Accumulation of Nicotinamide N-Methyltransferase (NNMT) in Cancer-associated Fibroblasts: A Potential Prognostic and Predictive Biomarker for Gastric Carcinoma

Nicotinamide N-methyltransferase (NNMT), a major metabolic regulator, has been identified as a predictor of cancer prognosis in ovarian and colorectal cancers. The study aims to evaluate the significance of stromal NNMT in gastric cancer (GC). Expression of stromal NNMT in 612 GC and 92 non-malignant tissues specimens was investigated by immunohistochemistry (IHC). The association between NNMT expression and occurrence of cancer or patient outcome was further analyzed, and the factors contributing to disease prognosis were evaluated by multiple Cox models. Stromal NNMT expression was higher in the malignant tissue (p<0.001). NNMT expression was significantly associated with GC stage (p=0.006). Compared to stromal “NNMT-low” cases, “NNMT-high” cases has lower disease-specific survival (hazard ratio [HR], 2.356; 95% confidence interval [CI] = 1.591–3.488; p<0.001) and disease-free survival (HR = 2.265; 95% CI = 1.529–3.354; p<0.001), as observed by multivariate Cox analysis after adjusting for stromal NNMT expression with other factors such as tumor grade and size. Notably, patients with stage II NNMT-low GC might be negatively affected by adjuvant chemotherapy, but lower stromal NNMT expression predicted a more favorable prognosis for GC. Our study confirmed that stromal NNMT expression is significantly increased in GC, which predicts an unfavorable post-operative prognosis for GC:     

Prognostic Significance of COX-2 Immunohistochemical Expression in Colorectal Cancer: A Meta-Analysis of the Literature

Background

Cyclooxygenase-2 (COX-2) is believed to be an important enzyme in the pathogenesis of colorectal cancer (CRC). Correlations between the expression of COX-2 with tumor growth and distant metastasis have become an issue; thus, attention has been paid to COX-2 as a prognostic factor. Various studies examined the relationship between COX-2 immunohistochemistry (IHC) overexpression with the clinical outcome in patients with colorectal cancer, but yielded conflicting results. The prognostic significance of COX-2 overexpression in colorectal cancer remains controversial.

Methods

Electronic databases updated to October 2012 were searched to find relevant studies. A meta-analysis was conducted with eligible studies which quantitatively evaluated the relationship between COX-2 overexpression and survival of patients with colorectal cancer. Survival data were aggregated and quantitatively analyzed.

Results

We performed a meta-analysis of 23 studies (n  =  4567 patients) that evaluated the correlation between COX-2 overexpression detected by IHC and survival in patients with colorectal cancer. Combined hazard ratios suggested that COX-2 overexpression had an unfavorable impact on overall survival (OS) (HR [hazard ratio]  =  1.193, 95% CI [confidence interval]: 1.02 ∼ 1.37), but not disease free survival (DFS) (HR  =  1.25, 95% CI: 0.99 ∼ 1.50) in patients with colorectal cancer.

Conclusions

Cox-2 overexpression in colorectal cancer detected by IHC appears to have slightly worse overall survival. However, the prognostic value of COX-2 on survival in colorectal cancer still needs further large-scale prospective trials to be clarified.     

Core Outcomes for Colorectal Cancer Surgery: A Consensus Study

BackgroundColorectal cancer (CRC) is a major cause of worldwide morbidity and mortality. Surgical treatment is common, and there is a great need to improve the delivery of such care. The gold standard for evaluating surgery is within well-designed randomized controlled trials (RCTs); however, the impact of RCTs is diminished by a lack of coordinated outcome measurement and reporting. A solution to these issues is to develop an agreed standard “core” set of outcomes to be measured in all trials to facilitate cross-study comparisons, meta-analysis, and minimize outcome reporting bias. This study defines a core outcome set for CRC surgery.ConclusionThis study used robust consensus methodology to develop a core outcome set for use in colorectal cancer surgical trials. It is now necessary to validate the use of this set in research practice.     

