the present staging system for melanoma , using breslow thickness , ulceration , mitotic rate , and the presence of regional and distant metastases , stratifies patients into heterogenous groups , with wide variability in outcome or response to therapy .
this results in applying more aggressive surgical and adjuvant therapies to large populations , diluting the impact of therapy while exposing more patients to toxicity .
better biomarkers in melanoma are needed to target both surgical and adjuvant therapies , but to date have been elusive . for many solid tumors , the large - scale analysis of gene expression at the rna level can provide patterns of gene expression that may stratify patients better than tnm staging and help guide therapy .
however , this approach requires fresh tissue from a large number of primary tumors , a unique challenge in melanoma where the primary is often only a few millimeters in size , with no residual tissue after the diagnosis has been made .   for this reason
, we chose to examine serum protein markers , hypothesizing that antibody discovery was ideal for the patient with malignant melanoma , as primary tumor tissue is not required and the presence of an immune response to melanoma - associated antigens has been well documented [ 14 ] .
the investigation of humoral response provides new perspective to focus on melanoma - associated antibodies , which are more sensitive and stable to become diagnostic biomarkers for early - stage melanoma .
we focused on glycoproteins , as most of the tumor - associated antigens are cell surface proteins or released to the extracellular matrix , where glycosylation is the major type of posttranslational modifications [ 5 , 6 ] .
moreover , glycoproteins are considered to be the linkage between t cells and antigen - presenting cells to help the orientation of binding , and play important roles in the generation and loading of antigenic peptides into mhc class i and mhc class ii [ 57 ] .   using this approach we sought biomarkers that correlated with the presence of regional metastases among melanoma patients .
using dual - lectin affinity chromatography and a natural protein microarray - based analysis to select a subproteome of target glycoproteins which were then used as baits to profile the antibodies against melanoma - associated antigens .
this significantly improved technology using lectin affinity chromatography allows us to concentrate low abundant glycoproteins which are typically undetectable in whole cell lysate .  
this approach led us to the discovery of antibodies to 5 interesting melanoma - associated antigens ( 75  kd glucose - regulated protein ( grp75 ) , 94  kd glucose - regulated protein ( grp94 ) , acid ceramidase ( asah1 ) , cathepsin d ( ctsd ) , and lactate dehydrogenase b ( ldhb ) ) that correlated with the presence of melanoma within the regional lymph nodes .  
a member of the heat shock protein-70 family , it also inactivates the tumor suppressor p53 . grp94 , also known as heat shock protein-90 , is a chaperone protein that is involved in the function and stability of many cell - signaling molecules .
asah1 is a catabolic lysosomal enzyme that deacylates ceramide , which when phosphorylated forms the poten mitogen s1p .
ctsd is a lysosomal acid proteinase which is involved in regulation of programmed cell death .
lactate dehydrogenase ( ldh ) is an enzyme that catalyzes the conversion of lactate to pyruvate , and serum levels are associated with outcome in stage iv melanoma .
we proposed that these autoantibodies may form the basis of a serum test that could select patients for sentinel lymph node biopsy .
however , many prognostic factors show limited utility when used clinically in the context of known prognostic factors . we therefore sought to examine the potential clinical utility of these novel serum markers for predicting regional involvement among patients with melanoma
in our previous work , we identified serum autoantibodies that recognized glycoproteins from a melanoma cell line and distinguished between 27 node - negative patients and 16 node - positive patients .   in that work , we subsequently validated these results using recombinant proteins among a larger sample set of 79 patients .
for this university of michigan institutional review board approved project , we used this latter sample set to examine the clinical utility of these serum autoantibodies as a predictor of regional node involvement .
serum samples were obtained from patients being evaluated at our melanoma multidisciplinary clinic , a few weeks after the diagnostic biopsy , but 2 to 3 weeks prior to undergoing wide local excision and lymph node surgery ( sln biopsy for clinically node - negative patients ( n = 71 ) or lymph node dissection for clinically node - positive patients ( n = 8) ) .
blood was allowed to clot at room temperature , after which the tubes were centrifugated at 2500  g for 10 minutes .
our initial discovery ( using extracted glycoproteins ) and validation ( using recombinant proteins ) of these serum autoantibodies have been previously described . for this study we used the results obtained with the recombinant proteins .
