EGFR-expression in primary urinary bladder cancer and corresponding metastases and the relation to HER2-expression. On the possibility to target these receptors with radionuclides.

BACKGROUND: There is limited effect of tyrosine kinase inhibitors or "naked" antibodies binding EGFR or HER2 for therapy of metastasized urinary bladder cancer and these methods are therefore not routinely used. Targeting radio-nuclides to the extracellular domain of the receptors is potentially a better possibility. METHODS: EGFR- and HER2-expression was analyzed for primary tumors and corresponding metastases from 72 patients using immunohistochemistry and the internationally recommended HercepTest. Intracellular mutations were not analyzed since only the receptors were considered as targets and intracellular abnormalities should have minor effect on radiation dose. RESULTS: EGFR was positive in 71% of the primary tumors and 69% of corresponding metastases. Local and distant metastases were EGFR-positive in 75% and 66% of the cases, respectively. The expression frequency of HER2 in related lesions was slightly higher (data from previous study). The EGFR-positive tumors expressed EGFR in metastases in 86% of the cases. The co-expression of EGFR and HER2 was 57% for tumors and 53% for metastases. Only 3% and 10% of the lesions were negative for both receptors in tumors and metastases, respectively. Thus, targeting these receptors with radionuclides might be applied for most patients. CONCLUSIONS: At least one of the EGFR- or HER2-receptors was present in most cases and co-expressed in more than half the cases. It is therefore interesting to deliver radionuclides for whole-body receptor-analysis, dosimetry and therapy. This can hopefully compensate for resistance to other therapies and more patients can hopefully be treated with curative instead of palliative intention.

Antibody performance in western blot applications is context-dependent.

An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.

Tumour expression of bladder cancer-associated urinary proteins.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The current basis for diagnosis and prognosis in urinary bladder cancer is based on the pathologists' assessment of a biopsy of the tumour. Urinary biomarkers are preferable as they can be non-invasively sampled. Urinary cytology is the only test with widespread use but is hampered by poor reproducibility and low sensitivity. By studying the protein expression in bladder tumour tissue samples of proteins previously found in elevated levels in the urine of patients with bladder cancer, we have been able to show that these proteins originate from the tumour. The immunoreactivity of three of the investigated proteins increased with higher stage. Also a serine peptidase inhibitor was found to be predictive of progression from non-muscle-invasive to muscle-invasive tumours. OBJECTIVES: To analyse the expression of five bladder cancer-associated urinary proteins and investigate if expression is related to the malignant phenotype of the tumour. To explore the possible prognostic value of these proteins. PATIENTS AND METHODS: Urine samples, 16 from patients with bladder cancer and 26 from controls, were used in Western Blotting experiments. Tissue microarrays with bladder tissue from 344 patients diagnosed with bladder cancer between 1984 and 2005 was used in immunohistochemistry experiments. The proteins apolipoprotein E (APOE), fibrinogen ß chain precursor (FGB), leucine-rich α2-glycoprotein (LRG1), polymerase (RNA) I polypeptide E (POLR1E), α1-antitrypsin (SERPINA1) and topoisomerase 2A (TOP2A) were probed with antibodies validated by the Human Protein Atlas. RESULTS: Increased expressions of APOE, FGB and POLR1E were correlated with increased tumour stage (P < 0.001). Expression of SERPINA1 in Ta and T1 tumours was found to increase the risk of tumour progression (hazard ratio 2.57, 95% confidence interval 1.13-5.87; P = 0.025) CONCLUSIONS: All proteins previously detected in urine from patients with bladder cancer were also expressed in bladder cancer tissue. The expression of APOE, FGB and POLR1E increased with stage and they are potential diagnostic markers. SERPINA1 was identified as a prognostic marker candidate.

Evaluation of real-time immunohistochemistry and interaction map as an alternative objective assessment of HER2 expression in human breast cancer tissue.

Immunohistochemical study (IHC) is a critical tool in the clinical diagnosis of breast cancer. One common assessment is the expression level of the HER2 receptor in breast cancer tissue samples with the aim of stratifying patients for applicability of the therapeutic antibody Herceptin. In this study, we aimed to investigate whether a novel assay, real-time IHC combined with Interaction Map analysis, offers the possibility of objective assessment of HER2 expression. Interaction Map presents real-time interaction data as a collection of peaks on a surface, and it was performed on 20 patient tissue samples previously scored for HER2 expression. The result shows that the relative weight of the peaks in the maps contains novel information that could discriminate between high and low HER2 expression in an operator-independent manner (P<0.001). We conclude that the real-time IHC assay has a promising potential to complement conventional IHC and may improve the precision in the future clinical diagnostics of breast cancer.