Lectin precipitation using phytohemagglutinin-L(4) coupled to avidin-agarose for serological biomarker discovery in colorectal cancer

N-acetylglucosaminyltransferase V (GnT-V) has been reported to be upregulated in malignant cancer cells, and its targets have been sought after with regard to biomarker identification. The low capacity and high false positive rates of 2-DE gel-based lectin blots using phytohemagglutinin-L(4) (L-PHA) prompted us to develop a novel protocol for identifying GnT-V targets, in which serum proteins were subjected to immunodepletion, alkylation, and lectin precipitation using L-PHA coupled to avidin-agarose bead complexes, and tryptic digestion. Proteins captured by L-PHA conjugates were analyzed by a nano-LC-FT-ICR/LTQ MS. Here, we report 26 candidate biomarkers for colorectal cancer (CRC) that show 100% specificity and sensitivities of greater than 50%. Not only can these candidate proteins be used as analytes for validation, but the novel protocol described herein can be applied to biomarker discovery in nonCRCs.     

Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems

A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads’ stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls (“IHControls”). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.     

Dynamic changes of interleukin-8 network along the colorectal adenoma–carcinoma sequence

The interleukin-8 (IL-8) network is involved in the colorectal cancer (CRC) progression. However, its role during the adenoma–carcinoma transition to date has not been fully investigated. To evaluate the dynamic changes of IL-8 network along the colorectal adenoma–carcinoma sequence, we examined the tissue IL-8 mRNA level in colorectal biopsies from 53 colorectal adenomas, 44 CRCs and 18 controls by quantitative real-time PCR (Q-PCR), and the expressions of IL-8 and its receptors (IL-8RA and IL-8RB) in the tumor microenvironment by immunohistochemistry (IHC) and double IHCs. The results showed that the tissue IL-8 mRNA level began to increase in the precancerous lesions (adenomas) as compared with the controls and became even higher in the CRCs. Significantly, the increase of IL-8 mRNA levels was associated with the increase of dysplastic grades in the adenomas, and also paralleled to the increase of Duke’s stages in the CRCs. IHC results revealed that IL-8 and its receptors, IL-8RA and IL-8RB, were observed both in the stroma and in the adenomatous/cancerous cells. By double IHCs, the IL-8 expression was characterized in macrophages, lymphocytes and myofibroblasts in the tumor stroma. Further double IHC identified the co-expression of IL-8 receptors (IL-8RA and IL-8RB) with CD34 positive tumor-associated microvessels in both the adenomas and CRCs. We, therefore, conclude that activated IL-8 network in the tumor microenvironment may function as a significant regulatory factor for the adenoma progression and the adenoma–carcinoma transition.     

Phospho-Akt Immunoreactivity in Prostate Cancer: Relationship to Disease Severity and Outcome,Ki67 and Phosphorylated EGFR Expression

Background

In the present study, we have investigated the prognostic usefulness of phosphorylated Akt immunoreactivity (pAkt-IR) in prostate cancer using a well-characterised tissue microarray from men who had undergone transurethral resection due to lower urinary tract symptoms.

Methodology/Principal Findings

pAkt-IR in prostate epithelial and tumour cells was assessed using a monoclonal anti-pAkt (Ser⁴⁷³) antibody. Immunoreactive intensity was determined for 282 (tumour) and 240 (non-mlignant tissue) cases. Tumour pAkt-IR scores correlated with Gleason score, tumour Ki67-IR (a marker of cell proliferation) and tumour phosphorylated epidermal growth factor receptor (pEGFR)-IR. For cases followed with expectancy, a high tumour pAkt-IR was associated with a poor disease-specific survival, and the prognostic information provided by this biomarker was additive to that provided by either (but not both) tumour pEFGR-IR or Ki67-IR. Upon division of the cases with respect to their Gleason scores, the prognostic value of pAkt-IR was seen for patients with Gleason score 8–10, but not for patients with Gleason score 6–7.

Conclusions/Significance

Tumour pAkt-IR is associated with both disease severity and disease-specific survival. However, its clinical use as a biomarker is limited, since it does not provide prognostic information in patients with Gleason scores 6–7.     

Elacestrant in metastatic breast cancer: Is the “standard of care” meeting standard requirements?

