Epithelial ovarian cancer (EOC) is the leading cause of death within gynecological cancers in the developed countries (http://seer.cancer.gov
). Due to the lack of specific symptoms, EOC is often detected at an advanced stage with a five-year survival rate less than 40% [1
]. However, 25% of EOC patients are diagnosed in early stage (I-II) as defined by Fédération Internationale de Gynécologie et d’Obstétrique (FIGO), where the disease is often cured by surgery alone, or in combination with platinum-based chemotherapy [2
]. Even though the prognosis of patients with FIGO stage I-II increases dramatically with treatment, with five-year survival rates between 80–90%, some subgroups of early-stage EOC will relapse and 20–30% of these patients will finally succumb to the disease [4
]. Older age, greater stage, higher grade and malignant cytology are independent prognostic factors for recurrence [7
]. Moreover, the prognosis differs between the histological subtypes with high-grade serous ovarian cancer (HGSOC) being the most common one, accounting for 70–80% of ovarian cancer-related deaths.
According to guidelines of the European Society for Medical Oncology (ESMO), bilateral salpingo-oophorectomy, hysterectomy, omentectomy, peritoneal stripping and lymph node sampling are recommended procedures for stage I and II HGSOC patients (https://www.esmo.org/guidelines/gynaecological-cancers/newly-diagnosed-and-relapsed-epithelial-ovarian-carcinoma/esmo-esgo-consensus-conference-recommendations-on-ovarian-cancer
]. However, fertilization-sparing surgery (FSS) for women of childbearing age could be considered, and be discussed individually [10
]. Different criteria for selecting patients have been applied and the debate over FSS in HGSOC is more than controversial as there are limited data on that issue. Preoperative screening methods and comprehensive surgical staging for accurate disease classification are mandatory [11
]. In this context, one third of presumed stage I ovarian cancers were found to be upstaged by the findings of dissemination in the peritoneal cavity [13
]. Patients with high-risk early-stage EOC, defined as stage I, grade 3, stage IC and II, as well as clear cell cancers, will require adjuvant chemotherapy which has been shown to reduce the relapse rate by >60% in stage IC EOC patients [14
]. Hence, platinum-based chemotherapy is an important factor in treating these patients with high-risk early-stage EOC with impact on both recurrence-free (RFS) and overall survival (OS). Prognostic markers are needed to stratify patients into low- and high-risk groups in order to select patients who will benefit from chemotherapy. The term EOC refers to at least four different histological subtypes which is an important issue to take into account in the risk assessment of clinical progression. The most aggressive histotype is HGSOC. Nevertheless, the optimal clinical management is still a controversial debate and patients with early-stage high-grade serous EOC might be over-treated which could potentially result in complications after radical surgical management and an increase in toxicity of chemotherapy [15
]. Hence, it is of utmost importance to identify novel diagnostic markers for this patient cohort in order to improve the risk assessment of tumor recurrence. An optimal evaluation of risk for progression would have the benefit of personalized chemotherapy, and reduced costs and treatment side effects in patients with little risk for progression. Commonly used tissue-based techniques, such as liquid chromatography-based mass spectrometry or gene expression profiling, require large amounts of tissue material. Moreover, these methods do not enable a direct correlation between differentially expressed molecular profiles and the tissue histology [17
]. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has the advantage of combining morphological features with protein expression in tissue. This technique enables spatially resolved tissue assessment via specific molecular signatures (e.g., proteins, peptides, lipids and molecules of cell metabolites) and allows their correlation with alterations in tissue histology [18
] as well as stages of ovarian cancer [21
This recently developed diagnostic method of imaging mass spectrometry (MALDI-imaging MS) has also been used for the rapid diagnosis and prognosis of patients [22
], and to identify peptide profiles spatially resolved directly on the paraffin-embedded tissue to depict and assign to the histological and clinical pathological subtypes of cancer.
Here, we have applied the method to detect a molecular signature of 13 peptides that predicts tumor recurrence in patients with early-stage HGSOC. According to their specific sequence, these peptides were allocated to a signature of proteins for risk stratification in support of clinical management of patients with early-stage HGSOC.
In general, HGSOC patients diagnosed at early-stage have an excellent prognosis and concern arises that some of the early-stage HGSOCs are over-treated. Hence, there has been a debate about the optimal duration and chemotherapy treatment strategy, e.g., Carboplatin only, or combination regimens, four cycles vs. six cycles. However, a subgroup of patients will relapse and need therapies that are more intensive at time of diagnosis. It is therefore of great importance to identify these high-risk patients in order to improve their clinical outcome.
Currently, there are no reliable markers at hand for standard immunohistochemical assessment of this subpopulation. Here, we have used a novel approach using MALDI-IMS technology to screen for a prognostic peptide signature to support the clinical management of these patients. For this purpose, standardized protocols for MALDI-IMS sample preparation have been developed [25
], which are intended to enable reliable exploration of molecular signatures as biomarkers and has been shown to provide valuable diagnostic and risk assessment capabilities for other diagnostically challenging neoplasms [27
]. Our recently published data, showed that IMS can reliably detect the histological subtypes of ovarian cancer [20
]. In this presented study, proteomic analysis results 151 discriminative m
values between early-stage HGSOC patients with either RD or non-RD. In order to identify MALDI-IMS-derived m
values, the “bottom-up”-nano liquid chromatography (nLC)-MS/MS approach was performed on adjacent tissue sections. According to the IMS guidelines [28
], the mass difference between MALDI-IMS and LC-MS/MS m
values should be less than 0.9 Da and requires the identification of more than one peptide.
Specific peptides linked to Keratin type1, Actin, cytoplasmic 1 and Collagen alpha-2(I) were observed to have the strongest expression levels in primary tumors from early-stage HGSOC patients with RD and indicated greatest prognostic values (AUC > 0.7). A published reference database of MALDI-IMS-derived peptide and protein values in various and in particular for ovarian cancer FFPE tissue [29
] was intended as support for the verification of protein identifications. The observed m
values 1562.8 ± 0.2 from Collagen alpha-2(I) and 1790.9 ± 0.2 Da from Actin, cytoplasmic 1 were also determined and identified in MALDI-IMS studies of biopsies from lung tumor patients [30
]. Through regulation of various signaling pathways in cancer cells, Keratins, the epithelial-predominant members of the intermediate filament superfamily, are involved in a number of processes in tumor progression [31
]. KRT9 is one of the most common contaminants in proteomic mass spectrometry analyses, both in ESI and MALDI mass spectrometry methods (see also reference [32
]. These contaminations may rarely have their source in the sample material (randomly distributed), but are more often introduced during sample preparation (e.g., contamination from the environment like dust in solvents, buffers or matrix) [32
]. However, the difference with MALDI imaging experiments is that the m
values can be represented spatially in the tissue, such that contaminations would be evenly distributed over the whole sample material. Therefore, the tissue microarrays (TMAs) are randomized and a control area outside the tissue is measured as a control to exclude such contamination. No peptides (Isotopic pattern) and salt adducts were detected in the control area. Only singles from alpha -Cyano-4-hydroxycinnamic acid matrix clusters could be found with no influence on the data evaluation.
Moreover, both patient groups’ cores were included and randomly distributed on the same cover slip. Therefore, it is unlikely to detect any significant differences. Furthermore, MALDI imaging experiments predominantly address structural proteins, such as ECM molecules, since methodically an enzymatic surface digestion of the tissue sections is performed. Excluding cytoskeleton proteins from the analysis would be premature, especially since KRT9 is a cellular component of the cytoskeleton, cytosol, extracellular region or membrane (see https://www.uniprot.org/uniprot/P35527—subcellular
location). Furthermore, a query of the kmplot.com (https://kmplot.com/analysis/
) database showed a significant decrease in overall survival (p
< 0.0052) associated with high expression of KRT9 considering only stage I EOC including HGSOC (p
< 0.0028) patients (Figure S2
). Therefore, our KRT9 MALDI-IMS measurement is unlikely a result of contamination.
The major sources of collagen expression are stromal cells with increased collagen production and disposition in the stromal compartment has been shown to be associated with breast cancer development and progression [33
]. Nevertheless, it was also demonstrated that expression of collagen by ovarian cancer cells, including Collagen alpha-2(I), could increase drug resistance by inhibiting the penetration of the drug into the cancer tissue as well as increase resistance to apoptosis [35
The analysis of three additional early-stage HGEC patients (two with RD; one without RD) showed comparable measured peptide intensities to the HGSOC patients. A multivariate regression was not feasible due to an insufficient number of observations [36
]. However, reduction in covariates (dimension reduction), such as in a PCA, showed the discriminative capacity of the proposed prognostic marker candidates for patients with early-stage of either HGEC or HGSOC (Figure 5
). Peptide markers separated into two distinct groups based on the correlation between them.
Even though the utilized sample size of four patients for each group is not sufficient for clinical validation, the purpose of this proof of concept study is to identify prognostic marker candidates. Consequently, validation of applicability of the proposed prognostic marker candidates, including for endometrioid carcinomas, necessitates subsequent high-sample size follow-up studies.
Moreover, changes in the tumor microenvironment in response to malignant transformation have been neglected in the past and need to be considered as a suitable compartment for biomarker discovery. So far, a major limitation of dissecting the stromal signature has been a lack of suitable methods. IMS is able to provide spatial information of protein signatures in both compartments. Unfortunately, the quality of the adjacent stroma in the majority of cores from the tumor tissue was not suitable for further assessment but should be included and subject of future prognostic biomarker research for early-stage HGSOC patients.
Eventually, a profound understanding of the biology in early-stage HGSOC might result in a redefinition of high-risk early-stage EOC to develop novel therapeutic approaches. However, this will need molecular characterization supported by RNA-Seq and high-resolution proteomics data from micro-dissected malignant and adjacent stroma compartments. Nevertheless, the identification of the subpopulation of patients developing recurrent tumors is an unmet clinical need. Here, we show that MALDI-IMS technology has the potential to make a meaningful impact for risk assessment and, hence, patient outcome.