Martina Stella

Proteomic profiles of thyroid tumors by mass spectrometry-imaging on tissue microarrays.

The current study proposes the successful use of a mass spectrometry-imaging technology that explores the composition of biomolecules and their spatial distribution directly on-tissue to differentially classify benign and malignant cases, as well as different histotypes. To identify new specific markers, we investigated with this technology a wide histological Tissue Microarray (TMA)-based thyroid lesion series. Results showed specific protein signatures for malignant and benign specimens and allowed to build clusters comprising several proteins with discriminant capabilities. Among them, FINC, ACTB1, LMNA, HSP7C and KAD1 were identified by LC-ESI-MS/MS and found up-expressed in malignant lesions. These findings represent the opening of further investigations for their translation into clinical practice, e.g. for setting up new immunohistochemical stainings, and for a better understanding of thyroid lesions. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging in the Study of Gastric Cancer: A Mini Review

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide and the disease outcome commonly depends upon the tumour stage at the time of diagnosis. However, this cancer can often be asymptomatic during the early stages and remain undetected until the later stages of tumour development, having a significant impact on patient prognosis. However, our comprehension of the mechanisms underlying the development of gastric malignancies is still lacking. For these reasons, the search for new diagnostic and prognostic markers for gastric cancer is an ongoing pursuit. Modern mass spectrometry imaging (MSI) techniques, in particular matrix-assisted laser desorption/ionisation (MALDI), have emerged as a plausible tool in clinical pathology as a whole. More specifically, MALDI-MSI is being increasingly employed in the study of gastric cancer and has already elucidated some important disease checkpoints that may help us to better understand the molecular mechanisms underpinning this aggressive cancer. Here we report the state of the art of MALDI-MSI approaches, ranging from sample preparation to statistical analysis, and provide a complete review of the key findings that have been reported in the literature thus far.

The proteomic landscape of renal tumors

ABSTRACT Introduction: Renal cell carcinoma (RCC) is the most fatal of the common urologic cancers, with approximately 35% of patients dying within 5 years following diagnosis. Therefore, there is a need for non-invasive markers that are capable of detecting and determining the severity of small renal masses at an early stage in order to tailor treatment and follow-up. Proteomic studies have proved to be very useful in the study of tumors. Areas covered: In this review, we will detail the current knowledge obtained by the different proteomic approaches, focusing on MS-based strategies, used to investigate RCC biology in order to identify diagnostic, prognostic and predictive biomarkers on tissue, cultured cells and biological fluids. Expert commentary: Currently, no reliable biomarkers or targets for RCC have been translated into the clinical setting. Moreover, despite the efforts of proteomics and other -omics disciplines, only a small number of them have been observed as shared targets between the different analytical platforms and biological specimens. The difficulty to define a specific molecular pattern for RCC and its subtypes highlights a peculiar profile and a heterogeneity that must be taken into account in future studies.

Molecular signatures of medullary thyroid carcinoma by matrix-assisted laser desorption/ionisation mass spectrometry imaging

