. Inhibition of expression of FGD4 using siRNAs showed reduced proliferation and cell cycle arrest in G2/M phase of androgen dependent LNCaP-104S and androgen refractory PC-3 cells. Inhibition of FGD4 also resulted in reduced cell migration and CDC42 activities in PC-3 cells whereas, ectopic expression of FGD4 induced cell migration, altered expression of mesenchymal and epithelial markers and activation of CDC42/PAK signaling pathway. Reduced expression of FGD4 improved sensitivity of LNCaP-104S cells to the anti-androgen drug Casodex and PC-3 cells to the microtubule stabilizing drug docetaxel. Conclusions Our data demonstrate a tumor promoting and a cell migratory function of FGD4 in prostate cancer cells and that inhibition of FGD4 expression enhances the response for both androgen-dependent and independent prostate cancer cells towards currently used prostate cancer drugs.

It is rare that stage IV colon cancer is cured with chemotherapy. Here we report the long-term survival of a patient who presented with highly advanced disease characterized by a papillary architecture as well as porta hepatis lymph nodes but responded extremely well to FOLFIRI/bevacizumab. His original tumor underwent comprehensive genomic testing that included whole genome DNA sequencing, targeted sequencing, and RNA sequencing. These genetic results suggest the patient’s tumor harbored mutations in APC, KRAS, and TP53 as well as in PIK3CB. Moreover, the RNA-seq data suggested that the tumor belonged to the consensus molecular subtype 4, the “inflamed, immune phenotype,” with increased angiogenesis. Deep sequencing of highly responsive cancers may yield molecular insights into mechanisms underpinning a remarkable response.

Abstract Background Forty years ago the actin cytoskeleton was determined to be disrupted in fibroblasts from persons with DNA repair-defective, hereditary colon cancer, with no clear connection between the cytoskeleton and DNA repair defects at that time. Recently, the large number of sequenced genomes has indicated that mammalian mutagenesis has a large stochastic component. As a result, large coding regions are large mutagen targets. Cytoskeletal protein-related coding regions (CPCRs), including extra-cellular matrix proteins, are among the largest coding regions in the genome and are indeed very commonly mutated in cancer. Methods To determine whether mutagen sensitivity of the actin cytoskeleton could be assessed experimentally, we treated tissue culture cells with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and quantified overall cytoskeleton integrity with rhodamine-phalloidin stains for F-actin. Results The above approach indicated cytoskeletal degradation with increasing mutagen exposure, consistent with increased mutagenesis of CPCRs in TCGA, smoker samples, where overall mutation rates correlate with CPCR mutation rates (R2 = 0.8694; p

Objective: Intra-abdominal fat is a risk factor for pancreatic cancer (PC), but little is known about its contribution to PC precursors known as intraductal papillary mucinous neoplasms (IPMNs). Our goal was to evaluate quantitative radiologic measures of abdominal/visceral obesity as possible diagnostic markers of IPMN severity/pathology.Methods: In a cohort of 34 surgically-resected, pathologically-confirmed IPMNs (17 benign; 17 malignant) with preoperative abdominal computed tomography (CT) images, we calculated body mass index (BMI) and four radiologic measures of obesity: total abdominal fat (TAF) area, visceral fat area (VFA), subcutaneous fat area (SFA), and visceral to subcutaneous fat ratio (V/S). Measures were compared between groups using Wilcoxon two-sample exact tests and other metrics.Results: Mean BMI for individuals with malignant IPMNs (28.9 kg/m2) was higher than mean BMI for those with benign IPMNs (25.8 kg/m2) (P=0.045). Mean VFA was higher for patients with malignant IPMNs (199.3 cm2) compared to benign IPMNs (120.4 cm2), P=0.092. V/S was significantly higher (P=0.013) for patients with malignant versus benign IPMNs (1.25 vs. 0.69 cm2), especially among females. The accuracy, sensitivity, specificity, and positive and negative predictive value of V/S in predicting malignant IPMN pathology were 74%, 71%, 76%, 75%, and 72%, respectively.Conclusions: Preliminary findings suggest measures of visceral fat from routine medical images may help predict IPMN pathology, acting as potential noninvasive diagnostic adjuncts for management and targets for intervention that may be more biologically-relevant than BMI. Further investigation of gender-specific associations in larger, prospective IPMN cohorts is warranted to validate and expand upon these observations.

