the early diagnosis of colorectal and pancreatic cancers remains an area of high unmet medical need , as underscored by the u s estimated combined , annual death rate of > 89,000 . although the serum marker ca19 - 9 is elevated in the majority of pancreatic cancer patients , the specificity of ca19 - 9 is limited . ca19 - 9 is frequently elevated in patients with various benign pancreaticobiliary disorders [ 24 ] . as a result of all of these issues , the american college of gastroenterology ( acg ) recommends colonoscopy as the preferred screening / prevention test for colorectal cancer . currently , there is no consensus for screening for the early detection of pancreatic cancer . unlike colorectal cancer , the majority cases of pancreatic cancer are detected when a patient is symptomatic which often times represents late stage cancer , resulting in an overall 5 year survival of less than 5% . the majority of colorectal and pancreatic cancer patients are diagnosed utilizing invasive procedures that are expensive , and usually reveal the diagnosis later in the disease process . newer approaches are being investigated that could allow for earlier detection of disease , in a cost - effective manner , that furthermore could result in better outcomes for patients with these diseases . as an alternative diagnostic approach , we developed an elisa using a promising novel tumor - specific monoclonal antibody generated against a clinically tested human colon cancer vaccine . npc-1 is a monoclonal antibody that was derived from a tumor associated antigen- ( taa- ) based vaccine that was previously tested in phase i - ii clinical trials performed in the united states [ 79 ] . the taa utilized in these studies was derived from pooled allogeneic colon cancer specimens from multiple patients , which was obtained postoperatively . cell membranes were isolated from the tumor , and proteins from solubilized membranes were prepared by sonication and sephadex g-200 chromatography . semipurified taas were identified by in vitro and in vivo testing in colon cancer patients and healthy volunteers for cell - mediated immunoreactivities . the colon taa was detected in fetal intestine and cell membranes , and was localized on tumor cell membranes . using discontinuous , gradient gel electrophoresis , both colon taa and cea were separated and cross - compared . the cdna encoding the npc-1 antibody was cloned from hybridoma cells , chimerized by genetic engineering , and expressed in a heterologous expression system ( chinese hamster ovary cells ) . the npc-1c antibody binds to a protein antigen biomarker expressed by human colorectal and pancreatic tumors . in immunohistochemical testing , npc-1c did not react significantly with tissues from healthy donors or other types of cancer . furthermore , as discussed below , the npc-1c antibody elisa developed can distinguish serum of patients with colorectal or pancreatic cancer from healthy volunteers , thereby providing the rationale for accelerated development and testing of the variant muc5ac ( npc-1c antigen ) detection assay . the test may have application in diagnosis and treatment monitoring of patients with pancreatic or colorectal cancers . this homologous antibody format was possible due to the discovery of multiple npc-1c antigen - binding sites expressed by the cancer - associated muc5ac - related ( npc-1c ) antigen . serum samples were procured from various commercial and private sources under appropriate irb - reviewed protocols . the assay developed here used serum from colorectal and pancreatic cancer patients , and serum from healthy blood donors . microtiter plates ( 96-well nunc maxisorp ) were coated with purified unlabeled npc-1c antibody at 10  g / ml in 0.5  m sodium carbonate ph 9.5 overnight at 25c . plates were then blocked with 1% skim milk made in tris - buffered saline ( tbs ) containing 5  mm edta and 1% sucrose for 4 hours at 25c . plates prepared in this manner could be stored dried and sealed for at least 12 months . all dilutions were made in immunobooster buffers ( bioworld consulting laboratories , llc ) supplemented with 20  mm edta . a detergent extract of cultured human ls174 t colorectal tumor cells was used as a source of npc-1c antigen to derive a standard curve . extracts derived from human pancreatic cfpac-1 tumor cells or human lung a549 tumor cells were generated similarly . all tumor cell lines were purchased from american type culture collection ( manassas , va ) and grown in rpmi medium containing 10% fbs ( heat - inactivated ) with 8  mm glutamine . to measure direct binding of npc-1c antibody to the variant muc5ac ( npc-1c ) antigen , cfpac-1 cells were grown in serum - free medium for 5 days and the conditioned medium was filtered and stored in one large lot at 4c . the sandwich elisas were performed by diluting the cell extract standard on each plate , next to patient or normal serum samples diluted 1  :  24 in the diluent . all incubations were performed at 25c and all volumes were 100  ul per well . the biotin - labeled npc-1c antibody was then added to the wells at 1  g / ml , incubated for 15 minutes , and plates were washed three times . peroxidase - conjugated streptavidin ( 1  :  5,000 dilution ) was added to the plates for 15 minutes , and plates were washed three times with wash buffer and two times with tbs . the assay was developed by the addition of tmb substrate ( biofx laboratories inc . ) to the plates , incubation for 15 minutes , then the color reaction was stopped with the addition of 0.5  m sulfuric acid . npc-1c antibody - binding competition assays were performed by coating microtiter plates with serum - free conditioned medium from cfpac-1 tumor cells shown to secrete the