Imaging mass spectrometry (IMS) using matrix-assisted laser desorption ionization (MALDI) is a new and effective tool for molecular studies of complex biological samples such as tissue sections. As histological features remain intact throughout the analysis of a section, distribution maps of multiple analytes can be correlated with histological and clinical features. Spatial molecular arrangements can be assessed without the need for target-specific reagents, allowing the discovery of diagnostic and prognostic markers of different cancer types and enabling the determination of effective therapies.

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Prostate cancer has become one of the most common malignancies worldwide. Although lacking in specificity its diagnosis is still based partially on the serumbased test for prostate-specific antigen. As its pathogenesis has not yet been deciphered, the ongoing search for new and more reliable biomarkers remains a challenge to stratify disease onset and progression. Matrix-assisted laser desorption/ ionization (MALDI)-Imaging is a promising technique to assist in this endeavor. It delivers accurate mass spectrometric information of the sample's proteins and enables the visualization of the spatial distribution of protein expression profiles and correlation of the information with the histomorphological features of the same tissue section. This study describes the analysis of 22 prostate sections (11 with and 11 without prostate cancer) by MALDI-Imaging. Specific protein expression patterns were obtained for normal and cancerous regions within the tissue sections. Applying a 'support vector machine' algorithm to classify the cancerous from the noncancerous regions, an overall cross-validation, a sensitivity and specificity of 88, 85.21 and 90.74%, respectively, was achieved. Additionally four distinctively overexpressed peaks were identified: 2,753 and 6,704 Da for non-cancerous glands, and 4,964 and 5,002 Da for cancerous glands. The results of this first clinical study utilizing the new technique of MALDI-Imaging underline its vast potential to identify candidates for more reliable prostate cancer tumor markers and to enlighten the pathogenesis of prostate cancer.

CXCR4 is a G-protein-coupled receptor that mediates recruitment of blood cells toward its ligand SDF-1. In cancer, high CXCR4 expression is frequently associated with tumor dissemination and poor prognosis. We evaluated the novel CXCR4 probe [68Ga]Pentixafor for in vivo mapping of CXCR4 expression density in mice xenografted with human CXCR4-positive MM cell lines and patients with advanced MM by means of positron emission tomography (PET). [68Ga]Pentixafor PET provided images with excellent specificity and contrast. In 10 of 14 patients with advanced MM [68Ga]Pentixafor PET/CT scans revealed MM manifestations, whereas only nine of 14 standard [18F]fluorodeoxyglucose PET/CT scans were rated visually positive. Assessment of blood counts and standard CD34+ flow cytometry did not reveal significant blood count changes associated with tracer application. Based on these highly encouraging data on clinical PET imaging of CXCR4 expression in a cohort of MM patients, we conclude that [68Ga]Pentixafor PET opens a broad field for clinical investigations on CXCR4 expression and for CXCR4-directed therapeutic approaches in MM and other diseases.

Purpose To facilitate the transition of MALDI–MS Imaging (MALDI–MSI) from basic science to clinical application, it is necessary to analyze formalin‐fixed paraffin‐embedded (FFPE) tissues. The aim is to improve in situ tryptic digestion for MALDI–MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites. Experimental Design FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) are prepared for MALDI–MSI. Samples are coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples are sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50 µm spatial resolution. Data are analyzed using flexImaging, SCiLS, and R. Results Trypsin application and digestion are identified as sources of variation and loss of spatial resolution in the MALDI–MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA. Conclusions and Clinical Relevance Spatial resolution and site‐to‐site reproducibility can be maintained by adhering to a standardized MALDI–MSI workflow.

Lung cancer is the leading cause of cancer related death worldwide among both, men and woman with about 1.59 million reported deaths in 2012 (1). Two major lung cancer categories are discerned, namely small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) 1 , with the latter comprising ϳ85% of all cases. The two predominant histological NSCLC entities are adenocarcinoma (ADC) and squamous cell carcinoma (SqCC) accounting for ϳ50 and 40% of all lung cancers, respectively (2).Because differentiation of NSCLC subtypes is crucial for the selection of chemotherapy regimens and subsequent molecular test strategies, precise subtyping is paramount. Therapeutic targets such as ALK translocations or activating mutations of the epidermal growth factor receptor (EGFR) have been found almost exclusively in ADC and these patients benefit from the respective molecularly tar-