Immune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3ʹuntranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3ʹUTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFβ), as evidenced by antisense TGFβ transfection into H1355 or H1299 tumor cells which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFβ expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells.

APC is a well-known tumour suppressor that is frequently inactivated in colorectal cancer. Here, the authors sequence more than 1000 cancer genes in 468 colorectal cancers and show that mutation signatures can be used to classify the tumours and that multiple mutations in APCare associated with a poor prognosis.

Prediction of lung cancer metastasis relies on post-resection assessment of tumor histology, which is a severe limitation since only a minority of lung cancer patients are diagnosed with resectable disease. Therefore, characterization of metastasis-predicting biomarkers in pre-resection small biopsy specimens is urgently needed. Here we report a biomarker consisting of the phosphorylation of the retinoblastoma protein (Rb) on serine 249 combined with elevated p39 expression. This biomarker correlates with epithelial-to-mesenchymal transition traits in non-small cell lung carcinoma (NSCLC) cells. Immunohistochemistry staining of NSCLC tumor microarrays showed that strong phospho-Rb S249 staining positively correlated with tumor grade specifically in the squamous cell carcinoma (SCC) subtype. Strong immunoreactivity for p39 positively correlated with tumor stage, lymph node invasion, and distant metastases, also in SCC. Linear regression analyses showed that the combined scoring for phospho-Rb S249, p39 and E-cadherin in SCC is even more accurate at predicting tumor staging, relative to each score individually. We propose that combined immunohistochemistry staining of NSCLC samples for Rb phosphorylation on S249, p39, and E-cadherin protein expression could aid in the assessment of tumor staging and metastatic potential when tested in small primary tumor biopsies. The intense staining for phospho-Rb S249 that we observed in high grade SCC could also aid in the precise sub-classification of poorly differentiated SCCs.

BACKGROUND:Intraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursors. Differentiating between high-risk IPMNs that warrant surgical resection and low-risk IPMNs that can be monitored is a significant clinical problem, and we sought to discover a panel of mi(cro)RNAs that accurately classify IPMN risk status. METHODOLOGY/PRINCIPAL FINDINGS:In a discovery phase, genome-wide miRNA expression profiling was performed on 28 surgically-resected, pathologically-confirmed IPMNs (19 high-risk, 9 low-risk) using Taqman MicroRNA Arrays. A validation phase was performed in 21 independent IPMNs (13 high-risk, 8 low-risk). We also explored associations between miRNA expression level and various clinical and pathological factors and examined genes and pathways regulated by the identified miRNAs by integrating data from bioinformatic analyses and microarray analysis of miRNA gene targets. Six miRNAs (miR-100, miR-99b, miR-99a, miR-342-3p, miR-126, miR-130a) were down-regulated in high-risk versus low-risk IPMNs and distinguished between groups (P

