
 osteosarcoma ( os ) , characterized by the production of osteoid material by malignant osteoblastic cells , is a primary malignant bone tumor deriving from primitive bone - forming mesenchymal cells .
overall , the treatment strategies are preoperative chemotherapy and surgical resection followed by postoperative chemotherapy and adjuvant therapy for several years .
the 5-year survival rates for patients with localized osteosarcoma have dramatically improved from less than 15% in the 1950s to greater than 60% since the 1980s . despite advances in diagnostic and treatment regiments , progress since
one of the reasons could be the presence of mutated oncogenes , which confer drug resistance of the cancer cell .
currently , the diagnosis of os is generally dependent on a comprehensive examination including clinical symptoms , imaging , laboratory examinations , biopsy , and immunohistochemistry .
clinically , os was always diagnosed at the middle or even at a late stage .
therefore , the long - term survival rate of os has not been improved in the past 20 years .
the histological examination of the biopsy specimens is still preferred by many orthopedic oncologists for the diagnosis of os . despite its invasive procedure , the accuracy of diagnosis may vary among different sample collection and different observers , making the clinical prediction questionable .
thus , we have focused our attention on the development of a noninvasive method for the early diagnosis of osteosarcoma .
many studies have identified that antigenic changes in cells can be recognized by the immune system of patients .
some early studies demonstrated the presence of serum autoantibodies to a panel of known tumor associated antigen ( taa ) in various human cancers .
the study indicated that autoantibody reactivity to individual taa rarely exceeded 20% in the cancer patient populations compared with normal human sera ( nhs ) which are usually less than 5% .
it is conceivable that specific autoantibody profiles can be identified with help for discriminating autoantibody reactivity between cancer patients and control individuals and distinguishing between some types of cancer .
so far , only a few studies have been performed in the field of using autoantibodies as diagnostic markers in osteosarcoma .
several different approaches based on mass spectrometry ( ms ) have been applied in the search for cancer biomarkers .
direct analysis of serum samples using ms is becoming more popular due to its high - throughput nature and increased sensitivity .
one of these proteomic approaches , surface - enhanced laser desorption / ionization - time - of - flight mass spectrometry ( seldi - tof - ms ) , is a rapid and sensitive proteomic technique to identify biomarkers in various forms of cancers [ 7 , 8 ] .
this technology has been effectively used in the validation of serum antigens for early - stage detection of various cancers , such as prostate , ovarian , and breast cancers .
therefore , exploitation of seldi - tof - ms for screening biomarkers in os is applicable .
the searching database tool , empirical proteomic ontology knowledge base ( epo - kb ) , including tens of thousands of mass to charge ratio ( m / z ) protein function , was used to identify proteins characterized from seldi - tof - ms . using seldi - tof - ms combined with epo - kb makes it possible to directly separate and identify the proteins from human osteosarcoma sera .
the major aim of this study using seldi - tof - ms combined with epo - kb is to identify serum markers from human osteosarcoma sera as potential os - associated markers for early diagnosis of os .
sera from 25 chinese patients , han ethnic ( fourteen males and eleven females , mean age 19.3 years , and range 250 ) , with high - grade os were collected at the time of diagnosis before biopsy and chemotherapy were analyzed .
all samples were provided by luoyang orthopedic - traumatological hospital and luoyang institute of orthopedic and traumatology between jan .
we randomly collected 26 human serum samples from normal age - matched donors in the same hospital as a normal control and 16 samples from osteochondroma ( oc ) ( nine males and six females , mean age 16.8 years , and range 448 years , chinese patients , han ethnic ) as benign tumor control .
all blood samples ( 5  ml from each patient or normal person ) were collected in edta - containing tubes at room temperature and immediately centrifuged at 1000  rpm for 10  min . the serum supernatant was collected and divided into aliquots and stored at 80c .
the study was approved by the ethics committee of luoyang orthopedic - traumatological hospital and luoyang institute of orthopedic and traumatology .
serum samples were diluted to a certain concentration and immediately centrifuged ( 20,000  rpm ) for 10  min at 4c .