Insulin-secreting INS-1E cells express functional TRPV1 channels.

We have studied whether functional TRPV1 channels exist in the INS-1E cells, a cell type used as a model for ß-cells, and in primary ß-cells from rat and human. The effects of the TRPV1 agonists capsaicin and AM404 on the intracellular free Ca (2+) concentration ([Ca (2+)]i) in the INS-1E cells were studied by fura-2 based microfluorometry. Capsaicin increased [Ca (2+)]i in a concentration-dependent manner, and the [Ca (2+)]i increase was dependent on extracellular Ca (2+). AM404 also increased [Ca (2+)]i in the INS-1E cells. Capsazepine, a specific antagonist of TRPV1, completely blocked the capsaicin- and AM404-induced [Ca (2+)]i increases. Capsaicin did not increase [Ca (2+)]i in the primary ß-cells from rat and human. Whole cell patch clamp configuration was used to record currents across the plasma membrane in the INS-1E cells. Capsaicin elicited inward currents that were inhibited by capsazepine. Western blot analysis detected TRPV1 proteins in the INS-1E cells and the human islets. Immunohistochemistry was used to study the expression of TRPV1, but no TRPV1 protein immunoreactivity was detected in the human islet cells and the human insulinoma cells. We conclude that the INS-1E cells, but not the primary ß-cells, express functional TRPV1 channels.

Proteomic analysis of urinary biomarker candidates for nonmuscle invasive bladder cancer.

Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen ß chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen ß chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.

Comparative biodistribution of imaging agents for in vivo molecular profiling of disseminated prostate cancer in mice bearing prostate cancer xenografts: focus on 111In- and 125I-labeled anti-HER2 humanized monoclonal trastuzumab and ABY-025 affibody.

INTRODUCTION: Human epidermal growth factor receptor type 2 (HER2) overexpression supports proliferation of androgen-independent prostate cancer (PC). Radionuclide molecular imaging of HER2 expression in disseminated PC would aid in the selection of patients who are likely responders to HER2 targeting therapy. In this study, we evaluated whether ABY-025 Affibody molecule, a small (∼ 7-kDa) HER2-binding scaffold protein, produces superior tumor-to-nontumor ratios compared with those obtained through the use of radiolabeled humanized anti-HER2 antibody, trastuzumab. The influence of (111)In vs. (125)I radiolabel was evaluated for both tracers. METHODS: ABY-025 was labeled with (111)In using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelator, site-specifically coupled to the C-terminus via the maleimido derivative. Trastuzumab was labeled with (111)In using a CHX-A″ diethylene triamine pentaacetic acid (DTPA) chelator. An indirect radioiodination with [(125)I]-N-succinimidyl-para-iodobenzoate was used for both targeting proteins. Biodistribution of all labeled targeting proteins was evaluated in mice bearing DU-145 PC xenografts. RESULTS: The use of residualizing (111)In-label facilitated better tumor uptake and better tumor-to-nontumor ratios for both targeting agents. [(111)In]-ABY-025 provided tumor uptake of 7.1±0.8% injected dose per gram of tissue (% ID/g) and tumor-to-blood ratio of 47±13 already at 6 h postinjection. The maximum tumor-to-nontumor ratios with [(111)In]-CHX-DTPA-trastuzumab were achieved at 72 h postinjection, whereas tumor uptake was 11±4% ID/g and tumor-to-blood ratio was 18±7. The biodistribution data were confirmed with gamma-camera imaging. CONCLUSIONS: Radiolabeled ABY-025 Affibody molecule provides higher contrast in imaging of HER2-expressing PC xenografts than radiolabeled trastuzumab. Residualizing radiometal label for ABY-025 provides better contrast in imaging of HER2-expressing PC xenografts than nonresidualizing radiohalogen.

SATB2 in combination with cytokeratin 20 identifies over 95% of all colorectal carcinomas.

