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Platelet protein biomarker panel for ovarian cancer diagnosis

  • Marta Lomnytska1, 2, 3Email authorView ORCID ID profile,
  • Rui Pinto4,
  • Susanne Becker3,
  • Ulla Engström5,
  • Sonja Gustafsson6,
  • Christina Björklund6,
  • Markus Templin7,
  • Jan Bergstrand8,
  • Lei Xu8,
  • Jerker Widengren8,
  • Elisabeth Epstein2, 9,
  • Bo Franzén3, 6 and
  • Gert Auer3, 6
Biomarker Research20186:2

https://doi.org/10.1186/s40364-018-0118-y

Received: 5 September 2017

Accepted: 3 January 2018

Published: 12 January 2018

Abstract

Background

Platelets support cancer growth and spread making platelet proteins candidates in the search for biomarkers.

Methods

Two-dimensional (2D) gel electrophoresis, Partial Least Squares Discriminant Analysis (PLS-DA), Western blot, DigiWest.

Results

PLS-DA of platelet protein expression in 2D gels suggested differences between the International Federation of Gynaecology and Obstetrics (FIGO) stages III-IV of ovarian cancer, compared to benign adnexal lesions with a sensitivity of 96% and a specificity of 88%. A PLS-DA-based model correctly predicted 7 out of 8 cases of FIGO stages I-II of ovarian cancer after verification by western blot. Receiver-operator curve (ROC) analysis indicated a sensitivity of 83% and specificity of 76% at cut-off >0.5 (area under the curve (AUC) = 0.831, p < 0.0001) for detecting these cases. Validation on an independent set of samples by DigiWest with PLS-DA differentiated benign adnexal lesions and ovarian cancer, FIGO stages III-IV, with a sensitivity of 70% and a specificity of 83%.

Conclusion

We identified a group of platelet protein biomarker candidates that can quantify the differential expression between ovarian cancer cases as compared to benign adnexal lesions.

Keywords

Ovarian cancerPlatelet proteomeBiomarkerLiquid biopsy

Background

Epithelial ovarian cancer is characterised by an asymptomatic growth in the abdominal cavity. In 75% of all cases, it is only detected at an advanced stage. The 5-year survival rate in FIGO stages I-II is over 90%, compared to around 30% in stages III-IV [1, 2]. The sensitivity of the CA-125 tumor marker for the detection of non-advanced epithelial ovarian cancer ranges from 50% to 70%, and this parameter alone is not recommended for differentiating between a benign and a malignant adnexal mass [3]. An expertly conducted transvaginal sonography (TVS) is a primary method for evaluation of ovarian and pelvic tumors, as it is able to discriminate between benign and malignant conditions with 90% sensitivity and 94% specificity using the International Ovarian Tumor Analysis (IOTA) pattern recognition [4, 5]. Despite high performance of TVS, the assessment will be inconclusive in around 8% of cases, also in the hands of an expert examiner [4]. Commonly used tumor markers, such as CA-125, HE4 and the Risk of Malignancy Index (RMI), did not improve, but rather deteriorated assessment in these difficult to classify tumors [6].

A several-fold increase in a patient’s platelet count is a common observation in cancer. A high preoperative platelet count is associated with early relapse in non-advanced epithelial ovarian cancer [7] and colorectal cancer [8]. Platelets influence angiogenic and immunological processes in cancer [9, 10], as well as directly protect tumor cells [7]. Proteomic analysis of platelets has identified several potential cancer markers [11]. Among them are angiogenic factors that have been shown to be sequestered by platelets [12] and delivered to the site of activated endothelium within an early tumor [11]. Detection of platelet-derived growth factor, platelet factor 4 (PF-4) and platelet-derived endothelial cell growth factor was suggested for diagnosis of several cancers [13]. Upon platelet activation, PF-4, vascular endothelial growth factor (VEGF) and fibrinogen undergo significant spatial rearrangements, detectable only by super resolution stimulated emission depletion (STED) microscopy [14].

The proteome of platelets in ovarian cancer has not been previously studied. The hypothesis of the current study is that knowledge of quantitative alterations of proteins in platelets can become the basis for non-invasive diagnosis of ovarian cancer and evaluation of the malignant potential of adnexal lesions.

Methods

The study comprised three phases:
  1. 1.

    Platelet pellets were prospectively collected from patients with benign adnexal lesions and ovarian cancer. This clinical material was subjected to two-dimensional (2D) gel electrophoresis, statistical analysis of protein expression, and subsequently, mass spectrometry-aided identification of protein biomarker candidates.

     
  2. 2.

    Antibody identification and confirmation of protein identification by western blot.

     
  3. 3.

    Verification of our biomarker candidate protein panel using western blot and DigiWest, and evaluation of sensitivity and specificity for detecting ovarian cancer.

     

Clinical material

Blood samples were obtained from volunteering women with adnexal lesions and suspected ovarian cancer. Approval for the study was given by the local ethical committee of Stockholm County Council, Dnr 2010/504–31. Cases were coded as “TR” followed by a number, and the coding was saved separately from the personal information of patients. Clinical material from 114 patients was prospectively collected between 2011 and 2014 at the Department of Obstetrics and Gynaecology, Karolinska University Hospital-Solna, Stockholm, Sweden (Table 1, Additional file 1: Table S1). Peripheral venous blood was drawn from the antecubital vein of each subject. The requirement for inclusion in the study was collection of a blood sample prior to any invasive diagnostic or treatment procedures. Patients with another known active cancer were excluded from the study. Patient records included age at diagnosis, co-morbidities, medication with coagulation and platelet aggregation blockers, optionally - TVS according to IOTA criteria, and post-operative histopathological conclusion. Randomization of the material was performed prior to experimental procedures.
Table 1