The EMERALD trial was an open label phase 3 trial evaluating elacestrant, the first oral selective estrogen receptor degrader (SERD), as compared to “standard of care”, in ER+/HER2- (hormone receptor positive, no HER2 overexpression) advanced or metastatic breast cancer.The EMERALD trial restricted the “standard of care” control arm to limited options that may have led to a substandard control arm. We describe how the EMERALD trial protocol allowed different clinically inappropriate scenarios in the control arm, according to prior therapy. The main relevant question remains the potential advantage of elacestrant over fulvestrant in fulvestrant-naive patients.Analyzing outcomes in subgroups according to prior and per-protocol therapy would help analyzing trial results. However, these subgroup results may be non-significant, and another randomized trial will be needed. Trials should be designed to answer directly clinical questions that are relevant.     

Using the “reverse Warburg effect” to identify high-risk breast cancer patients: Stromal MCT4 predicts poor clinical outcome in triple-negative breast cancers

We have recently proposed a new model of cancer metabolism to explain the role of aerobic glycolysis and L-lactate production in fueling tumor growth and metastasis. In this model, cancer cells secrete hydrogen peroxide (H₂O₂), initiating oxidative stress and aerobic glycolysis in the tumor stroma. This, in turn, drives L-lactate secretion from cancer-associated fibroblasts. Secreted L-lactate then fuels oxidative mitochondrial metabolism (OXPHOS) in epithelial cancer cells, by acting as a paracrine onco-metabolite. We have previously termed this type of two-compartment tumor metabolism the “reverse Warburg effect,” as aerobic glycolysis takes place in stromal fibroblasts, rather than epithelial cancer cells. Here, we used MCT4 immunostaining of human breast cancer tissue microarrays (TMAs; >180 triple-negative patients) to directly assess the prognostic value of the “reverse Warburg effect.” MCT4 expression is a functional marker of hypoxia, oxidative stress, aerobic glycolysis and L-lactate efflux. Remarkably, high stromal MCT4 levels (score = 2) were specifically associated with decreased overall survival (<18% survival at 10 years post-diagnosis). In contrast, patients with absent stromal MCT4 expression (score = 0), had 10-year survival rates of ∼97% (p-value < 10⁻³²). High stromal levels of MCT4 were strictly correlated with a loss of stromal Cav-1 (p-value < 10⁻¹⁴), a known marker of early tumor recurrence and metastasis. In fact, the combined use of stromal Cav-1 and stromal MCT4 allowed us to more precisely identify high-risk triple-negative breast cancer patients, consistent with the goal of individualized risk-assessment and personalized cancer treatment. However, epithelial MCT4 staining had no prognostic value, indicating that the “conventional” Warburg effect does not predict clinical outcome. Thus, the “reverse Warburg effect” or “parasitic” energy-transfer is a key determinant of poor overall patient survival. As MCT4 is a druggable target, MCT4 inhibitors should be developed for the treatment of aggressive breast cancers, and possibly other types of human cancers. Similarly, we discuss how stromal MCT4 could be used as a biomarker for identifying high-risk cancer patients that could likely benefit from treatment with FDA-approved drugs or existing MCT-inhibitors (such as, AR-C155858, AR-C117977 and AZD-3965).Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A3, monocarboxylic acid transporter, two-compartment tumor metabolism     

Impact of Preanalytical Factors on the Measurement of Tumor Tissue Biomarkers Using Immunohistochemistry

Analysis of formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemistry (IHC) is commonplace in clinical and research laboratories. However, reports suggest that IHC results can be compromised by biospecimen preanalytical factors. The National Cancer Institute’s Biospecimen Preanalytical Variables Program conducted a systematic study to examine the potential effects of delay to fixation (DTF) and time in fixative (TIF) on IHC using 24 cancer biomarkers. Differences in IHC staining, relative to controls with a DTF of 1 hr, were observed in FFPE kidney tumor specimens after a DTF of ≥2 hr. Reductions in H-score and/or staining intensity were observed for c-MET, p53, PAX2, PAX8, pAKT, and survivin, whereas increases were observed for RCC1, EGFR, and CD10. Prolonged TIF of 72 hr resulted in significantly reduced H-scores of CD44 and c-Met in kidney tumor specimens, compared with controls with 12-hr TIF. An elevated probability of altered staining intensity due to DTF was observed for nine antigens, whereas for prolonged TIF an elevated probability was observed for one antigen. Results reported here and elsewhere across tumor types and antigens support limiting DTF to ≤1 hr when possible and fixing tissues in formalin for 12–24 hr to avoid confounding effects of these preanalytical factors on IHC.     