twenty microliters of each serum was denatured by adding 40  ml of u9 buffer containing 9  mol / l uric acid , 2% chaps ( ph 9.0 ) , and 50  mmol / l tris - hcl ( ph 9.0 ) and shaken for 20  min at 4c .
twenty microliters of the denatured samples were fractionated by 240  l of u1 buffer and shaken for 30  min .
each chip was added with bluestone ( 50  l , 100  mmol / l ) and shaken ( 200  rpm ) for 5  min at room temperature , and the bluestone solution was immediately poured out . after being washed using ionized water for 5 times
, the chip was added with 50  l of cm low stringency buffer ( 0.1  m sodium acetate , ph 4.0 ) , shaken ( 200  rpm ) for 5  min .
then , each chip was added with 150  l of standard buffer containing 100  mol / l sodium phosphate and 500  mmol / l sodium chloride ( ph 7.0 ) and incubated on the oscillator for 5  min at room temperature , and the buffer solution was removed .
this process was repeated one more time . each serum sample ( 50  l )
was randomly assigned to a spot on each protein - chip array and incubated on the oscillator for 60  min at 4c .
the samples were denatured by 150  l of standard buffer , washed and oscillated for 3 times each for 5  min , and quickly washed with 1  mmol / l hepes ( ph 7.0 ) .
chip eam ( sinapinic acid ( spa ) ) was added with 100  mmol / l acetonitrile ( 75  l ) and trifluoroacetic acid ( tfa ) ( 75  l ) .
the reagents such as uric acid , acetonitrile , tfa , and spa were purchased from sigma .
the pbsiic and imac3-cu were calibrated externally by using the all - in - one peptide and protein ii molecular mass standards ( ciphergen biosystems ) .
each sample was analyzed in duplicate to minimize the effects of intra - assay variation .
proteomic profiles on the protein - chip arrays were detected by a protein biology system ( model pbsiic ) .
statistical signification peaks were recorded by x-s and identified by using parametric t - test with p value of 0.001 with biomarker wizard and biomarker pattern system software .
the diagnostic accuracy was measured by external leave - one - out cross - validation , which is one of the feature selection steps .
sixty - seven serum samples were assayed by seldi - tof - ms . from a total of 67 serum samples ,
25 serum samples are from os patients compared with the control group of 42 serum samples ( 16 from osteochondroma and 26 from normal human serum ) .
one protein peak at 3954  da was overexpressed and another one at 6438  da ( table 1 ) .
the mass spectrum shows the comparison of three serum samples from patients with os and oc as well as a serum from a normal individual ( figures 1 and 2 ) . in order to take a marked contrast
, we apply the biomarker pattern software to analyze protein differences in the template group to m / z , respectively , 3954  da and 6438  da proteins composed of two different diagnostic classification tree models ( figure 3 ) .
the 67 samples were repeated sampling in the learning mode , and the diagnostic sensitivity was 98.51% and specificity was 98.51% , while , in test mode , the diagnostic sensitivity was 98.51% , and specificity was 100.00% .
it was verified by leave - one - out and 3-fold cross - validation that correct grouping rate was 100% ( 67/67 ) , good response rate was 100% ( 25/25 ) , and specificity was 100% ( 42/42 ) .
we searched the m / z of 3954  da and 6438  da in the first classification tree models and got related results in the epo - kb database .
the relevant information of 6438  da protein was retrieved ; however , the 3954  da protein needs further identification ( http://www.expasy.org/cgi-bin/prosite-search-ac?pdoc50099 and http://us.expasy.org/prosite/ps50324 ) .
it is serine rich protein sequences involved in cell signal transduction and carcinogenesis . in some microtubule plus end tracking proteins , there are some basic amino acid serine rich sequences .
these basic amino acids serine rich sequences are often mediated by the microtubule plus end tracking proteins with microtubules and eb protein and the role of other proteins .
protein peak of m / z at 6438  da in the b - cell lymphoma is highly expressed and , in the low expression of t cell lymphoma , it is closely related to the human immune system regulation .