MALDI-imaging MS is a new molecular imaging technologyfor direct in situ analysis of thin tissue sections. Multiple analytes can be monitored simultaneously without prior knowledge of their identities and without the need for target-specific reagents such as antibodies. Imaging MS provides important insights into biological processes because the native distributions of molecules are minimally disturbed, and histological features remain intact throughout the analysis. A wide variety of molecules can be imaged, including proteins, peptides, lipids, drugs, and metabolites. Several specific examples are presented to highlight the utility of the technology. MALDI imaging MS (IMS)4 is an emerging new tool for the analysis of biological and clinical tissue samples. It has been shown to be amenable for the analysis of proteins, peptides (both endogenous and enzymatically produced), lipids, and small molecules (such as drugs and endogenous metabolites). Spatial relationships of molecules within a specimen are preserved because intact tissue is directly analyzed without homogenization. In this way, molecules can be interrogated in their native environments, providing new insights into the biological processes involved.IMS requires minimal sample preparation for analysis. Thin sections of tissue samples (typically, 5-10 m thick) are obtained from frozen or formalin-fixed paraffin-embedded (FFPE) tissue blocks and collected on conductive MALDI targets. A matrix compound (typically, a small organic acid as well as a proteolytic enzyme when necessary) is applied to the surface of the tissue sample. Mass spectra are subsequently collected by firing a laser in an ordered pattern of thousands of ablated spots on the tissue section, and a discrete spectrum is collected from each location on the sample. Each ablated spot is analogous to a pixel in a digital photograph. Each pixel (spectrum) contains many analytes that can be individually displayed as a function of their position and relative intensity within the tissue section. In this way, hundreds of images from specific molecular species can be generated simultaneously from a single tissue section without prior knowledge of their identities. Specific reagents such as antibodies are not needed. Alternatively, IMS can be carried out in a profiling mode in which only relatively small selected areas from each tissue section are targeted for analysis. A stained serial section is typically used to guide the analysis. The sample preparation and analysis process of fresh-frozen and FFPE tissues are summarized in Fig. 1.IMS can be very high-throughput in nature. Often, 10 -20 tissue sections can be collected on a single target plate and analyzed concurrently. With currently available high repetition rate lasers (1 kHz or greater), the entire target plate can be analyzed in a matter of a few minutes to a few hours, depending on the desired spatial resolution. It has recently been shown that, through the use of a 5 kHz laser and continuous laser raster, a rat brain measuring 185 mm 2 can ...

In prostate cancer (PC) patients, the differentiation between lung metastases and lesions of different origin, for example, primary lung cancer, is a common clinical question. Herein, we investigated the use of Glu-NH-CO-NH-Lys(Ahx)-HBED-CC ( 68 Ga-PSMA-HBED-CC) for this purpose. Methods: PC patients (n 5 1,889) undergoing 68 Ga-PSMA PET/CT or PET/MR scans were evaluated retrospectively for suggestive lung lesions. For up to 5 lesions per patient, location, CT diameter, CT morphology, and SUV max were determined. The standard for classification was either histopathologic evaluation or, in the case of PC metastases, responsivity to antihormone therapy. A comparison of the different classes was executed by Student t test. Prostate-specific antigen and prostate-specific membrane antigen (PSMA) immunohistochemistry were performed if histologic samples were available; 68 Ga-PSMA autoradiography was performed on an exemplary case of PET-positive lung cancer. Results: Eighty-nine lesions in 45 patients were identified, of which 76 were classified as PC (39 proven, 37 highly probable), 7 as primary lung cancer, and 2 as activated tuberculosis; 4 lesions remained unclear. The mean SUV max was 4.4 ± 3.9 for PC metastases and 5.6 ± 1.6 for primary lung cancer (P 5 0.408). Additionally, substantial differences in SUV max intraindividually were detected. The 2 tuberculous lesions showed an SUV max of 7.8 and 2.5. Using immunohistochemistry, we could demonstrate PSMA expression in the neovasculature of several PSMA PET-positive lung cancers as well as in tuberculous lesions from our histologic database. Conclusion: Quantitative (SUV) analysis of 68 Ga-PSMA PET was not able to discriminate reliably between pulmonary metastases and primary lung cancer in PC patients. The reason for the unexpectedly high tracer uptake in non-PC lesions is not completely clear. PSMA expression in neovasculature provides a possible explanation for this finding; however, other contributing factors, such as tracer binding to proteins other than PSMA, cannot be excluded at present.