Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems ...through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings.
Normalization is critically important for the proper interpretation of matrix-assisted laser desorption/ionization (MALDI) imaging datasets. The effects of the commonly used normalization techniques ...based on total ion count (TIC) or vector norm normalization are significant, and they are frequently beneficial. In certain cases, however, these normalization algorithms may produce misleading results and possibly lead to wrong conclusions, e.g. regarding to potential biomarker distributions. This is typical for tissues in which signals of prominent abundance are present in confined areas, such as insulin in the pancreas or β-amyloid peptides in the brain. In this work, we investigated whether normalization can be improved if dominant signals are excluded from the calculation. Because manual interaction with the data (e.g., defining the abundant signals) is not desired for routine analysis, we investigated two alternatives: normalization on the spectra noise level or on the median of signal intensities in the spectrum. Normalization on the median and the noise level was found to be significantly more robust against artifact generation compared to normalization on the TIC. Therefore, we propose to include these normalization methods in the standard “toolbox” of MALDI imaging for reliable results under conditions of automation.
Clinical laboratory testing for HER2 status in breast cancer tissues is critically important for therapeutic decision making. Matrix-assisted laser desorption/ionization (MALDI) imaging mass ...spectrometry (IMS) is a powerful tool for investigating proteins through the direct and morphology-driven analysis of tissue sections. We hypothesized that MALDI-IMS may determine HER2 status directly from breast cancer tissues. Breast cancer tissues (n = 48) predefined for HER2 status were subjected to MALDI-IMS, and protein profiles were obtained through direct analysis of tissue sections. Protein identification was performed by tissue microextraction and fractionation followed by top-down tandem mass spectrometry. A discovery and an independent validation set were used to predict HER2 status by applying proteomic classification algorithms. We found that specific protein/peptide expression changes strongly correlated with the HER2 overexpression. Among these, we identified m/z 8404 as cysteine-rich intestinal protein 1. The proteomic signature was able to accurately define HER2-positive from HER2-negative tissues, achieving high values for sensitivity of 83%, for specificity of 92%, and an overall accuracy of 89%. Our results underscore the potential of MALDI-IMS proteomic algorithms for morphology-driven tissue diagnostics such as HER2 testing and show that MALDI-IMS can reveal biologically significant molecular details from tissues which are not limited to traditional high-abundance proteins.
In clinical diagnostics, it is of outmost importance to correctly identify the source of a metastatic tumor, especially if no apparent primary tumor is present. Tissue-based proteomics might allow ...correct tumor classification. As a result, we performed MALDI imaging to generate proteomic signatures for different tumors. These signatures were used to classify common cancer types. At first, a cohort comprised of tissue samples from six adenocarcinoma entities located at different organ sites (esophagus, breast, colon, liver, stomach, thyroid gland, n = 171) was classified using two algorithms for a training and test set. For the test set, Support Vector Machine and Random Forest yielded overall accuracies of 82.74 and 81.18%, respectively. Then, colon cancer liver metastasis samples (n = 19) were introduced into the classification. The liver metastasis samples could be discriminated with high accuracy from primary tumors of colon cancer and hepatocellular carcinoma. Additionally, colon cancer liver metastasis samples could be successfully classified by using colon cancer primary tumor samples for the training of the classifier. These findings demonstrate that MALDI imaging-derived proteomic classifiers can discriminate between different tumor types at different organ sites and in the same site.
The activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab combined with oxaliplatin/leucovorin/5-fluorouracil (FUFOX) was assessed in first-line metastatic ...gastric and oesophago-gastric junction (OGJ) cancer in a prospective phase II study showing a promising objective tumour response rate of 65% and a low mutation frequency of KRAS (3%). The aim of the correlative tumour tissue studies was to investigate the relationship between EGFR gene copy numbers, activation of the EGFR pathway, expression and mutation of E-cadherin, V600E BRAF mutation and clinical outcome of patients with gastric and OGJ cancer treated with cetuximab combined with FUFOX.
Patients included in this correlative study (n = 39) were a subset of patients from the clinical phase II study. The association between EGFR gene copy number, activation of the EGFR pathway, abundance and mutation of E-cadherin which plays an important role in these disorders, BRAF mutation and clinical outcome of patients was studied. EGFR gene copy number was assessed by FISH. Expression of the phosphorylated forms of EGFR and its downstream effectors Akt and MAPK, in addition to E-cadherin was analysed by immunohistochemistry. The frequency of mutant V600E BRAF was evaluated by allele-specific PCR and the mutation profile of the E-cadherin gene CDH1 was examined by DHPLC followed by direct sequence analysis. Correlations with overall survival (OS), time to progression (TTP) and overall response rate (ORR) were assessed.
Our study showed a significant association between increased EGFR gene copy number (≥ 4.0) and OS in gastric and OGJ cancer, indicating the possibility that patients may be selected for treatment on a genetic basis. Furthermore, a significant correlation was shown between activated EGFR and shorter TTP and ORR, but not between activated EGFR and OS. No V600E BRAF mutations were identified. On the other hand, an interesting trend between high E-cadherin expression levels and better OS was observed and two CDH1 exon 9 missense mutations (A408V and D402H) were detected.