Description of the clinical material

Diagnosis and International classification of disease (ICD) coding

Benign lesions

 

Epithelial ovarian cancer, C56.9

n

stages I-II

n

stages III-IV

n

Serous ovarian cyst, N82.3

19

serous

2

serous

47

Ovarian fibrom, N82.3

10

mucinous

1

endometrioid

1

Dermoid ovarian cyst, N82.3

5

endometrioid

2

clear-cell

1

Endometriosis cyst, N80.1

8

clear-cell

3

  

Mucinous ovarian cyst, N83.2

10

    

Non-cancer ascites, R18.9

1

    

Paratubar cyst, Q50.5

2

    

Uterine myom, D25.9

2

    

Total

57

 

8

 

49

Transvaginal sonography, IOTA classification

Ultrasound assessment

Certainty in the assessment

Histopathology

n

Benign

certainly benign

 

benign

7

Benign

probably benign

 

benign

16

Benign

uncertain

 

benign

6

Borderline tumor

uncertain

 

benign

4

Malignant

probably malignant

 

benign

2

Malignant

certainly malignant

 

malignant

1a

Malignant

certainly malignant

 

malignant

13b

no IOTA-based examination

   

benign

22

no IOTA-based examination

   

malignant

7a

no IOTA-based examination

   

malignant

36b

Total

    

114

  

Medication:

   

Comorbidities

n

coagulation/aggregation blockers

n

None

99

 

none

 

111

One or few following diseases:

15

 

Warfarin

 

1

 Breast cancer remission

4

 

Dabigatran

1

 Cardiovascular

14

 

Aspirin

 

1

 Rheumatic

2

 

Total

 

114

 Endocrine

4

 

Experimental setup, n

 

 Astma

2

Method/Statistics

Benign

Ovarian cancer, stage

 Hepatitis C

1

 

lesions

I-II

III-IV

Total

114

2D/PCA

28

8

32

 

2D/PLS-DA

25

8

30

 

Western blot/PLS-DA

20

8

20

 

DigiWest/PLS-DA

29

0

30

astages I-II

bstages III-IV

Isolation of platelets from peripheral blood

Peripheral venous blood was drawn into 4.5 ml vacutainer plastic whole blood collection tubes with spray-coated K2EDTA (Vacutainer, BD, Franklin Lakes, NJ, USA) and processed within 30 min. Isolation of platelets was performed by three centrifugation steps. Exclusion of erythrocytes and leucocytes was achieved by centrifugation of whole blood at 1500 relative centrifugal force (RCF) for 10 min at +4 °C, and the platelet-rich plasma obtained was subjected to a second centrifugation at 3000 RCF for 10 min at +4 °C [15]. The resulting platelet pellet was re-suspended in 500 μL 0.9% NaCl and centrifuged at 3000 RCF for 10 min at +6 °C. The quality and purity of the platelet isolation was confirmed by light microscopy after immunostaining with an antibody against CD61 (119,992, Abcam, Cambridge, UK). In addition, fluorescence-activated cell sorting (FACS) of selected cases using the antibody against p-selectin, or CD62 (348,107, BD Biosciences-Europe) was also used to assess quality and purity. Platelet pellets were aliquoted to avoid excess freeze-thaw cycles and stored at −70 °C.

Two-dimensional gel electrophoresis

The platelet fractions for 2D gel electrophoresis were lyophilized and resuspended in lysis buffer; the protein concentration was determined using the Bradford protein analysis protocol [16] and the Versa Max Microplate reader (Molecular Devices, Sunnyvale CA, USA). Samples of 75.0 μg protein were subjected to 2D gel electrophoresis as previously described [17]. One 2D gel per clinical case was included into analysis after evaluation of the gel quality, i.e., absence of protein degradation protein spot smearing or overstraining. The expression level of protein spots in 2D gels was analysed using Progenesis SameSpot software (Nonlinear Dynamics, London, UK). The cut-off for selection of protein spots was a relative expression difference of 1.5-fold, p < 0.05, power > 0.8, q < 0.05, as evaluated by analysis of variance (ANOVA) by the SameSpot software.

Search parameters and acceptance criteria for MS/MS and peptide mass fingerprint (PMF)

Protein spots selected for identification were excised from the gels, treated for in-gel digestion, and subjected to MALDI TOF mass spectrometry carried out on the Ultraflex III TOF/TOF (Bruker Daltonics, Bremen, Germany). Peptide spectra were internally calibrated using trypsin autolytic peptides. A peak list generating software, Data Analysis 3.2 (Bruker Daltonics, Bremen, Germany), was used. In selected cases, MS/MS was performed with acceptance score exceeding 30. Mass tolerance for fragment ions was 0.5 Da. Based on the obtained peptide spectra, identification of the proteins was performed using the MASCOT (Matrix Science, London, England) and the “NCBInr” database. The deviation of mass did not exceed 0.05 Da. Probability of identification was evaluated according to score value, sequence coverage, and matched peptides. All other steps were performed as previously described [17].