75  kd glucose - regulated protein ( grp75 ) , 94  kd glucose - regulated protein ( grp94 ) , cathepsin d ( ctsd ) , and lactate dehydrogenase b ( ldhb ) were purchased from abcam ( cambridge , ma , usa ) .
these 5 recombinant proteins were chosen because the amino acid sequences described in the manufacturers ' instructions are perfectly matched with the sequences acquired from swiss - prot database .  
the sequences and the purity of purchased recombinant proteins were reconfirmed by maldi - qit ( shimadzu , ca , usa ) .   to summarize our previous work ,
the recombinant proteins were dissolved in the printing buffer ( 62.5  mm tris - hcl ( ph 6.8 ) , 1%  w / v sodium dodecyl sulfate ( sds ) , 5%  w / v dithiothreitol ( dtt ) , and 1% glycerol in 1x pbs ) to reach a final concentration of 100  g / ml , respectively .
each protein solution was then transferred to a well in a 200  l 96-well clear printing plate ( bio - rad ) .
the recombinant proteins from the printing plate were spotted onto nitrocellulose ( whatman , usa ) slides using a noncontact piezoelectric printer ( nanoplotter 2 gesim ) .
each spot contains five spotting events of 500  pl each so that the total volume of each protein solution was 2.5  nl .
each spot was found to be ~450  m in diameter , with the distance between spots maintained at 600  m .
printed slides were dried on the printer deck overnight and stored in a refrigerator desiccated at 4c if the slides were not used immediately .
each recombinant protein was printed in triplicate , and 14 identical blocks were printed on each slide .   the slides were washed three times with 0.1% tween-20 in pbs buffer ( pbst ) and then blocked with 1% bovine serum albumin ( roche ) in pbst for 1  hr .
the blocked slides were dried by centrifugation and inserted into a simplex ( gentel bioscience ) multiarray device which divides each slide by 16 wells .
each serum sample was diluted 1  :  200 in probe buffer which consisted of 1% bsa , 0.05% triton x-100 , 0.1% brij-30 ( sigma - aldrich , usa ) in 1x pbs .
the sample hybridization was totally randomized on each slide in no specific order to prevent bias .
each block was hybridized in 100 microliter of diluted serum for 2  hrs at 4c .
then goat - anti - human igg ( h+l ) conjugated with alexa fluor 647 ( 1  g / ml , invitrogen , carlsbad , ca ) was applied to each block to bind with the antibodies attached on the protein array .
anti - human igg was printed on the array as positive control and printing buffer served as the negative control .
all processed slides were immediately scanned using an axon 4000b microarray scanner ( axon instruments , foster city , usa ) .
genepix pro 6.0 was used to extract the numerical data from each spot on the slides .
the background subtracted median intensity of each spot was taken as a single data point .
then the mean intensity of each protein from the triplicate was used for the further analysis .
for this study , patient and tumor characteristics were collected for our sample of 79 melanoma cases , it included patient 's age at surgery , gender , tumor thickness ( breslow ) , mitotic rate , presence of ulceration , and nodal status .
for use as a potential clinical test , the sample distribution of each auto - antibody classified as over- or underexpressed , defined as a serum antibody level one standard deviation increment above the sample mean value .  
association between the levels of the autoantibodies is summarized by the spearman rank correlation coefficient with p values testing for significant correlations reported .  
the associations between patient , tumor , and antibody covariates with nodal disease was compared using the two - sample t - test for continuous covariates and the chi - square or fisher 's exact test for categorical covariates .  
the magnitude of the association between each serum antibody level and nodal disease is reported categorically as the odds ratio and 95% confidence interval for cases with overexpression versus cases without .
odds ratios and confidence intervals were reported separately for the univariate associations and after adjustment for the patient and tumor characteristics using standard logistic regression .  
all statistical analyses were conducted using sas version 9.2 ( sas institute , inc . , cary , nc , usa ) with p values less than 5% considered statistically significant .
in the previous work , we used the native proteins extracted by a dual - lectin column from the melanoma cell line as bait to detect the presence of autoantibodies in the sera of melanoma patients , identifying 5 antigens including 75  kd glucose - regulated protein ( grp75 ) , 94  kd glucose - regulated protein ( grp94 ) , acid ceramidase ( asah1 ) , cathepsin d ( ctsd ) , and lactate dehydrogenase b ( ldhb ) , and we investigated the humoral response against the recombinant proteins using a larger sample set of 79 melanoma patients ( figure 1 ) .
the clinical characteristics of the patient population are shown in table 1 for the total population and stratified by nodal disease status .
of note , one patient with a negative sln subsequently recurred in a regional basin , changing the population from 48 node - negative and 31 node - positive to 47 node - negative and 32 node - positive patients . among the 32 node - positive patients ,
8 were clinically node - positive , 23 were sln positive , and 1 represented a regional recurrence after false - negative sln .  
each glycoprotein was summarized by over- ( > 1sd ) , standard ( 1sd ) , and underexpression ( < 1sd ) , relative to the sample mean .  
asah1 , ctsd , and ldhb all had significant negative associations with the presence of nodal disease , with overexpression associated with a lower risk .