The main aim of the study was to assess the feasibility of matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in the pathological investigation of Medullary Thyroid Carcinoma (MTC). Formalin-fixed paraffin-embedded (FFPE) samples from seven MTC patients were analysed by MALDI-MSI in order to detect proteomic alterations within tumour lesions and to define the molecular profiles of specific findings, such as amyloid deposition and C cell hyperplasia (CCH). nLC-ESI MS/MS was employed for the identification of amyloid components and to select alternative proteomic markers of MTC pathogenesis. Results highlighted the potential of MALDI-MSI to confirm the classic immunohistochemical methods employed for the diagnosis of MTC, with good sensitivity and specificity. Intratumoural amyloid components were also detected and identified, and were characterised by calcitonin, apolipoprotein E, apolipoprotein IV, and vitronectin. The tryptic peptide profiles representative of MTC and CCH were distinctly different, with four alternative markers for MTC being detected; K1C18, and three histones (H2A, H3C, and H4). Finally, a further 115 proteins were identified through the nLC-ESI-MS/MS analysis alone, with moesin, veriscan, and lumican being selected due to their potential involvement in MTC pathogenesis. This approach represents a complimentary strategy that could be employed to detect new proteomic markers of MTC. STATEMENT OF SIGNIFICANCE: Medullary thyroid carcinoma (MTC) is a rare endocrine malignancy that originates from the parafollicular C-cells of the thyroid. The diagnosis is typically established using a combination of fine-needle aspiration biopsy (FNAB) of a suspicious nodule along with the demonstrable elevation of serum biomarkers, such as calcitonin and carcinoembryonic antigen (CEA). Unfortunately, this combination is often associated with a high degree of false-positive results and this can lead to misdiagnosis and avoidable total thyroidectomy. The current study presents the potential role of MALDI-MSI in the search for new proteomic markers of MTC with diagnostic and prognostic significance. MALDI-MSI was capable of detecting the classic immunohistochemical markers employed for the diagnosis of MTC, with good sensitivity and specificity. Furthermore, the complementary combination of MALDI-MSI and nLC-ESI-MS/MS analysis, using a single tissue section, enabled further potential markers to be identified and their spatial localisation visualised within tumoural regions. Such findings could be a valuable starting point for further studies focused on confirming the data presented here using thyroid FNABs, with the final objective being to provide complimentary assistance for the detection of MTC during the pre-operative phase.

Proteomics and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging as a Modern Diagnostic Tool in Kidney Diseases

Внаслідок швидкого розвитку сучасної науки ми постійно, день у день, удосконалюємо наші знання про патогенез і морфологію захворювань. У зв’язку з настанням ери наукомік, таких як геноміка, транскріптоміка та протеоміка, є велика необхідність у розумінні молекулярних механізмів хвороб і організмів. Кінцевими цілями повинні бути такі: більш успішні діагностика та прогноз, виявлення потенційних терапевтичних мішеней і прогнозування відповіді на лікування. Хронічна хвороба нирок (ХХН) є всесвітньою проблемою охорони здоров’я, що характеризується швидкозростаючою захворюваністю, a сам термін «ХХН» охоплює велику підмножину захворювань. Останнім часом для вивчення ХХН використовуються сучасні технології протеоміки, такі як мас-спектрометрія з матрично-активованою лазерною десорбцією/іонізацією. Незважаючи на ранній етап розвитку подібних методик, уже існує значна кількість досліджень і публікацій, присвячених цій темі.

The putative role of MALDI-MSI in the study of Membranous Nephropathy

Membranous Nephropathy (MN) is an immunocomplex mediated renal disease that represents one of the most frequent glomerulopathies worldwide. This glomerular disease can manifest as primary (idiopathic) or secondary and this distinction is crucial when choosing the most appropriate course of treatment. In secondary cases, the best strategy involves treating the underlying disease, whereas in primary forms, the identification of confirmatory markers of the idiopathic etiology underlining the process is requested by clinicians. Among those currently reported, the positivity to circulating antigens (PLA2R, IgG4 and THSD7A) was demonstrated in approximately 75% of iMN patients, while approximately 1 in 4 patients with iMN still lack a putative diagnostic marker. Ultimately, the discovery of biomarkers to help further stratify these two different forms of glomerulopathy seems mandatory. Here, MALDI-MSI was applied to FFPE renal biopsies from histologically diagnosed primary and secondary MN patients (n=20) in order to detect alterations in their tissue proteome. MALDI-MSI was able to generate molecular signatures of primary and secondary MN, with one particular signal (m/z 1459), identified as Serine/threonine-protein kinase MRCK gamma, being over-expressed in the glomeruli of primary MN patients with respect to secondary MN. Furthermore, a number of signals that could differentiate the different forms of iMN that were positive to PLA2R or IgG4 were detected, as well as a further set of signals (m/z 1094, 1116, 1381 and 1459) that could distinguish these patients from those who were negative to both. These signals could potentially represent future targets for the further stratification of iMN patients. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

MALDI-MSI Pilot Study Highlights Glomerular Deposits of Macrophage Migration Inhibitory Factor (MIF) as a Possible Indicator of Response to Therapy in Membranous Nephropathy

Membranous nephropathy (MN) is the most frequent cause of nephrotic syndrome in adults and the disease course is characterized by the “rule of third”, with one‐third of patients experiencing complete remission and the remaining experiencing relapses or progression of the disease. Additionally, the therapeutic approach is not standardized, leading to further heterogeneity in terms of response and outcome.