Western blot analysis

A semi-quantitative dual label fluorescent detection western blot analysis was performed using the same patient cases that were subjected to 2D gel electrophoresis (Table 1). Samples were diluted in 4× lithium dodecyl sulfate (LDS) buffer and 10× reducing agent to a concentration of 1 μg/μl, and then incubated for 10 min at 70 °C. Four NuPAGE 4–12% Bis-Tris Gel, 1.0 mm × 15 wells were run in parallel; a total of 10 μg protein per case was loaded. Gel running conditions were as follows: 200 V for 60 min in XCell SureLock Mini-Cell EI0001 (Life Technologies, Stockholm, Sweden), followed by incubation of gels in transfer buffer containing 10% methanol and blotting to a nitrocellulose membrane for 1 h at 30 V (BioRad Power Pac 100 and Hoefer EPS 2A200). Membranes were incubated with Odyssey blocking buffer (Li-Cor Biosciences, East Chesterton Ward, UK) with addition of 0.1% Tween 20 for one hour, and then with the combination of commercially available primary antibodies, GAPDH and anti-14-3-3-gamma for loading controls [18] (Additional file 1: Table S2) with rotation at +4 °C overnight. After washing with PBS, each membrane was incubated with secondary antibodies (IRDye 2nd Ab Goat anti-Rabbit 680, 1:10,000 and IRDye 2nd Ab Goat anti-Mouse 800, 1:15,000, Li-Cor Biosciences, East Chesterton Ward, UK) at room temperature for 1 h. After washing with PBS, each membrane was scanned using the Odyssey SA Infrared Imaging System (Li-Cor Biosciences, East Chesterton Ward, UK).

DigiWest analysis

For each platelet lysate, two technical repeats (20 μg protein per lane) were subjected to SDS-PAGE using 4–12% Bis-Tris gradient gels. After blotting with primary antibodies (Additional file 1: Table S2) and biotinylation of the proteins, individual sample lanes were cut into 96 molecular weight fractions (0.5 mm each) and proteins were eluted. Eluted proteins from each molecular weight fraction were loaded onto color-coded neutravidin-coated Luminex bead sets (MagPlex, Luminex, Austin TX, USA), and the antibody specific signals were analysed (DigiWest analysis, version 3.8.5.2, Excel-based) [19].

Multivariate methods (PCA, PLS-DA, OPLS-DA)

Principal components analysis (PCA) [20] is the most widely used (non-supervised) multivariate method, and the root to most others. By finding covariance between multiple (correlated) variables in a single dataset X, it sequentially defines (uncorrelated, or orthogonal) principal components, with decreasing amount of variance explained, built from weighted initial variables. A number of interesting components can be selected, as variation in the dataset is reframed as structured (e.g. biological information) or residual (noise). Each component defines a percentage of variation of the original dataset, and is described by two vectors: scores, representing the score of each sample in the newly created principal component; loadings, representing the weight of each initial variable in the principal component. PCA performs dimensionality reduction, thus allowing one to condense most of the information in a large dataset into a small number of components. Its scores and loadings can be plotted and used as an exploratory technique, to find relations between variables, detect groups and trends in the samples, as well as outliers.

Projection to latent structures (PLS) is a well established (supervised) method for the analysis of complex multivariate datasets, as found in the –omics fields, including proteomics [21, 22]. In originates from partial least squares regression, a multivariate analysis method which relates two matrices (X with the actual data or independent variables, and Y/y as responses or dependent variables) by maximizing the covariance of their latent variables. The 2-class PLS-DA is simply a particular type of PLS in which the dependent variable is a “dummy” binary (0/1) class y-vector. PLS-DA targets complementary objectives: as a discriminant method, it allows discrimination/prediction of class for test samples; as a multivariate method it shows the relationship among variables in the dataset through the creation of latent variables, built using weighted original variables; as a linear method, it allows one to visualize and understand which variables in the data are more relevant for the class discrimination.

Orthogonal PLS (OPLS) [23] is a modification of the PLS method, and while both models have the same model statistics and prediction capability, OPLS allows for easier interpretation of the relevant variables than PLS. The reason is because apart from dividing variation into systematic and residual (as PLS), OPLS also divides the systematic variation into predictive (related to the phenomenon in study) and orthogonal (structured variation related to other factors, such as age and gender). This property is advantageous when interpretation of the results, rather than prediction, is the main objective of the analysis. In the context of OPLS-DA the statistically significant predictive loadings show which initial variables are important in the discrimination of class, and in which class these variables have higher values.

(O)PLS-DA number of latent variables and validation [24]

(O)PLS-DA models can separate data variability into systematic and random, and use the systematic one for building the actual model while discarding the random variation, or noise. In order to attain that objective, it is critical to select an appropriate number of latent variables for the model, which is achieved in general through the use sampling methods such as cross-validation (CV). In this strategy, one calculates multiple (O)PLS-DA models using subsets of the X data, while predicting the class (y) of the samples left out in each round. By doing this for models with different number of latent variables, one can evaluate how well predicted by the model are the samples that were left out in each of those models, and select the best one. It yields the following cross-validation’s statistics: R2X- fraction of the variance in X explained by each latent variable; R2- fraction of variance of y (class) explained by each latent variable; and most importantly Q2- the fraction of variance of y predicted by the model. A model with good modelling and predictive power is desired, and while R2X may be low (due to low number of variables in X explaining class discrimination), R2Y and Q2 should be the closest possible to each other, as well as close to a maximum of 1.

After a model is built, containing the appropriate number of latent variables, it undergoes appropriate validation procedures, notably cross-validation analysis of variance (CV-ANOVA), permutation test, and evaluation of cross-validation scores. CV-ANOVA compares the y predicted residuals of the model of interest with the variation around the global average using an F-test, resulting in a significance p-value for decisional purposes. Permutation test evaluates the statistical significance of estimated predictive power, by comparing R2 and Q2 of multiple models (where y was randomized) with R2 and Q2 of the actual model of interest. If the model is significant, its values are expected to be higher than for the y-randomized models. Cross-validation scores are scores that are calculated for each sample during cross validation, and can be visually or systematically evaluated to indicate over-fitting in case they differ from the regular scores.

Prediction of test samples and variable relevance for class discrimination

After proper choice of number of latent variables and method validation, the model can be used for prediction of test samples, and to find out which variables are relevant for class discrimination.