Promoter DNA Methylation of Oncostatin M receptor-β as a Novel Diagnostic and Therapeutic Marker in Colon Cancer

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging “cancer methylome”. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-β (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.     

A Further Investigation of Combined Mismatch Repair and BRAFV600E Mutation Specific Immunohistochemistry as a Predictor of Overall Survival in Colorectal Carcinoma

Mutation specific immunohistochemistry (IHC) is a promising new technique to detect the presence of the BRAFV600E mutation in colorectal carcinoma (CRC). When performed in conjunction with mismatch repair (MMR) IHC, BRAFV600E IHC can help to further triage genetic testing for Lynch Syndrome. In a cohort of 1426 patients undergoing surgery from 2004 to 2009 we recently demonstrated that the combination of MMR and BRAFV600E IHC holds promise as a prognostic marker in CRC, particularly because of its ability to identify the poor prognosis MMR proficient (MMRp) BRAFV600E mutant subgroup. We attempted to validate combined MMR and BRAFV600E IHC as a prognostic indicator in a separate cohort comprising consecutive CRC patients undergoing surgery from 1998 to 2003. IHC was performed on a tissue microarray containing tissue from 1109 patients with CRC. The 5 year survivals stratified by staining patterns were: MMRd/BRAFwt 64%, MMRd/BRAFV600E 64%, MMRp/BRAFwt 60% and MMRp/BRAFV600E 53%. Using the poor prognosis MMRp/BRAFV600E phenotype as baseline, univariate Cox regression modelling demonstrated the following hazard ratios for death: MMRd/BRAFwt HR = 0.71 (95%CI = 0.40–1.27), p = 0.31; MMRd/BRAFV600E HR = 0.74 (95%CI = 0.51–1.07), p = 0.11 and MMRp/BRAFwt HR = 0.79 (95%CI = 0.60–1.04), p = 0.09. Although the findings did not reach statistical significance, this study supports the potential role of combined MMR and BRAF IHC as prognostic markers in CRC.     

Quantitative Analysis of BTF3, HINT1, NDRG1 and ODC1 Protein Over-Expression in Human Prostate Cancer Tissue

Prostate carcinoma is the most common cancer in men with few, quantifiable, biomarkers. Prostate cancer biomarker discovery has been hampered due to subjective analysis of protein expression in tissue sections. An unbiased, quantitative immunohistochemical approach provided here, for the diagnosis and stratification of prostate cancer could overcome this problem. Antibodies against four proteins BTF3, HINT1, NDRG1 and ODC1 were used in a prostate tissue array (> 500 individual tissue cores from 82 patients, 41 case pairs matched with one patient in each pair had biochemical recurrence). Protein expression, quantified in an unbiased manner using an automated analysis protocol in ImageJ software, was increased in malignant vs non-malignant prostate (by 2-2.5 fold, p<0.0001). Operating characteristics indicate sensitivity in the range of 0.68 to 0.74; combination of markers in a logistic regression model demonstrates further improvement in diagnostic power. Triple-labeled immunofluorescence (BTF3, HINT1 and NDRG1) in tissue array showed a significant (p<0.02) change in co-localization coefficients for BTF3 and NDRG1 co-expression in biochemical relapse vs non-relapse cancer epithelium. BTF3, HINT1, NDRG1 and ODC1 could be developed as epithelial specific biomarkers for tissue based diagnosis and stratification of prostate cancer.     

Spatiotemporal quantification of metastatic tumour cell growth and distribution in lymph nodes by whole-mount tissue 3D imaging

Lymph nodes (LNs) are a common site of metastasis in many solid cancers. Tumour cells can migrate to LNs for further metastatic colonization of distant organs, indicating poor prognosis and requiring different clinical interventions. The histopathological diagnostic methods currently used to detect clinical lymph node metastasis (LNM) have limitations, such as incomplete visualization. To obtain a complete picture of metastatic LNs on the spatial and temporal scales, we used ultimate 3D imaging of solvent-cleared organs (uDISCO) and 3D rapid immunostaining. MC38 cells labelled with EGFP were injected into the left footpads of C57BL/6 mice. Draining lymph nodes (DLNs) harvested from these mice were cleared using the uDISCO method. Metastatic colorectal cancer (CRC) cells in various regions of DLNs from mice at different time points were quantified using 3D imaging of whole-mount tissue. Several stages of tumour cell growth and distribution in LNs were identified: 1) invasion of lymphatic vessels (LVs) and blood vessels (BVs); 2) dispersion outside LVs and BVs for proliferation and expansion; and 3) re-entry into BVs and efferent lymphatic vessels (ELVs) for further distant metastasis. Moreover, these data demonstrated that mouse fibroblast cells (MFCs) could not only promote LNM of tumour cells but also metastasize to LNs together with tumour cells, thus providing a “soil” for tumour cell colonization. In conclusion, 3D imaging of whole-mount tissue and spatiotemporal analysis of LNM may collectively constitute an auxiliary method to improve the accuracy of clinical LNM detection.     

The Use of P63 Immunohistochemistry for the Identification of Squamous Cell Carcinoma of the Lung

Introduction

While some targeted agents should not be used in squamous cell carcinomas (SCCs), other agents might preferably target SCCs. In a previous microarray study, one of the top differentially expressed genes between adenocarcinomas (ACs) and SCCs is P63. It is a well-known marker of squamous differentiation, but surprisingly, its expression is not widely used for this purpose. Our goals in this study were (1) to further confirm our microarray data, (2) to analize the value of P63 immunohistochemistry (IHC) in reducing the number of large cell carcinoma (LCC) diagnoses in surgical specimens, and (3) to investigate the potential of P63 IHC to minimize the proportion of “carcinoma NOS (not otherwise specified)” in a prospective series of small tumor samples.

Methods

With these goals in mind, we studied (1) a tissue-microarray comprising 33 ACs and 99 SCCs on which we performed P63 IHC, (2) a series of 20 surgically resected LCCs studied for P63 and TTF-1 IHC, and (3) a prospective cohort of 66 small thoracic samples, including 32 carcinoma NOS, that were further classified by the result of P63 and TTF-1 IHC.

Results

The results in the three independent cohorts were as follows: (1) P63 IHC was differentially expressed in SCCs when compared to ACs (p<0.0001); (2) half of the 20 (50%) LCCs were positive for P63 and were reclassified as SCCs; and (3) all P63 positive cases (34%) were diagnosed as SCCs.

Conclusions

P63 IHC is useful for the identification of lung SCCs.     

Stathmin 1 is a biomarker for diagnosis of microvascular invasion to predict prognosis of early hepatocellular carcinoma

Microvascular invasion (MVI) is presently evaluated as a high-risk factor to be directly relative to postoperative prognosis of hepatocellular carcinoma (HCC). Up to now, diagnosis of MVI mainly depends on the postoperative pathological analyses with H&E staining assay, based on numbers and distribution characteristics of MVI to classify the risk levels of MVI. However, such pathological analyses lack the specificity to discriminate MVI in HCC specimens, especially in complicated pathological tissues. In addition, the efficiency to precisely define stages of MVI is not satisfied. Thus, any biomarker for both conforming diagnosis of MVI and staging its levels will efficiently and effectively promote the prediction of early postoperative recurrence and metastasis for HCC. Through bioinformatics analysis and clinical sample verification, we discovered that Stathmin 1 (STMN1) gene was significantly up-regulated at the locations of MVI. Combining STMN1 immunostaining with classic H&E staining assays, we established a new protocol for MVI pathological diagnosis. Next, we found that the degrees of MVI risk could be graded according to expression levels of STMN1 for prognosis prediction on recurrence rates and overall survival in early HCC patients. STMN1 affected epithelial-mesenchymal transformation (EMT) of HCC cells by regulating the dynamic balance of microtubules through signaling of “STMN1-Microtubule-EMT” axis. Inhibition of STMN1 expression in HCC cells reduced their lung metastatic ability in recipients of mouse model, suggesting that STMN1 also could be a potential therapeutic target for inhibiting HCC metastasis. Therefore, we conclude that STMN1 has potentials for clinical applications as a biomarker for both pathological diagnosis and prognostic prediction, as well as a therapeutic target for HCC.Subject terms: Tumour biomarkers, Diagnostic markers