Our finding that increased EGFR gene copy numbers, activated EGFR and the E-cadherin status are potentially interesting biomarkers needs to be confirmed in larger randomized clinical trials.
Multicentre clinical study with the European Clinical Trials Database number 2004-004024-12.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background The costimulatory molecule B7-H1 (programmed death-1 ligand-1, PD-L1) has been implicated as a potential regulator of antitumor immunity in various human cancers. To date, no data are ...available on the role of B7-H1 in Barrett carcinoma. Therefore, we investigated the expression pattern and clinical significance of B7-H1 in a large cohort of patients with Barrett carcinoma. Methods Expression of B7-H1 was evaluated by immunohistochemistry in 101 patients with Barrett carcinoma. Expression data were correlated with clinicopathologic features, including TNM stage, UICC (Union Internationale Contre le Cancer) tumor stage, tumor grade, resection status, and survival, and with the number of tumor-infiltrating CD3+ , CD8+ , and CD45RO+ T lymphocytes. Results Aberrant B7-H1 expression was found in Barrett carcinoma cells. High tumor B7-H1 expression was significantly associated with advanced T stage ( p = 0.002), advanced UICC tumor stage ( p = 0.022), and incomplete resection status ( p = 0.009). The median survival of patients with high tumor B7-H1 expression was 38 months compared with 136 months for patients with no or low tumor B7-H1 expression. In the multivariable analysis, high tumor B7-H1 expression was significantly associated with an increased risk of death from Barrett carcinoma (hazard ratio, 3.50; 95% confidence interval, 1.66 to 7.38; p < 0.001). Conclusions Our data suggest that B7-H1 may represent a new prognostic marker for patients with Barrett carcinoma. Furthermore, given its immune-inhibitory function, B7-H1 may represent a potential target in the treatment of Barrett carcinoma.
Purpose: Aurora kinase A (AURKA/STK15/BTAK) encodes a serine/threonine kinase associated with chromosomal distribution and its up-regulation
induces chromosomal instability, thereby leading to ...aneuploidy and cell transformation in several types of cancer. In this
study, we investigated the role of AURKA in head and neck squamous cell carcinoma (HNSCC).
Experimental Design: The mRNA expression levels of AURKA were compared in tumor tissues of 66 HNSCC patients with those in corresponding normal
squamous epithelium by real-time quantitative reverse transcriptase-PCR. In addition, the association between AURKA mRNA and
protein expression, centrosome abnormalities, and aneuploidy was studied in a subset of cases ( n = 34). All molecular variables were correlated to histomorphologic findings and clinical follow-up data of the patients.
Results: AURKA mRNA up-regulation was significantly associated with tumor stage and the occurrence of regional lymph node, as well
as distant metastasis ( P < 0.0001 for all). Similarly, a correlation was found for protein expression and the occurrence of regional lymph node ( P = 0.0183) and distant metastasis ( P = 0.03). The mRNA was positively associated with protein expression ( P = 0.003) and centrosome abnormalities ( P = 0.03). Cox regression analysis revealed that AURKA mRNA up-regulation correlated with disease-free survival of the patients
( P = 0.03) as well as shorter overall survival ( P < 0.001).
Conclusions: We conclude that the up-regulation of AURKA mRNA may play a critical role in the tumor progression of HNSCC and provides
useful information as a prognostic factor for HNSCC patients.
The validation of novel prognostic indicators is of greatest interest for the management of esophageal adenocarcinoma (Barrett's cancer), particularly for non-metastasized (stage I-IIA) disease. The ...prognostic role of tumor infiltrating T-lymphocytes (TILs) in Barrett's cancer has not been reported so far. Here we evaluated the impact of TILs on survival, recurrence, and metastasis in Barrett's cancer, particularly in stage I-IIA patients.
The levels of the adaptive immune markers CD3, CD8, and CD45RO were analyzed by immunohistochemistry and image analysis in tissue microarrays consisting of tumor tissues of 118 patients with primary resected Barrett's cancer. The findings were correlated with clinicopathological parameters including patient outcome.
In multivariate analysis, a low density of intratumoral CD45RO+ immune cells was an independent unfavorable factor for disease-free survival in stages I-IIA patients (P = 0.004, RR = 4.7, 95% CI = 1.6-13.5) as well in the entire cohort (P = 0.048, RR = 2.0, 95% CI = 1.0-4.0). High CD3+ and CD45RO+ levels were associated with prolonged disease-free survival and overall survival as well with low recurrence rates of disease (P = 0.005 and P = 0.018, respectively). In addition, low CD3+ levels were correlated with a higher frequency of lymph node metastasis (P = 0.025).
This study demonstrates that the density of CD45RO+ TILs is an independent prognostic factor in non-metastasized (stage I-IIA) Barrett's cancer patients and indicates an important role for the adaptive immunologic microenvironment. The inclusion of CD45RO+ density may help to improve the management of stage I-IIA Barrett's cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