As class membership is defined by the 0/1 dependent variable y vector in the training set, class membership of “new” samples in a test set is dependent on their predicted y value. Samples similar to class “1” are expected to yield values of y similar to 1, thus above a certain threshold, e.g. 0.5, while samples similar to the ones in class “0” will be predicted below that threshold.

To find out which variables are relevant for class discrimination, two methods are commonly used: the variable importance on the projection (VIP) method, which is an (unsigned) compact parameter to summarize the importance of each of the variables in PLS-type models with more than one latent variable; the other method can be used with OPLS-type models and checks if the confidence interval for the mean of the CV-calculated loadings crosses zero or not (in which case it is consistently positive or negative for each of the cross validation models).

PCA, PLS-DA and OPLS-DA were performed using SIMCA P v13.0 software (Umetrics AB).

Experimental design and statistical rationale

Normalized expression values exported from SameSpot software of all protein spots (approximately 2000 spots in one 2D gel) were subjected to PCA analysis and PLS-DA.

PCA analysis was performed using the material from patients with benign adnexal lesions (28 cases), ovarian cancer, International Federation of Gynaecology and Obstetrics (FIGO) stages I-II (8 cases), and ovarian cancer, FIGO stages III-IV (32 cases) (Table 1). The PLS-DA model was based on the expression of all platelet protein spots in 2D gels in benign ovarian lesions (16 cases) and ovarian cancer, FIGO stages III-IV (20 cases). The predictive ability of the model was tested using 8 cases of the ovarian cancer, FIGO stages I-II, 9 cases of benign adnexal lesions and 10 cases of the ovarian cancer, FIGO stages III-IV. Protein spots selected by PLS-DA were ranked as variables of importance in the projection (VIP) by the strength of their input into the model. An analysis of the influence of co-morbidities and the intake of coagulation and platelet aggregation blockers was performed.

Western blot analysis for antibody identification and confirmation of protein identification was performed using 20 cases of benign adnexal lesions, 20 cases of ovarian cancer, FIGO stages III-IV and 8 cases of stages I-II (Table 1). The same cases were used for 2D electrophoresis (Additional file 1: Table S1). For protein panel verification, all analysed samples were randomized and distributed to four sets of gel separation batches. Each batch produced four equal gels, each of which, after transferring, was incubated with four different mixtures of primary antibodies against 16 proteins (Additional file 1: Table S2) for a total of 16 gels per data set. This experiment was repeated three times (sets 1–3). Protein expression levels were subjected to OPLS-DA, logistic regression and receiver-operator curve (ROC) analysis (MedCalc Software, Ostend, Belgium).

Verification using DigiWest analysis [19] was performed on an independent set of prospectively collected randomized platelet samples from patients with ovarian cancer, FIGO stages III-IV (n = 30), and benign adnexal control lesions (n = 29) (Table 1, Additional file 1: Table S1). Statistical analysis was OPLS-DA based.

Results

Cancer-related expression of platelet proteins in 2D gels

Each 2D gel contained approximately 2000 protein spots (Fig. 1a). Initial PCA (Fig. 1c) of expression levels of significantly differentially expressed protein spots in 2D gels indicated a degree of separation between the three analysed patient groups: benign adnexal lesions, ovarian cancer, FIGO stages I-II and FIGO stages III-IV. PCA scores showing these groups are presented in Fig. 1c.
Figure 1
Fig. 1

Proteomics-based analysis of platelet proteins was based on the separation of proteins according to mass (Mr, kDa) and charge (pI) by 2D gel electrophoresis with further analysis of the expression of protein spots for marker identification. a 2D gel electrophoresis diagram of platelet proteins. Circles and numbers indicate the identified biomarkers. b The PLS-DA-based cross-validated model based on the partial least squares discriminate analysis of 2D gels for benign adnexal lesions (white circle) and ovarian cancer, FIGO stage III-IV (black circle) in accordance to the expression of all protein spots in the gel. c Principal component analysis (PCA) showing separation of the generated 2D gels for benign adnexal lesions (white circle), ovarian cancer, FIGO stage I-II (triangle) and FIGO stage III-IV (black circle) in accordance to the expression of selected biomarkers; percentage of variance X explained by the two PCA components shown

The PLS-DA cross-validated model based on the expression of all protein spots in 2D gels of benign adnexal lesions and ovarian cancer (FIGO stages III-IV) presented 96% sensitivity and 88% specificity for discrimination between these groups (Table 2a, Fig. 1b). The predictive ability of the built PLS-DA model was tested. We observed that all 8 cases ovarian cancer, FIGO stages I-II were recognised as cancer (1.00 sensitivity), and also 8 of 10 cases of ovarian cancer, FIGO stages III-IV (80%). Four out of 9 cases of benign adnexal lesions were recognised as benign (0.44 specificity) (Table 2a). Forty-three protein variables with VIP > 2 were selected for protein identification.
Table 2

Statistics for PLS-DA, OPLS-DA model, details for predictive and orthogonal parts; ROC analysis

A) PLS-DA-based analysis of protein spots expression in 2D

 Component

Latent variables

R2X (cum)

Q2 (cum)

CV-ANOVA, p-value

permutation test, p-value

  

Sensitivity

Specificity

 Model

3

0.318

0.72

8,5 * 10–9

<0.001

 

calibration set

0.96

0.88

       

validation set

1.00

0.44

B) OPLS-DA-based analysis of protein expression in western blot.

 Component

Latent variables

R2X (cum)

R2 (cum)

Q2 (cum)

CV-ANOVA, p-value

permutation test, p-value

 

Sensitivity

Specificity

 Model

1 + 1

0.203

0.632

0.477

4.41E-14

<0.001

calibration set

0.83

0.89

 Predictive

1

0.0717

0.632

0.477

  

validation set

0.88

not tested

 Orthogonal

1

0.131

0

      

C) ROC analysis of protein expression in western blot.

 Compared groups

AUC

standart deviation

95% confidence interval

z statistics

p-value

  

Sensitivity

Specificity

 ROC 1

0.777

0.0418

0,695 to 0,859

6.639

<0,0001

 

ROC1

60

83.33

 ROC 2

0.831

0.0501

0,733 to 0,930

6.615

<0,0001

 

ROC2

83.33

76.19

D) OPLS-DA-based analysis of protein expression in Digi west.

 Component

Latent variables

R2X (cum)

R2 (cum)

Q2 (cum)

CV-ANOVA, p-value

permutation test, p-value

 

Sensitivity

Specificity

 Model

1 + 2

0.24

0.785

0.345

4.50E-03

<0.001

test set

0.7

0.83

 Predictive

1

0.037

0.785

0.345

     

 Orthogonal

2

0.203

       

R2X cumulative percentage of X variance explained, R2 cumulative percentage of Y variance explained, Q2 cumulative percentage of variance of Y predicted, CV-ANOVA p-value p-value of cross-validation ANOVA, permutaion test p-value p-value of (1000 iterations) permutation test

Co-morbidities and use of of coagulation and platelet aggregation blockers did not influence the predictive ability of the model. The PLS-DA model suggested a list of protein spots for protein identification as potential biomarkers, which could be evaluated from the VIP plot.

Identification of platelet protein biomarkers and confirmation of identification

A total of 35 proteins were identified in 37 protein spots excised from 2D gels (Table 3).
Table 3

Protein identification

Protein spot number on 2D

Gene ontology name

Protein name

theoretical

experimental

NCBI accession number

Sequence coverage, %

Matched peptides

Total peptide count

score

msms

pI

Mr, kDa

pI

Mr, kDa

1

2

3

4

5

6

7

8

9

10

11

12

13

285

ACTN1

Alpha-actinin-1 isoform b

5.2

104

5.5

100

NP_001093

25

19

75

98

1836

ACTN4

Alpha actinin 4, partial

5.2

74

5.3

72

AAH15620

14

9

30

68

1153

ACTB

beta actin variant, partial

5.4

42

6.2

40

BAD96752.1

31

9

48

71

1803

ACTB

actin beta

5.2

39

5.3

42

BAG62914.1

30

10

54

81

1772

AP4A

Bis(5′-nucleosyl)- tetraphosphatase

5.1

17

5.4

17

NP_001152

27

5

36

52

1

1274

CAPZA1

capping actin protein of muscle Z-line alpha subunit 1

6

32

5.4

40

XP_005542394.1

54

11

60

106

911

CD41

Integrin alpha-IIb precursor variant

5.7

60

5.4

60

BAD92789.1

46

1

276

CD41

Integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41), isoform CRA_a

5.4

104

5.3

110

EAW51594.1

24

17

49

122

1761

CD42A

glycoprotein IX platelet

5.4

18

5.4

20

AAH30229

27

5

43

53

2

792

CD61

Platelet glycoprotein IIIa, partial

5

87

5.6

70

AAA52600

15

1

942

CNDP2

Cytosolic non-specific dipeptidase

5.6

53

6

55

BAG53426

22

8

48

51

3

1539

CRKL

Crk-lke protein

6.3

34

6.5

26

NP_005198

29

9

56

64

1

1525

ERP29

Erp28, endoplasmic reticulum resident protein 29 isoform 1 precursor

6.8

29

6.5

27

NP_006808

28

6

68

44

2

1854

FBG

Fibrinogen gamma chain

5.5

48

5.6

50

EAX04921

40

13

53

113

397

GELS

Gelsolin isoform a precursor (782 aa)

5.9

86

5.4

95

NP_000168

61

1

401

GELS

Gelsolin isoform b (731 aa)

5.6

81

5.7

90

NP_937895

12

9

50

45

2

311

HSPA9

Stress-70 potein, mitochondrial precursor

5.8

74

5.7

120

NP_001126860

27

17

66

100

3

1155

HP

Haptoglobin

6.3

38

5.4

42

AAI07588.1

25

10

61

71

482

HSPA8

Heat shock 70 kDa protein 1A/1B

5.4

70

5.7

80

P08107

13

6

56

29

1115

ILEU

Leukocyte elastase inhibitor

5.9

43

6.3

43

NP_109591

21

7

43

72

1177

MPST

3-mercaptopyruvate sulfurtransferase

6.8

35

6.4

37

BAD92061.1

33

8

39

75

1231

NSFL1C

NSFL1 cofactor p47 isoform X8

5

31

6.2

38

XP_011527607.1

25

5

46

38

1

1850

PGM1

phosphoglucomutase-1 isoform 1

6.3

62

6.8

70

NP_002624.2

11

5

32

33

1464

PHB

Prohibitin

5.5

28

5.7

30

XP_003912755.3

49

12

46

136

1054

RNH1

ribonuclease/angiogenin inhibitor 1

4.8

50

4.8

50

AAH11186.1

11

5

25

45

999

PRKAR1A

protein kinase cAMP-dependent type I regulatory subunit alpha

5.2

43

5.5

53

XP_004041145.1

35

13

72

102

 

939

PRKAR2B

protein kinase cAMP-dependent type II regulatory subunit beta

5

45

4.8

55

BAG54705.1

19

6

27

41

2

1175

SRB6

Serpin B6

4.2

39

5.6

40

XP_011512978.1

34

10

45

76

1821

SRC

Protooncogene tyrosine-protein kinase Src

7.1

60

6.6

60

NP_005408.1

13

8

54

39

1801

TUBA4A

Tubulin alpha-4A chain

4.9

49

5.1

55

NP_001265481.1

23

10

56

73

1801

TUBA1C

tubulin alpha 1c

4.9

59

5.1

55

BAH11541.1

23

10

56

68

 

758

TLN1

Talin 1

5.8

272

5.9

70

AAF27330

7

13

43

42

1

755

TLN1

Talin 1

5.8

272

5.8

70

AAF27330

9

15

46

54

292

TLN1

Talin 1

5.8

272

5.8

110

AAF27330

14

33

84

106

774

TLN1

Talin 1

5.8

272

6.4

70

AAF27330

25

1

1409

TLN1

Talin 1

5.8

272

6.4

34

AAF23322.1

6

15

39

47

1640

TPM1

Tropomyosin 1, alpha

5

20

4.7

20

XP_005254707

23

6

64

30

1894

TUBA6

Tubulin alpha-6 chain, partial

5

47

5.3

56

EHH66249.1

34

10

56

80

1894

TUBA8

tubulin alpha-8

5

43

5.3

56

XP_007469555.1

33

10

56

82

 

1894

TUBA1C

tubulin alpha-1C chain

5

47

5.3

56

XP_004646666.1

35

10

56

81

 

999

TUBB1

tubulin beta-1 chain isoform X7

5.1

43

5.5

53

XP_018872841.1

42

12

72

93

1850

WDR1

WD repeat-containing protein 1 iso 1

6.4

59

6.8

70

AAD05045

35

18

60

135

1 - number of a protein spot on 2-D gel,

2 - gene ontology name,

3 - protein name,

4 - isoelectric point of a protein spots according to the position on 2D gel,

5 - protein mass of a protein according to the position of a spot on 2D gel,

6 - isoelectric point of a protein spot as provided by Mascot search database,

7 - protein mass as provided by Mascot search database,

8 - protein accession number according to NCBI,

9 - matching of the experimental peptide sequence to a peptide sequence provided by NCBI,

10 - number of identified peptides that matched to the peptides of a peptide sequence provided by NCBI,

11 - total amount of peptides in a peptide sequence provided by NCBI,

12 - score provided by Mascot search engine,

13 - performed msms for the certainty of protein identification.

A number of proteins were excluded due to low quality and/or low expression level that made identification by mass spectrometry unfeasible. Visual representation of the location of the identified spots may be found in Fig. 1a.

Confirmation of the protein identification was performed by western blot analysis of the samples used for 2D electrophoresis. Antibodies against 16 proteins were analysed. Protein identities with the highest VIP < 20 were prioritised for analysis. Antibodies were selected based on the qualitative and quantitative recognition of protein bands, and protein detection was confirmed using positive control cell lines (Additional file 1: Table S2). Initially, we analysed several antibodies against each protein and identified useful antibodies for detection of biomarkers from the 2D gels (Fig. 2).
Figure 2
Fig. 2

Western blot analysis. a Confirmation of protein identification by western blot using antibodies against 15 selected proteins, and normalisation against14–3-3-gamma (loading control). b Western blot detection of ERP29. This example compares Mr/pI from 2D gel and Western blot where the mouse anti-ERP29 detects protein spot #1525, while the rabbit anti-ERP29 detects an additional protein spot, possibly an isoform of ERP29

Several isoforms of the identified proteins were detected by western blot. ERP29 was detected in both 2D and western blot at around 29 kDa as a double band, however only the upper band corresponded to protein spot #1525 from the 2D analysis. The identified isoform, with NCBI accession number NP-006808, had both experimental and theoretical pI of around 6.5. The particular band could be singled out by a mouse monoclonal antibody (83,073, Abcam, Cambridge, UK) and was confirmed by 2D Western blot (Fig. 2b). CRKL was located at 25 kDa on 2D and in western blot the correct isoform was identified among four detected bands. Finally, both the 60 kDa and 53 kDa isoforms of SRC were recognised as part of the biomarker panel.

Verification of platelet biomarkers by western blot and DigiWest data

Expression of proteins in western blot was normalized against the levels of 14–3-3-gamma, which is a more stable and relevant loading control for platelet proteins compared to GAPDH [18]. PCA was performed individually in each set of blotted membranes in order to look for major trends and grouping of the samples by observing the scores plots. These represented three repetitions of the experiment, with four groups per experiment. The PCA scores plots showed consistent separation between benign adnexal lesions from the cases of ovarian cancer, FIGO stages III-IV, as it was observed in 10 of the 12 PCA’s scores plots.

There was nonetheless some intrinsic batch variability, which could potentially be explained primarily by differences in the gels prepared for each batch, uneven protein transfer from gel to membrane, and suboptimal quantification of the protein bands expressed. Due to such variability, data were centred per batch previous to OPLS-DA modeling. After proper validation of the OPLS-DA model as described in the statistical section, results of the verification by western blot revealed a sensitivity of 83% (50/60) and a specificity of 89% (53/60) for separation between benign adnexal lesions and ovarian cancer, FIGO stages III-IV (Fig. 3a, Table 2b).
Figure 3
Fig. 3

Statistical analysis of the protein expression levels. a Cross-validated model built upon protein expression in benign and ovarian cancer, FIGO stage III-IV – western blot data, b Test model detecting the cases of ovarian cancer, FIGO stage I-II – western blot data, c Cross-validated model built upon protein expression in benign and of ovarian cancer, FIGO stage III-IV – DigiWest data, d Relative contribution of variables within the model on the separation between benign adnexal lesions and ovarian cancer, FIGO stage III-IV – DigiWest data

Next, model was tested for its predictive capacity. The correct prediction of two out of three samples per case was set as an acceptance criterion. Seven out of eight cases of ovarian cancer, FIGO stages I-II were predicted correctly (88% sensitivity). Sample 30 was reclassified by histology as benign and the prediction model confirmed this reclassification (Fig. 3b). Nine protein variables were suggested as a biomarker panel. In ovarian cancer, FIGO stages III-IV, seven protein variables were up-regulated and two variables were down-regulated. The ROC analysis of the detected nine-variable panel suggested discrimination between benign adnexal lesions and ovarian cancer, FIGO stages III-IV with a sensitivity of 60% and a specificity of 83% (Table 2c – ROC1) and between benign adnexal lesions and ovarian cancer, FIGO stages I-II with a sensitivity of 83% and a specificity of 76% (Table 2c – ROC2).

Next, the panel was verified by DigiWest with inclusion of a new, larger set of samples that were not previously used for 2D gel electrophoresis and western blot analysis. The expression profiles were analysed by OPLS-DA and the DigiWest based prediction model suggested a sensitivity of 70% (21/30) and a specificity of 83% (24/29) for the separation between benign adnexal lesions and ovarian cancer, FIGO stages III-IV (Fig. 3c, Table 2d). The verification of biomarker candidates by DigiWest suggested seven protein variables as seen in the loadings plot in Fig. 3d; three protein variables were increased in ovarian cancer, FIGO stages III-IV and four were decreased. We observed a correspondence between western blot and DigiWest analysis regarding the potential value of six identified proteins as biomarkers, of which three were increased in ovarian cancer, FIGO stages III-IV and three were decreased in both western blot and DigiWest.

Discussion

In this study, we analysed the proteome of platelets isolated from peripheral blood of patients with ovarian cancer, FIGO stages I-II and III-IV, and benign adnexal lesions. By PLS-DA modelling of protein expression in 2D gels we observed the possibility to correctly differentiate between cases of benign adnexal lesions and ovarian cancer, FIGO stages III-IV, and correctly predict cases of FIGO stages I-II. These findings were verified by western blot and DigiWest.

In the prospectively collected clinical material, there were 57 cases of benign adnexal lesions, 49 cases of ovarian cancer, FIGO stages III-IV, and 8 cases of ovarian cancer, FIGO stages I-II. Overrepresentation of advanced ovarian cancer patients (80% of all cases) in our study reflects the worldwide situation regarding the detection rate of the disease at its advanced stages [1]. The initial PCA provided an indication of degree of correspondence between protein profiles of the compared groups. PLS-DA allowed us to build a multivariate regression model. Through PLS-DA it became possible to 1) “filter” the high level of biological background noise by using only the latent variables related to the biological variation of interest, 2) build a training model and use it to predict the class of earlier cancer test samples, and 3) identify the most discriminant variables within the multiplex profile by observing their variable importance in projection (VIP), i.e. ranking in the model [2224].

Our proteome analysis was based on 2D gel electrophoresis, which made it possible to identify full-length proteins and also their isoforms with regards to their relevance for the protein marker panel. The verification of identified platelet proteins suggested the possibility for separating benign adnexal lesions, ovarian cancer, FIGO stages I-II and III-IV. However, verification by western blot has inherently several disadvantages, such as gel batch variability, protein transfer limitations, and semi-quantitive output. DigiWest, a recently developed system that represents a high-throughput multiplex version of the classical western blot method, uses a bead-based microarray platform for signal generation [19]. The sensitivity, signal linearity, and reproducibility of DigiWest are comparable to or exceed the best available western blot systems. Compared to the western blot method, DigiWest provides higher reproducibility with simultaneous analysis of up to 100 samples and quantification of individual cases.

The function of the 16 identified and immuno-validated platelet proteins was described using the Platelet Web database [25] (Additional file 1: Table S3). Most of these proteins are reported to have a functional relevance to ovarian cancer, normal platelet biology and platelet-associated pathological conditions (Table 4). Published proteomics and molecular biology studies suggest up-regulation of ACTN4, CRKL, GELS, HSPA8, talin-1, tubulins and WDR1 in ovarian cancer. Expression changes and possible functional impact of ACTN1 [26], MPST [27], ERP29 [28] and SERPINB6 [29] are reported for several cancers, but not for ovarian cancer. PHB, SRC, talin-1, tubulins and WDR1 are related to development of ovarian cancer resistance to paclitaxel [30, 31] and cis-platin [3234]. An interesting observation is an increased expression level of CD41/CD61 in platelets associated with tumor xenografts as well as enhanced tumor growth in the presence of platelets [35].
Table 4

Function of proteins biomarkers in relation to ovarian cancer and platelets

Protein name

Associated pathologic conditions

ovarian cancer

platelet disorders

ACTN1

not studied

macrotrombocytopenia [37, 38]

ACTN4

↑ in ovarian cancer [39]

myelodysplastic syndrome [38, 40]

CD41/CD61

platelets contribute to ovarian cancer growth [35]

deficient in Glanzmann thrombasthenia type II [41]

CRKL

↑ in ovarian cancer [42]

ST - elevated myocadial infarction [43]

ERP29

not studied

mediator of thrombus formation [44]

GELS

↑ in serum in ovarian cancer [45]

↑ in megakaryoblastic leukemia [46], ↑ in thrombin-activated platelets [47]

HSPA8

↑ in ovarian cancer, a potential therapy target [48]

mediator of thromboembolism [49]

MPST

not studied

not clarified

PHB

↑ in paclitaxel-resistant ovarian cancer [30] ↓ in platinum-resistant ovarian cancer [32]

mediator of platelet aggregation [50]

SRC

therapy-target for thyrosine-kinase inhibitor in ovarian cancer [51], resistance to cis-platin [33]

ST - elevated myocadial infarction [43]

SERPINB6

not studied

inhibitor of thrombin [25]

TLN1

↑ in ovarian cancer [52], resistance to cis-platin [33]

myelodysplastic syndrome [40]

TUBB1, TUBA4

↑ in ovarian cancer, mediator of paclitaxel resistance [31]

↑ in thrombin-activated platelets [47], gene mutation in autosomal dominant macrothrombocytopenia [25]

WDR1

↑ in ovarian cancer [53], resistance to cis-platin [34]

mediator of TLN1-induced activation of CD41/CD61 [54]

↑ corresponds up-regulation, ↓ corresponds down-regulation

Using the STRING database [36], we analyzed potential interactions and functional impact of the identified platelet proteins on other known protein targets (Additional file 1: Table S4, Fig. 4). Most described protein interactions are related to CRKL, SRC, CD61, GELS (GSN), and ACTN1, while information is limited regarding the interaction profile of MPST, ERP29, HSPA8, and WDR1.
Figure 4
Fig. 4

Functional interaction profile of identified platelet proteins [36]. Lines represent interaction, where thick lines suggest a substantial number of references, thin lines correspond to single studies, arrows point the direction of activation influence, and block signs describe inhibitory actions

Limitations of the current study include dependency of sensitivity and specificity on the quantitative properties of the verification method and analysis of relatively small groups of clinical material. Multivariate PLS-DA-based statistics partially allowed us to overcome the problem of small clinical groups, however, further validation using clinical material from patients with early-stage ovarian cancer and borderline ovarian tumors is expected to strengthen the diagnostic sensitivity and specificity. Combination of the marker panel with the TVS protocol, which evaluates malignant potential of adnexal masses, towards a biochemical-instrumental test is the final goal. The objective diagnosis of adnexal lesions will help clinicians to correctly refer patients to a gynaecological cancer specialist, thus minimizing the cost of additional examinations and delays for patients needing advanced treatment. The panel detected is currently being developed as a new non-invasive in vitro diagnostic multivariate index-based analytical method for differentiation of adnexal lesions.

Conclusion

The identified platelet protein panel allows for differentiation between benign adnexal lesions and ovarian cancers of FIGO stages III-IV. Correct prediction of seven out of eight cases of ovarian cancer, FIGO stages I-II was possible through multivariate prediction modeling based on platelet protein expression profiles.

Abbreviations

2D: 

Two-dimensional

ANOVA: 

Analysis of variance

AUC: 

Area under the curve

CV: 

Cross validation

DA: 

Discriminant analysis

FIGO: 

International Federation of Gynaecology and Obstetrics

IOTA: 

International Ovarian Tumor Association.

LDS: 

Lithium dodecyl sulfate.

PCA: 

Principal components analysis.

PF-4: 

Platelet factor 4.

PLS: 

Projection to latent structures (or partial least squares).

PMF: 

Peptide mass fingerprint.

RCF: 

Relative centrifugal force.

ROC: 

Receiver-operator curve.

RMI: 

Risk of malignancy index.

STED: 

Stimulated emission depletion.

TVS: 

Transvaginal ultrasound.

VEGF: 

Vascular endothelial growth factor.

VIP: 

Variable importance in projection.

X: 

Data matrix (number of rows equal to number of samples, number of columns equal to number of variables).

Y: 

Column vector (with one element per sample).

Declarations

Acknowledgements

We acknowledge Teddy Primack and Michael Vanlandewijck for language assistance.

Funding

This work was supported by grants from the Cancer Research Foundations of Radiumhemmet, Stockholm Count County: HMT 20130704 and HMT 20140754, KI Fonder 2013fobi37433, Dagmar Ferbs Minnesfond 2012–2016, Vinnova 2014–03458 to NeoProteomics AB.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its Additional files.

Authors’ contributions

LM, FB, AG, WJ, EE planned the project, selected patients, interpreted results, LM and EE recruited patients for the study, BS, BC, GS performed preparation of the platelet fraction, 2D gel electrophoresis, western blot, EU performed mass-spectrometry, TM performed Digi west analysis, XL, BJ performed analysis of platelets using high-resolution microscopy, PR performed statistical analysis, LM performed ROC curve analysis, LM, FB, PR and AG were the major contributors in writing the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Approval for the study was given by the local ethical committee of Stockholm County Council, Dnr 2010/504–31. Written consent for participation and data presentation was received from the patients who volunteered to take part in the study.

Consent for publication

Not applicable.

Competing interests

Authors Gustafsson Sonja, Björklund Christina, Franzén Bo and Auer Gert are affiliated to NeoProteomics AB. Other authors declare no competing interests.

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Obstetrics and Gynaecology, Academical Uppsala University Hospital, Uppsala University, Uppsala, Sweden
(2)
Institute of Women’s and Children’s Health, Karolinska Institute, Stockholm, Sweden
(3)
Department of Oncology and Pathology, Cancer Centre Karolinska, Karolinska Institute, Stockholm, Sweden
(4)
Department of Epidemiology and Biostatistics, MRC-PHE Centre for Environment and Health, School of Public Health, Imperial College London, St. Mary’s Campus, London, UK
(5)
Ludwig Institute for Cancer Research Ltd, Uppsala, Sweden
(6)
NeoProteomics AB, Cancer Centre Karolinska, Stockholm, Sweden
(7)
NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany
(8)
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden
(9)
Department of Obstetrics and Gynaecology, Department of Clinical Science and Education, Stockholm, Sweden

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