Effects of Hematuria on the Proteomic Profile of Urinary Extracellular Vesicles: Technical Challenges

Hematuria is a common sign of many renal and urologic pathologic conditions and it may affect the proteomic analysis of urinary extracellular vesicles (UEv), nanovesicles released from all cells in contact with the urinary space. This condition hinders UEv based proteomic studies aiming to discover biomarkers. Therefore, we studied the effects of hematuria on the proteome of UEv and introduced a possible solution to reduce its misleading impact. We mimicked hematuria by adding increasing amount of blood to nonaffected urine and investigated its effects on UEv isolation, purity, and proteomic composition. We proposed a trypsin treatment able to reduce the impact of hematuria on UEv. The effects of the treatment were investigated by evaluating the UEv proteomic profile, the enrichment of known UEv markers, and by assessing differential protein content by MS-based label-free quantification. Results showed that as the blood contamination increased, it affected both the proteome profile and the yield of UEv isolated from urine. Our treatment with trypsin was able to counteract completely these effects for low/medium levels of hematuria, which are most commonly encountered. This promising finding could lead to the reliable use of hematuria samples for UEv proteomic investigation.

Proteomic and Bioinformatic Studies for the Characterization of Response to Pemetrexed in Platinum Drug Resistant Ovarian Cancer

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

Histoproteomic Characterization of Localized Cutaneous Amyloidosis in X-Linked Reticulate Pigmentary Disorder

tion 0.4 mg/mL, dilution 1: 300; Altas Antibodies, AlbaNova University Center, Stockholm, Sweden), light chain negative (DAKO, Glostrup, Denmark) with intense immunoreactivity of the amorphous deposits for IgG (DAKO, Glostrup, Denmark), and CK AE1/AE3 positive (concentration 0.4 mg/mL, dilution 1: 300; Altas Antibodies; Fig. 1 e, f). Further proteomic analysis detected the overexpression of a group of proteins that were potentially involved in the pathogenesis of the localized cutaneous amyloidosis (LCA; Table 1 ) [6– 9] . In particular, one of these proteins was identified as apolipoprotein E, a chaperon protein found in the dermis of patients affected by lichen amyloidosis and macular amyloidosis, thus suggesting that a dysregulation of the apoptosis system is an initial cause of the disorder [8] . Another candidate from the same group of proteins is galectin-7, a proapoptotic protein that is expressed by the damaged keratinocytes [6, 7] and is related to the cathepsin and trypsin-like families. Furthermore, SAP (serum amyloid P component), a member of the pentraxin family, has been identified Dear Editor, Skin involvement is an inconsistent but characteristic feature of the X-linked reticulate pigmentary disorder (XLRPD; OMIM 301220), a rare entity that is characterized by recurrent infections and autoimmune reactions against various organs with respiratory, gastrointestinal, and neurological manifestations [1] . Given that many patients require transplantation [2] , dermatologists should consider graft versus host disease (GVHD) during a differential diagnosis. In XLRPD, histology essentially reveals the presence of amyloid deposits that are mainly localized within the dermoepidermal junction of adult patients, leading to the hypothesis that this is an age-related condition [3–5] . In their report, Pezzani et al. [2] described a case of genetically confirmed XLRPD in which no dermopathological symptoms were present at diagnosis. However, during 4 years of follow-up, the same child developed a peculiar skin lesion that was clinically suggestive of GVHD ( Fig. 1 a, b). A skin biopsy showed a prominent amyloid deposition, whilst also being Congo red positive ( Fig. 1 c, d), anti-AAP negative (concentraReceived: August 5, 2016 Accepted after revision: February 17, 2017 Published online: April 4, 2017