higher grp94 levels were associated with a higher risk of nodal disease ; however , the level of grp75 was not significantly associated with nodal status .
correlation between antibody levels and age , gender , breslow thickness , mitotic rate , or ulceration are shown in table 2 .
correlations included asah1 which negatively correlated with patient age ( although the magnitude of the correlation was mild , r < 0.3 ) and grp75 which was higher in females than males ( p = 0.03 ) . the magnitude of the association of the glycoproteins with nodal disease status is reported in table 3 .  
even after adjustment for clinical parameters ( age , gender , ulceration , and breslow thickness ) asah1 , ctsd , and ldhb remained significantly negatively associated with nodal disease , and grp94 positively associated ( table 4 ) .    
a composite measure for the overexpression of any of the three proteins shown to have a negative association with nodal disease ( asah1 , ctsd , and ldhb ) was constructed with 22 ( 27.9% ) of the total population with composite overexpression .   only 2 ( 6.2% ) of the node - positive patients had composite overexpression in contrast to 20 ( 42.5% ) node - negative patients .   for the 9 of 32 node - positive patients ( 28% ) who had overexpression of grp94 , 5 ( 16% )
patients had clinically involved nodes while the remaining 4 ( 14% ) had microscopic disease ( < 2% surface area ) in clinically negative nodes .  
overexpression of anti - asah1 , anti - ldhb , or anti - ctsd ( decreased risk ) , the overexpression of anti - grp94 ( increased risk ) and breslow thickness ( increased risk ) significantly correlated with the likelihood of regional metastases .
the roc curve for this model is presented in figure 2 , with an area under the curve of 0.8690 .
using dual - lectin affinity chromatography to generate a subproteome of glycoproteins from a melanoma cell line generated from a metastatic deposit , we discovered 4 antibodies in the serum of melanoma patients recently diagnosed with melanoma that were strongly correlated with the presence or absence of nodal metastases . in this analysis
, we demonstrate that overexpression of these antibodies were independent of other known prognostic factors in melanoma , and on multivariate analysis maintained highly significant , independent prognostic value .  
these results demonstrate the potential of these 4 autoantibodies as a serum test for the purpose of selecting clinically node - negative patients for sln biopsy .  
elevation ( defined as > 1  sd above the mean ) of anti - asah1 , anti - ctsd , or anti - ldhb was highly significantly associated with being sln negative , with an odds ratio of 0.05 ( 0.010.31 , p = 0.002 ) after adjusting for age , gender , ulceration , mitotic rate , and breslow thickness .
elevation of one of these three antibodies was not uncommon ; among the 70 clinically node - negative patients in this study , 22 patients ( 33% ) had elevation of one of these antibodies .
if it were validated that patients with elevation of one of these antibodies had a very low risk of sln metastases , then potentially these patients could be treated by wide excision alone , reducing the cost and morbidity of melanoma treatment .
in contrast , elevation of anti - grp94 was associated with an increased risk of regional metastases , and was elevated in both patients with clinically evident disease ( 5 of 8 patients ) and microscopic disease ( 4 of 23 patients ) . while detection of serum anti - grp94 levels would be less useful clinically , it could potentially identify some patients with thin melanoma for whom sln might otherwise be omitted .  
a bigger question is the role these proteins may play in the development and progrsion of melanoma . beyond the development of serum - based diagnostic tests , proteomics may identify targets for therapeutic intervention .
indeed , 3 of the 4 proteins have strong associations with melanoma progression and prognosis .
lactate dehydrogenase ( ldh ) is an enzyme that catalyzes the conversion of lactate to pyruvate , and serum levels of ldh are strongly associated with melanoma prognosis .
serum ldh levels strongly correlate with outcome among stage iv patients and serum ldh measurements are part of the american joint cancer commission ( ajcc ) staging system for melanoma [ 913 ] . however , serum ldh levels are rarely elevated and of no clinical utility in nonmetastatic melanoma [ 1416 ] .
cathepsin d ( ctsd ) is a lysosomal acid proteinase which degrades proteins , peptides , and peptide precursors .
in addition , it appears to be involved in other biological processes including regulation of programmed cell death , tissue remodeling and renewal , activation of proteolytic enzymes , and fibrinolysis .
many tumors have altered processing , secretion , and activity levels of ctsd , and they are often associated with aggressive behavior , stimulating tumor cell proliferation , invasion , and metastases [ 17 , 18 ] .
immunohistochemical studies have shown that ctsd is markedly expressed in melanoma cell lines and tissue biopsies from primary and metastatic melanoma , and these correlate with poor outcome [ 1924 ] .
