Ion mobility mass spectrometry of human melanoma gangliosides

Highlights

• IMS MS was applied to a native human melanoma ganglioside (GG) mixture.

• IMS MS generated the first inventory of malignant human melanoma GGs.

• IMS MS revealed the elevated incidence of GM3 and GD3 bearing different ceramides.

• De-N-acetyl GM3, de-N-acetyl GD3and N-glycolyl GM3 were detected in melanoma.

• IMS CID MS/MS confirmed the structure of GD3 (d18:1/16:0) and GD3 (d18:1/24:1).

Abstract

Malignant melanoma is an aggressive type of skin cancer, rarely detected in the early stages. Various sets of methods and techniques, including dermatoscopical inspection of the “ABCDE” signs of the lesion, imaging techniques or microscopical, immunohistochemical and serological biomarkers are available and used nowadays to diagnose malignant melanoma. To date, different biomarkers were proposed for melanoma, but only a few, including circulating proteins, such as lactate dehydrogenase, molecular and metabolite biomarkers, have reached clinical applications. Gangliosides represent an emerging class, being used as tumor markers and targets of antibody therapy in melanomas, based on their elevated abundance in melanoma, especially of GM3 and GD3, when compared with the corresponding normal tissues. The conjunction of mass spectrometry (MS) with ion mobility separation (IMS) demonstrated an elevated potential in detection and identification of low abundant components, with biomarker role, in extremely complex biological mixtures. Therefore, here, a native ganglioside extract originating from human melanoma was investigated for the first time by IMS MS to provide the first profiling of gangliosides in this type of cancer. The present approach revealed the high incidence of species belonging to GD3 and GM3 classes, as well as of de-N-acetyl GM3 (d-GM3) and de-N-acetyl GD3 (d-GD3), characteristic for human melanoma. Additionally, the structure of two molecules characterized by shorter glycan chains associated to melanoma, were investigated in detail. The present approach brings valuable data related to this type of cancer, completing the existing inventory of melanoma-associated biomarkers and opens new directions for further research in this field.

Introduction

Melanoma, the most serious type of skin cancer, represents a malignant tumor of melanocytes, the melanin-producing cells. The controlled and ordered way of skin cell development, if interrupted by the DNA damage of some of the cells, determines the uncontrollably division of the new cells and, eventually, the formation of a cancerous cells mass. Unlike other forms of skin cancer, such as basal cell carcinoma or squamos cell carcinoma, which are more frequent but more benign, malignant melanoma remains one of the fastest growing cancers worldwide, with a very rapid methastasis, and once the spreading happens, it can be difficult to treat.

The incidence increased dramatically over the past 30 years [1], being the most common cancer in young adults aged 25–29 years and the second most common cancer in those aged 15–29 years [2]. Though the majority of the patients are treated by surgical excision of the primary tumor, still many patients develop metastases [3]. The exact cause of what damages DNA in skin cells and how this leads to melanoma is not clear, but it is believed to be a process that requires a complex interaction between exogenous and endogenous triggers as well as tumor-intrinsic and immune-related factors [4].

The exposure to ultraviolet (UV) radiation from the sunlight or tanning lamps and beds, the familiar history, the number of melanocytic nevi, along with several phenotypic characteristics, among which red or blonde hair, fair skin, frequent freckles, light eyes and sun sensitivity increase the risk by approximately 50% for developing cutaneous melanoma [[4], [5], [6]]. Relatively rare in people with darker skin, melanomas can develop anywhere on the body, the most affected areas being the chest and back, legs, arms and face. However, there are several hidden melanoma, more common in people with darker skin, which occur in areas unexposed to sun, such as the soles of the feet, palms of the hand, fingernail beds, eyes, or even inside the body, such as in the nose or throat. Given this abnormal places for melanoma development, it is likely that other factors, including the genetic factors, are responsible for melanoma.

The American Cancer Society (ACS) has calculated the 5-year relative survival rates for melanoma and estimated for 2020 that there would be about 100,350 new diagnoses of melanoma, and that around 6850 people will die due to melanoma in USA [1]. The 5-year relative survival rate can drop off up to 64% if the melanoma spreads to deeper tissues or nearby lymph nodes, and to 23% if it reaches distant organs or tissues [7]. Therefore, it is extremely important to examine any changing moles and seek medical attention for any modification of them.

According to the frequency, speed of expansion and the area of occurrence, there are four major forms of melanoma. The most common form, accounting for about 70% of melanomas is the superficial spreading melanoma, which often appears on the trunk or limb, followed by nodular melanoma, the most aggressive form, characteristic for trunk, head, or neck and having an incidence of about 15–30% of all melanoma cases. The lentigo maligna and acral lentiginous forms represent less than 10% of melanoma cases [8].

Of extreme importance in the diagnosis process is the assignment of a melanoma stage, from stage 0, known as “melanoma in situ”, in which the cancer is present only in the outermost layer of skin, up to stage 4 where the cancer is spread to brain, lungs, or liver. A key factor in lowering the mortality caused by malignant melanoma remains the early detection. Compared to other cancers, malignant melanoma has the benefit of being cutaneous localized, which facilitates its early detection through non-invasive methods [6]. The general screening for melanoma includes a total body skin examination according to the “ABCDE rule”, the acronyms for Asymmetry, Border irregularity, Color variations, Diameter >6 mm, and Elevated surface [8], followed by dermoscopy or other imaging techniques, such as in-vivo reflectance confocal laser microscopy, computer-aided multispectral digital analysis, and electrical impedance spectroscopy [4]. Subsequent to the histopathological diagnosis, palpatory and sonographical inspection of the regional lymph-node basin is performed prior further surgical procedures in order to eliminate macroscopic lymphogenic metastatic spread [4].

Research in melanoma diagnostics is focused on identifying specific tissue and blood-based biomarkers. According to Lim et al. [9], the cancer biomarkers are classified in diagnostic, prognostic or predictive. While the diagnostic biomarkers facilitate early detection of cancer by identifying and confirming its presence, the prognostic markers forecast the feasible course and likely outcomes of a disease regardless of treatment, and the predictive biomarkers evaluate the chance of benefit from a precise treatment [8,10].

Currently, various prognostic and predictive biomarkers were proposed for melanoma, but only a limited number of molecular biomarkers have reached clinical application and are included in the American Joint Committee on Cancer (AJCC) melanoma staging system [11]. Lactate dehydrogenase (LDH), followed by S100B, C reactive protein (CRP) and melanoma-inhibiting activity (MIA) protein, all circulating proteins [8,12] have significant prognostic value in the AJCC staging system. Besides the protein biomarkers, the molecular (nucleic acids and microphthalmia transcription factor) [12,13] and metabolite [14] biomarkers have also provided valuable prognostic information about tumor outcome and significant predictive data about tumor response to therapy.

Gangliosides (GGs), a family of sialic acid-containing glycosphingolipids are highly expressed in nervous tissues of mammals, where are involved in the development and function of nervous systems. Gangliosides with relatively simple structures were identified as cancer-associated antigens, since they are regularly over expressed in the external membrane of cancer cells. Hence, GGs were used as tumor markers [[15], [16], [17], [18], [19]], and as targets of antibody therapy in melanomas [[20], [21], [22]] based on their elevated abundance in melanoma when compared with the corresponding normal tissues, especially of GM3 and GD3 species. Either for proteome, lipidome, metabolome or gangliosidome profiling, mass spectrometry (MS) in conjunction or not with different separation techniques demonstrated over the years to be the best option for the identification and validation of biomarkers in both tissue and body fluids [10,[15], [16], [17], [18],[23], [24], [25], [26], [27], [28]].

Moreover, the conjunction with MS of ion mobility separation (IMS), a robust and reliable analytical system, demonstrated an elevated potential in detection and identification of low abundant components in extremely complex biological mixtures [[29], [30], [31], [32]]. While broadly applied for the investigation of peptides, proteins, lipids, carbohydrates and small molecules, IMS MS was less exploited in glycolipidomics, being introduced for the first time in brain ganglioside research only in 2016 [33].

Since then, IMS MS demonstrated its feasibility for mapping and detailed structural analysis of GG mixtures, revealing besides a larger inventory of glycoforms in various human brain regions, compared with studies where no separation prior to MS was involved [[33], [34], [35]], the incidence of species bearing unusual type of attachments [34]. The applicability of IMS MS in the study of human melanoma, is still limited, up to now to our knowledge, only two studies being conducted and published, one on proteins [36], and one on metabolites [14].

Considering the record number of newly diagnosed melanoma worldwide, the best impact on reducing this incidence implies the identification and further, the validation of potential biomarkers by simple, rapid and highly sensitivity profiling technologies. Up to now, only low sensitivity methods, such as histopathological or chromatographic ones were applied in order to determine the ganglioside profile of melanoma tumors, all pointing out the elevated incidence of GM3, GD3 and GD2 classes, but not the exact structure of them.

In this context, in the present study, the native ganglioside extract originating from human melanoma (HME) was investigated for the first time by IMS MS and collision induced dissociation (CID) tandem MS (MS/MS) to provide the first reliable mapping and structural analysis of melanoma GGs at high sensitivity, reproducibility and data accuracy. The present approach and results offer new and valuable information related to this type of cancer at the molecular level, completing with novel species the existing inventory of melanoma-associated biomarkers.