seldi - tof - ms is a relatively new proteomic technology that has impacted many areas of biological research [ 13 , 14 ] .
the high - throughput nature of this technology allows the processing of many samples simultaneously in a relatively short period of time .
it requires only small amounts of samples for profiling , which makes it particularly suitable for clinical or translational studies .
seldi - tof - ms has been successfully applied in identifying early detection biomarkers in multiple cancers , including ovarian , prostate , and breast cancers .
however , seldi - tof - ms technology is not widely used in bone oncology to identify biomarkers and perform molecular classification , partly due to the limited sample size when compared to other cancers .
the current study shows that patients with os and oc and normal individuals can be distinguished from each other by the special proteins with different m / z characters screened by seldi - tof - ms .
the results showed three samples determined by the difference of the following standards ( m3954  0.396 ( normal ) , m6438  1.329 ( os ) , and m6438 > 1.329 ( oc ) ) .
it was verified by leave - one - out and 3-fold cross - validation that correct grouping rate was 100% ( 67/67 ) , which proved that the results is reliable .
the diagnosis of os generally depended on a comprehensive way including clinical symptoms , signs , image , laboratory examinations , biopsy , immunohistochemistry , and other methods .
the surgical biopsy is also the terminal way for final diagnosis , which is an invasive procedure and has risks of enhancing tumor dissemination and metastases .
if we can perform specific markers with high sensitivity and specificity using blood specimens from the patients , it will be more convenient and noninvasive to the patients at the initial diagnosis .
these specific markers combined with x - ray exam could be used to monitor os development in coordination with chemotherapy or radiography . and
the present study has found the m / z of 3954  da and 6438  da proteins in the classification tree models and gotten 6438  da in the epo - kb database , which is a serine rich protein sequences involved in cell signal transduction and carcinogenesis .
these basic amino acids serine rich sequences are often mediated by the microtubule plus end tracking proteins with microtubules and eb protein and the role of other proteins .
since protein peak of m / z at 6438 da showed high expression in the b - cell lymphoma and low expression in t - cell lymphoma , it is closely related to the human immune system regulation .
we thought that the 6438  da protein identified by seldi - tof - ms may potentially be used for early detection , diagnosis , therapeutically monitoring , and prognosis of os .
in addition , randomized clinical trials are required to investigate its effect ( or lack thereof ) .
seldi - tof - ms technology is still more constraining in clinical research , because the only result we can get is m / z , rather than the molecular weight .
after finding the different proteins by seldi - tof - ms , further studies with chromatography , two - dimensional electrophoresis , spectroscopy , and other techniques need to be performed to identify these proteins immensely .
however , these laboratory approaches could not be used for large - scale screening of biomarkers in clinical application .
additionally , its wide acceptance is still in question due to some concerns regarding reproducibility and reduced sensitivity at high molecular weight range .
these findings suggest that the m / z of 3954  da and 6438  da proteins could potentially be biomarkers for early diagnosis of osteosarcoma
. it would be interesting and important to conduct other independent studies in large populations for comparison .
future functional studies are warranted to verify these findings and to improve our understanding of the underling molecular mechanism of genetic contribution to os carcinogenesis .
 osteosarcoma ( os ) is the most common malignant bone tumor . to identify os - related specific proteins for early diagnosis of os , a novel approach , surface - enhanced laser desorption / ionization - time - of - flight mass spectrometry ( seldi - tof - ms ) to serum samples from 25 os patients , 16 osteochondroma , and 26 age - matched normal human volunteers as controls , was performed . 
 two proteins showed a significantly different expression in os serum samples from control groups . 
 proteomic profiles and external leave - one - out cross - validation analysis showed that the correct rate of allocation , the sensitivity , and the specificity of diagnosis were 100% . 
 these two proteins were further identified by searching the epo - kb database , and one of the proteins identified as serine rich region profile is involved in various cellular signaling cascades and tumor genesis . 
 the presence of these two proteins in os patients but absence from premalignant and normal human controls implied that they can be potential biomarkers for early diagnosis of os . 
1. Introduction 
2. Materials and Methods
3. Results
4. Discussion 