The special AT-rich sequence-binding protein 2 (SATB2), a nuclear matrix-associated transcription factor and epigenetic regulator, was identified as a tissue type-specific protein when screening protein expression patterns in human normal and cancer tissues using an antibody-based proteomics approach. In this respect, the SATB2 protein shows a selective pattern of expression and, within cells of epithelial lineages, SATB2 expression is restricted to glandular cells lining the lower gastrointestinal tract. The expression of SATB2 protein is primarily preserved in cancer cells of colorectal origin, indicating that SATB2 could function as a clinically useful diagnostic marker to distinguish colorectal cancer (CRC) from other types of cancer. The aim of this study was to further explore and validate the specific expression pattern of SATB2 as a clinical biomarker and to compare SATB2 with the well-known cytokeratin 20 (CK20). Immunohistochemistry was used to analyze the extent of SATB2 expression in tissue microarrays with tumors from 9 independent cohorts of patients with primary and metastatic CRCs (n=1882). Our results show that SATB2 is a sensitive and highly specific marker for CRC with distinct positivity in 85% of all CRCs, and that SATB2 and/or CK20 was positive in 97% of CRCs. In conclusion, the specific expression of SATB2 in a large majority of CRCs suggests that SATB2 can be used as an important complementary tool for the differential diagnosis of carcinoma of unknown primary origin.

Diagnostic biomarkers of prostate cancer.

OBJECTIVE: Diagnostic tissue biomarkers for prostate cancer (PC) include basal cell markers and α-methylacyl-coenzyme A-racemase (AMACR), often used in combination. Their sensitivity and specificity are not perfect and there is a need for additional diagnostic biomarkers for PC in cases that are difficult to diagnose on routine stained sections. MATERIAL AND METHODS: This study investigated the diagnostic accuracy of three novel tissue biomarkers for PC found through a search in the Human Protein Atlas database ( www.proteinatlas.com ): somatic cytochrome c (CYCS), intestinal cell kinase (ICK) and inhibitor of nuclear factor-κB kinase subunit beta (IKBKB), and compared the results with AMACR. A tissue microarray was constructed from 40 consecutive radical prostatectomy (RP) specimens including benign prostatic tissue, atrophy, high-grade prostatic intraepithelial neoplasia (HGPIN) and PC. Immunoreactivity was scored based on staining intensity and extent. Real-time polymerase chain reaction (PCR) was performed on malignant and benign frozen tissue samples from 32 RP specimens. RESULTS: All four biomarkers showed a stronger expression in PC and HGPIN than in benign tissue (p < 0.001). The highest diagnostic accuracy for PC was achieved with ICK and AMACR at 97%. The area under the curve for CYCS, ICK, IKBKB and AMACR was 0.859, 0.997, 0.865 and 0.983, respectively. The presence of mRNA transcripts of the genes was confirmed by real-time PCR in benign and malignant prostatic tissue. CONCLUSIONS: AMACR is an accurate diagnostic tissue marker for PC. However, in some PCs AMACR is false negative and a panel of CYCS, ICK and IKBKB may serve as ancillary diagnostic tool.

GAD1 is a biomarker for benign and malignant prostatic tissue.

OBJECTIVE: Tissue-specific markers are useful for identification of tumour type in advanced cancers of unknown origin. This study investigated the expression of glutamate decarboxylase 1 (GAD1) in prostate and control tissue compared with the established prostate-specific markers prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). MATERIAL AND METHODS: A tissue microarray was constructed of 36 prostate adenocarcinomas, eight benign prostate samples and benign and malignant control tissues from urinary bladder, lung and rectum. Immunohistochemistry for GAD1, PSA and PSMA was performed. The products of staining intensity and extent were analysed. The GAD1 antibody was validated by Western blot. Real-time polymerase chain reaction (RT-PCR) was performed on malignant and benign samples from each tissue type. RESULTS: GAD1 and PSA immunostains were significantly stronger in malignant and benign prostatic tissue than in controls. PSMA was stronger in prostate cancer than in urothelial and rectal cancer but had a lower specificity than GAD1 and PSA. GAD1 expression decreased with increasing Gleason score. RT-PCR confirmed the presence of mRNA for GAD1, PSA and PSMA in prostate samples. CONCLUSION: GAD1 is expressed in benign and malignant prostatic tissue and may serve as a highly prostate-specific tissue biomarker.