It is established that histone modifications like acetylation, methylation, phosphorylation and ubiquitination affect chromatin structure and modulate gene expression. Lysine methylation/demethylation on Histone H3 and H4 is known to affect transcription and is mediated by histone methyl transferases and histone demethylases. KDM2A/JHDM1A/FBXL11 is a JmjC-containing histone demethylase that targets mono- and dimethylated Lys36 residues of Histone H3; its function in breast cancer is not fully understood. Here we show that KDM2A is strongly expressed in myoepithelial cells (MEPC) in breast cancer tissues by immunohistochemistry. Ductal cells from ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) show positive staining for KDM2A, the expression decreases with disease progression to metastasis. Since breast MEPCs have tumor-suppressive and anti-angiogenic properties, we hypothesized that KDM2A could be contributing to some of these functions. Silencing KDM2A with small interfering RNAs demonstrated increased invasion and migration of breast cancer cells by suppressing a subset of matrix metalloproteinases (MMP-2, -9, -14 and -15), as seen by real-time PCR. HUVEC cells showed increased angiogenic tubule formation ability in the absence of KDM2A, with a concomitant increase in the expression of VEGF receptors, FLT-1 and KDR. KDM2A physically bound to both Rb and E2F1 in a cell cycle dependent manner and repressed E2F1 transcriptional activity. Chromatin immunoprecipitation (ChIP) assays revealed that KDM2A associates with E2F1-regulated proliferative promoters CDC25A and TS in early G-phase and dissociates in S-phase. Further, KDM2A could also be detected on MMP9, 14 and 15 promoters, as well as promoters of FLT1 and KDR. KDM2A could suppress E2F1-mediated induction of these promoters in transient transfection experiments. These results suggest a regulatory role for KDM2A in breast cancer cell invasion and migration, through the regulation of E2F1 function.

Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA) cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G(1) cell-cycle arrest and increased p27(Kip1) nuclear accumulation. This finding is significant considering that loss of nuclear p27(Kip1) expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27(Kip1) expression. To determine whether p27(Kip1) induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27(Kip1), in MIAPaCa-2 PDCA cells and demonstrated that p27(Kip1) silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27(Kip1) mRNA expression but not its protein degradation. p27(Kip1) gene promoter activity was induced by δ-tocotrienol through the promoter's E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27(Kip1) expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27(Kip1) induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27(Kip1) as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.

Changes in host tumor genome DNA methylation patterns are among the molecular alterations associated with HPV-related carcinogenesis. However, there is little known about the epigenetic changes associated specifically with the development of anal squamous cell cancer (SCC). We sought to characterize broad methylation profiles across the spectrum of anal squamous neoplasia.Twenty-nine formalin-fixed paraffin embedded samples from 24 patients were evaluated and included adjacent histologically normal anal mucosa (NM; n = 3), SCC-in situ (SCC-IS; n = 11) and invasive SCC (n = 15). Thirteen women and 11 men with a median age of 44 years (range 26-81) were included in the study. Using the SFP(10) LiPA HPV-typing system, HPV was detected in at least one tissue from all patients with 93% (27/29) being positive for high-risk HPV types and 14 (93%) of 15 invasive SCC tissues testing positive for HPV 16. Bisulfite-modified DNA was interrogated for methylation at 1,505 CpG loci representing 807 genes using the Illumina GoldenGate Methylation Array. When comparing the progression from normal anal mucosa and SCC-IS to invasive SCC, 22 CpG loci representing 20 genes demonstrated significant differential methylation (p

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use with a wide variety of diseases. Alone, this technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. In this study we evaluated the MEA for its use in DNA vaccination using Hepatitis B virus as the infectious model. We utilized the guinea pig model because their skin is similar in thickness and morphology to humans. The plasmid encoding Hepatitis B surface antigen (HBsAg) was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to injection only groups. While this electrode requires further study, our results suggest that the MEA has potential for use in electrically mediated intradermal DNA vaccination.

The AKT/PKB kinase is a key signaling component of one of the most frequently activated pathways in cancer and is a major target of cancer drug development. Most studies have focused on its activation by Receptor Tyrosine Kinase (RTK) mediated Phosphatidylinositol-3-OH kinase (PI3K) activation or loss of Phosphatase and Tensin homolog (PTEN). We have uncovered that growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1 (also known as ACK or TNK2), which directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473-phosphorylation leading to AKT activation. Mice expressing activated Ack1 specifically in the prostate exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia (mPINs). Further, expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 were positively correlated with the severity of disease progression, and inversely correlated with the survival of breast cancer patients. Thus, RTK/Ack1/AKT pathway provides a novel target for drug discovery.