as with ldh , measuring plasma levels of ctsd was not of clinical value for identifying patients at risk of recurrence [ 17 , 25 ] . as we can detect very low levels of antibodies in the serum , measuring antibody levels may be more sensitive than measuring protein levels , allowing transition of known serum markers from utility in stage iv disease only to the setting of early - stage disease .
prior to this publication , the third protein , acid ceramidase ( asah1 ) , had not been strongly associated with melanoma progression , but has been associated with cancers of the breast , prostate , and thyroid [ 2629 ] .
asah1 is a catabolic lysosomal enzyme that deacylates ceramide and yields sphingosine , which when phosphorylated , forms the potent mitogen s1p .
the cellular levels of ceramide , sphingosine , and s1p are integral in determining cell survival and growth [ 3032 ] . targeting this pathway holds promise for anticancer therapies [ 3234 ] .   in the case of these three proteins ,
expression of the proteins is associated with advanced stage , but the presence of antibodies to these proteins is associated with lower stage .
the presence of the antibody may be due to increased exposure of the proteins ( overexpression ) , immune recognition of protein alteration , or the antibodies may be functionally blocking critical pathways .   in the case of these three proteins / antibodies
, it remains unclear whether this represents specific functional inhibition by the antibodies , or increased progression in the face of decreased immune recognition of overexpressed proteins ( disease advancement in the face of decreased immune surveillance ) .
as these proteins are identified , further analysis of their role in melanoma progression , and their posttranslational structure is necessary .
in contrast to these three proteins , for which the detection of autoantibodies was a favorable prognostic sign , the presence of autoantibodies to grp94 , or heat shock protein-90 ( hsp90 ) , was associated with an increased risk of regional metastases .
although this protein is highly conserved ( and should not trigger a significant immune response ) , our primary data and validation studies using the recombinant proteins demonstrate the presence of anti - grp94 antibodies in the serum of nearly between 1/4 and 1/3 of node - positive patients .
hsp90 is a chaperone protein that is crucially involved in the function and stability of many oncogene products and cell - signaling molecules , including craf , erb - b2 , bcr - abl , cdk4 , cdk6 , akt , mutated p53 , mek , vegfr , and importantly to melanoma mutated ( but not wildtype ) braf .
hsp90 protects these proteins from deterioration caused by environmental stress , which includes cancer therapy .
expression of hsp90 is elevated in melanoma , correlates with increasing breslow thickness , and is associated with advanced disease . because hsp90 chaperones so many proteins implicated in carcinogenesis , inhibiting hsp90 could inhibit several pathways at once , hsp90 inhibitors are presently in clinical trial in metastatic melanoma . while our data suggest that autoantibodies do little to inhibit hsp90 functionally , their presence as a response to overexpression is clearly related to melanoma progression . in conclusion ,
the creation of a glycoprotein microarray to screen melanoma patient serum samples for autoantibodies yielded four autoantibodies that show promise in predicting regional metastases , and could potentially form the basis of a blood test to select clinically node - negative patients for sln biopsy .  
if validated , this test could greatly minimize the cost and morbidity associated with the surgical treatment of melanoma , as well as identify patients with thin melanomas who should undergo the procedure .
in addition , glycoproteins recognized by these antibodies may have important roles in the development and progression of melanoma and may serve as targets for intervention . on a broader scale , this approach could be used to identify additional serum autoantibodies that can identify patients at high risk of distant metastases and those unlikely to respond to treatment , allowing a more tailored use of adjuvant therapies .
 better prognostic and predictive markers in melanoma are needed to select patients for therapy . 
 we utilized a dual - lectin affinity chromatography and a natural protein microarray - based analysis to select a subproteome of target glycoproteins to profile serum antibodies against melanoma associated antigens that may predict nodal positivity . 
 we identified 5 melanoma - associated antigens using this microarray coupled to mass spectrometry ; grp75 , grp94 , asah1 , ctsd and ldhb . 
 we evaluated their predictive value for nodal status adjusting for age , gender , breslow thickness , mitotic rate and ulceration using standard logistic regression . 
 after adjustment , asah1 , ctsd and ldhb were significantly negatively associated with nodal status ( p = 0.0008 ) and grp94 was significantly positively associated ( p = 0.014 ) . 
 our best multivariate model for nodal positivity included breslow thickness , presence of serum anti - asah1 , anti - ldhb or anti - ctsd , and presence of serum anti - grp94 , with an area under the roc curve of 0.869 .   
 if validated , these results show promise for selecting clinically node negative patients for sln biopsy . in addition 
 , there is strong potential for glycoprotein microarray to screen serum autoantibodies that may identify patients at high risk of distant metastases or those likely or unlikely to respond to treatment , and these proteins may serve as targets for intervention . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion