Downregulation of C 3 and C 4 A B complement factor fragments in plasma from patients with squamous cell carcinoma of the penis
ARTICLE INFO _______________________________________________________________________________ IBJU DOWNREGULATION OF COMPLEMENT FACTOR FRAGMENTS IN PLASMA FROM PATIENTS WITH CARCINOMA OF THE PENIS 740 patients with penile carcinoma present inguinal involvement at diagnosis and physical examination is a not a reliable predictor or lymph nodes status (2) Therefore, reliable staging information can only be acquired through surgical procedures with subsequent histologic examination of the inguinal lymph nodes. The pathologic factors with known prognostic value, other than the presence of lymph node metastasis, include tumor thickness, grade, histologic type, lymphovascular embolization, presence of koilocytosis and stage (4 6) On the clinical nodal status there is a dilemma: a signifi cant number of patients with palpable lymph nodes do not have metastasis, in the other hand, 20% of patients with clinically non suspicious nodes present micrometastasis at pathological examination. Thus, prophylactic bilateral inguinal lymph node dissection is considered unnecessary in up to 80% of penile carcinoma patients with clinically negative regional lymph nodes (7) While DNA is the information archive, proteins do the work for the cell. It has been shown that direct gene expression is only responsible for a small part of the complexity of a living organism (8) Proteomics is the large scale study of proteins, and is associated traditionally with displaying a large number of proteins from a given cell line or organism. Curiously, there is no strict linear relationship between genes and the protein complement or 'proteome' of a cell. Proteomics is complementary to genomics, because it focuses on the gene products, which are the active agents in cells. There are two strategies for fi nding protein biomarkers in tissues or in biological fl uids. Several possible biomarkers have been identifi ed using a gel based approach in bi dimensional electrophoresis (with and without stable isotopic labeling) and mass spectrometry (9,10) However, since this approach is very laborious and time consuming, it is not practicable in clinical chemistry laboratories. Other methods based on the biological fl uid proteome prefractionation have been recently made available (11,12) The successful discovery of a proteomic profi le correlated to an altered state by the ClinProt method has been reported in various human diseases, such as oral, bladder, nasopharyngeal and neck cancer (13 15) Recently, we have used this approach to fi nd possible ccRCC biomarkers in urine and serum (12,16) In view of these results, we investigated the possibility of using the ClinProt technique to fi nd possible plasma diagnostic markers that can better distinguish healthy subjects from patients affected by SCCP. MATERIALD AND METHODS Chemicals The C8 Hydrophobic kit, a cyano 4hydroxycinnamic acid (HCCA) and Protmix1 were purchased from Bruker Daltonics, GmbH (Bremen, Germany) acetonitrile from Merck KGaA (Darmstadt, Germany) and methanol from Sigma Aldrich, Inc. (St. Louis, MO) Patients and Blood Sample Collection Twenty fi ve patients with penile cancer being treated by the National Cancer Institute and Mario Kroeff Hospital were enrolled in this study after permission of the hospital's ethic committee. In addition, we used blood samples from thirty six healthy subjects (blood donor volunteers) who underwent circumcision in Santa Veronica Hospital. Informed consent was obtained from all patients. After diagnosis of penile cancer, 5 ml of blood was collected before surgery and stored in a BD vacutainer with EDTA. About 2.5 ml of plasma was obtained after centrifugation at 1500 g for 10 min. within 4 hours of collection. The samples were frozen on dry ice and sent to Milano Bicocca University in Italy. All the samples were stored at 80 oC until being sent to Italy. The mean age of the patients and controls was 63.56 (range, 38 90) and 60 years (range, 2383) respectively. Follow up was evaluated in all patients. Median follow up was 9 months (range 1 to 36) The data collected from the patients' medical records are shown in Table 1. Due to the small number of patients, no stratifi cation according to risk factors for penile cancer was performed. Study Design The entire data set, composed of 36 controls and 25 penile cancer patients, was randomly split into two groups. The fi rst group (training data set: 28 controls and 17 penile cancer patients) was IBJU DOWNREGULATION OF COMPLEMENT FACTOR FRAGMENTS IN PLASMA FROM PATIENTS WITH CARCINOMA OF THE PENIS 741 used for the identifi cation of signals related to peptides expressed differentially in penile cancer patients compared with controls (pattern recognition) The second group (test data set: 8 controls and 8 penile cancer patients) was used for preliminary pattern validation of the cluster. Sample Purifi cation ClintProt was applied to analyze the plasma samples collected from SCCP patients. Healthy subjects and SCCP patients were randomly split into two groups for the training and the test experiments. Because of the high complexity of spectrum profi les, two algorithms were tested for biomarker discovery. After the basic statistical analysis, the two algorithms were used for the selection of a signal cluster that was able to differentiate patients from controls. Peptides were extracted from the plasma by ClintProt C8 magnetic beads according to the kit instructions. Plasma aliquots (40 mL each) were mixed with 5 mL of magnetic beads and 40 mL of the kit buffer, allowing peptides and proteins to bind to hydrophobic C8 surface of the magnetic micro particles. The supernatant was removed after 1 minute of incubation and the beads were washed twice with 45 mL and once with 15 mL of the recommend washing solution. Peptides were then eluted with 10 mL of 50% acetonitrile. The procedure was automatically carried out using the ClinProt robot (Bruker Daltonics, GmbH, Bremen, Germany) thus reducing variability. Mass Spectrometry Analysis Aliquots of eluted peptides (5 mL) were mixed with 10 mL of HCCA matrix solution (6.2 g/L HCCA in methanol/acetonitrile/water 50/40/10) About 1 mL of this mixture was spotted four times onto a MALDI ToF MTP 384 target plate ground steel F (Bruker Daltonics, GmbH, Bremen, Germany) All preparation steps were performed automatically by a robot (ClinProt robot, Bruker Daltonics, GmbH, Bremen, Germany) Mass spectra were acquired by an Ultrafl eXtreme MALDI TOF/TOF MS instrument (Bruker Daltonics, GmbH, Bremen, Germany) operated in positive ion linear or refl ectron mode, recording m/z values from 1000 10000 Da. External calibration was performed using a set of peptide/protein standards (ProtMix1) MALDI TOF acquisition parameters were as follows: total of 1200 shots (200 x 6) laser power about 70% (linear mode) and 40% (refl ectron mode) laser movement hexagonal. Data Analysis Data analysis was performed using the ClinProtTools 2.1 software package (17) (Bruker Daltonics, GmbH, Bremen, Germany) ClinProTools was used for multiple spectra comparison and protein pattern identifi cation with the following workfl ow: (i) spectra normalization to their total ion current; (ii) spectra recalibration using the prominent peaks; (iii) baseline subtraction and peak detection; and (iv) calculation of peak areas for each spectrum. Peak detection was done with signal to noise ratio of 5 and peak areas were calculated using endpoint level integration type. Spectra were also "top hat" baseline subtracted with a minimum baseline width of 10% and processed in a range of 1 10KDa. Only 1 of the 8 spectra obtained from the plasma of each subject by MALDI TOF was used for the next statistical analysis, with the help of "Support Spectra Grouping" and "Enable Similarity Selection" options. The program, after calculation of the mean spectrum of each subject's data set, selects the spectrum that is most similar to the average for subsequent statistical analysis. Basic statistical analysis, genetic algorithm (GA) (18) and Support Vector Machine (SVM) (19) were then used for the selection of signal clusters that were able to differentiate controls from penile cancer patients. These signals were preliminarily tested for their diagnostic capability using the two different data sets separately. The receiver operating characteristic curve analysis and area under curve (AUC) calculation were performed by the ClinProTools 2.1 software to determine the diagnostic effi cacy of each marker. The cut off value (P 0.001) corresponding to the highest accuracy (the lowest false negative and false positive results) was also calculated, along with specifi city and sensitivity. Peptide Identifi cation by MALDI TOF/TOF The peptides were identifi ed from the C8 MB eluted fraction of patients' and control subIBJU DOWNREGULATION OF COMPLEMENT FACTOR FRAGMENTS IN PLASMA FROM PATIENTS WITH CARCINOMA OF THE PENIS 742 Ta bl e 1 P at ie nt c ha ra ct er is tic s, h is to pa th ol og ic fi nd in gs p at ho lo gi c st ag in g, ty pe o f s ur ge ry a nd fo llo w u p. Pt s. Ag e Ra ce Gr ad e Va sc ul ar In va si on St ag e TN M Su rg er y Fo llo w u p (m on th s) De at hs b y Di se as e