Nicotine is the major addictive component of tobacco smoke. Although nicotine is generally thought to have limited ability to initiate cancer, it can induce cell proliferation and angiogenesis in a variety of systems. These properties might enable nicotine to facilitate the growth of tumors already initiated. Here we show that nicotine significantly promotes the progression and metastasis of tumors in mouse models of lung cancer. This effect was observed when nicotine was administered through intraperitoneal injections, or through over-the-counter transdermal patches.In the present study, Line1 mouse adenocarcinoma cells were implanted subcutaneously into syngenic BALB/c mice. Nicotine administration either by intraperitoneal (i.p.) injection or transdermal patches caused a remarkable increase in the size of implanted Line1 tumors. Once the tumors were surgically removed, nicotine treated mice had a markedly higher tumor recurrence (59.7%) as compared to the vehicle treated mice (19.5%). Nicotine also increased metastasis of dorsally implanted Line1 tumors to the lungs by 9 folds. These studies on transplanted tumors were extended to a mouse model where the tumors were induced by the tobacco carcinogen, NNK. Lung tumors were initiated in A/J mice by i.p. injection of NNK; administration of 1 mg/kg nicotine three times a week led to an increase in the size and the number of tumors formed in the lungs. In addition, nicotine significantly reduced the expression of epithelial markers, E-Cadherin and beta-Catenin as well as the tight junction protein ZO-1; these tumors also showed an increased expression of the alpha(7) nAChR subunit. We believe that exposure to nicotine either by tobacco smoke or nicotine supplements might facilitate increased tumor growth and metastasis.Our earlier results indicated that nicotine could induce invasion and epithelial-mesenchymal transition (EMT) in cultured lung, breast and pancreatic cancer cells. This study demonstrates for the first time that administration of nicotine either by i.p. injection or through over-the-counter dermal patches can promote tumor growth and metastasis in immunocompetent mice. These results suggest that while nicotine has only limited capacity to initiate tumor formation, it can facilitate the progression and metastasis of tumors pre-initiated by tobacco carcinogens.

Background : Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) are important and well-established prognostic and predictive biomarkers for breast cancers and routinely tested on patient′s tumor samples by immunohistochemical (IHC) study. The accuracy of these test results has substantial impact on patient management. A critical factor that contributes to the result is the interpretation (scoring) of IHC. This study investigates how computerized image analysis can play a role in a reliable scoring, and identifies potential pitfalls with common methods. Materials and Methods : Whole slide images of 33 invasive ductal carcinoma (IDC) (10 ER and 23 HER2) were scored by pathologist under the light microscope and confirmed by another pathologist. The HER2 results were additionally confirmed by fluorescence in situ hybridization (FISH). The scoring criteria were adherent to the guidelines recommended by the American Society of Clinical Oncology/College of American Pathologists. Whole slide stains were then scored by commercially available image analysis algorithms from Definiens (Munich, Germany) and Aperio Technologies (Vista, CA, USA). Each algorithm was modified specifically for each marker and tissue. The results were compared with the semi-quantitative manual scoring, which was considered the gold standard in this study. Results : For HER2 positive group, each algorithm scored 23/23 cases within the range established by the pathologist. For ER, both algorithms scored 10/10 cases within range. The performance of each algorithm varies somewhat from the percentage of staining as compared to the pathologist′s reading. Conclusions : Commercially available computerized image analysis can be useful in the evaluation of ER and HER2 IHC results. In order to achieve accurate results either manual pathologist region selection is necessary, or an automated region selection tool must be employed. Specificity can also be gained when strict quality assurance by a pathologist is invested. Quality assurance of image analysis by pathologists is always warranted. Automated image analysis should only be used as adjunct to pathologist′s evaluation.