variant muc5ac antigen into the culture medium . following a blocking step as described above , a solution of 1  g / ml npc-1c antibody was mixed with serial dilutions of conditioned medium from cfpac-1 , ls174 t , and a549 tumor cells . the elisa was developed using anti - human igg peroxidase - conjugated antibody followed by tmb substrate incubation as described above . tumor biopsy specimens from colorectal , pancreatic , or lung cancer patients were deparaffinized at 60c for 30 minutes prior to staining with npc-1c antibody . slides ( 4 microns thick ) were blocked with peroxo - bloc inhibitor ( zymed laboratories ) for 2 minutes , rinsed with phosphate - buffered saline ( pbs ) , and blocked with cas ( zymed laboratories ) for an additional 10 minutes . slides were stained with 10  g / ml of biotin - labeled npc-1c antibody for 1 hour , and washed three times with pbs containing 0.05% tween-20 nonionic detergent . previous titration of biotinylated - npc-1c antibody demonstrated 10  g / ml to be an optimal concentration for immunohistochemical detection of the variant muc5ac antigen . a 1  :  400 dilution of peroxidase - conjugated streptavidin ( dako north america , inc . ) was then applied to the slides for 30 minutes and slides were washed 3 times . a solution of dab ( zymed laboratories ) a solution of hematoxylin was then applied for 3 minutes and rinsed with tap water until clear . the slides were dehydrated with xylene and a coverslip was added using permount mounting medium . additional consecutive slides were stained with human cytokeratin ae1/ae3 ( abcam plc ) as a positive control , and human igg1 isotype as a negative control ( axxora , llc ) . the anti - muc5ac antibody ( clone 45m1 ) used to stain lung tumor tissue ( 10  g / ml ) was purchased from abcam plc . the npc-1 antibody was generated in mice immunized with a preparation of pooled human colon tumor tissue extract . hundreds of hybridomas were screened for cancer - specific characteristics such as binding to tumor tissues and cell lines , with no cross - reactivity to normal human tissues . the original murine igg , npc-1 , was cloned and chimerized by genetically engineering the mouse variable regions of heavy and light chains with human igg1 constant regions of the heavy and light chains . the resulting chimeric antibody , npc-1c , was then expressed in a recombinant chinese hamster ovary cell clone for further preclinical and clinical development . the npc-1c antibody was used as an affinity chromatography tool to isolate and identify the target antigen expressed by colorectal and pancreatic tumor cells . it was shown early that although the murine npc-1 antibody was generated against a preparation of colorectal tumor - associated antigens , the npc-1 antibody cross - reacted with pancreatic cancer tissues and cell lines . the target antigen recognized by npc-1c was determined to be related to the mucin 5ac ( muc5ac ) protein , a member of the secreted mucin family of glycoproteins ( reviewed in ) . we rapidly determined that the variant muc5ac antigen was shed into the supernatant of many cultured human colorectal and pancreatic tumor cells , but not by other tumor cell lines such as prostate or lung tumor cells . we therefore examined whether the npc-1c antibody could be useful to detect the target muc5ac - related antigen in humans afflicted with colorectal or pancreatic cancer . to demonstrate by example that the secreted variant muc5ac antigen recognized by npc-1c antibody was expressed specifically by colorectal and pancreatic tumor cells , supernates from ls174 t , and cfpac-1 were admixed with npc-1c in a competitive elisa format . figure 1 shows that the soluble variant muc5ac antigen secreted by ls174 t and cfpac-1 tumor cells could compete effectively with binding to npc-1c antibody when the variant muc5ac antigen was coated on microtiter plates . in contrast , supernates from human lung a549 tumor cells that are known to secrete normal muc5ac did not compete for binding to npc-1c antibody . similar competition curves were shown with other colorectal and pancreatic tumor cell lines , but not with another squamous tumor cell line ( data not shown ) . together the results demonstrate that npc-1c binds specifically to a variant muc5ac antigen expressed by colorectal and pancreatic tumor cells , but not muc5ac secreted by other tumor cell types . chemical and enzymatic digestion of the npc-1c antibody purified variant muc5ac antigen revealed that each molecule of the npc-1c target contained multiple epitopes for npc-1c binding ( data not shown ) . therefore , we reasoned that it would be possible to use npc-1c as a specific reagent for both the capture and detection antibody in a homologous format elisa . biotin - labeled npc-1c antibody was prepared as the detection reagent and tested with variant muc5ac antigen expressed by ls174 t cells . figure 2 shows that npc-1c antibody was capable of measuring the cognate antigen expressed by ls174 t in this elisa format . in contrast , muc5ac expressed by lung a549 tumor cells was not detected in the elisa . thus , a surrogate standard muc5ac - related antigen reagent was generated , frozen , and used for all subsequent elisa tests . the npc-1c antibody immunoreactive antigen is reported here in units of ls174 t cells / well equivalent . the proof - of - concept to demonstrate that the npc-1c antibody elisa test could be utilized to detect the variant muc5ac antigen in human serum specimens was demonstrated with a small number of serum samples from colorectal cancer patients . figure 3 shows the results from five serum specimens collected from colorectal cancer patients compared to pooled ab serum from healthy donors . the results demonstrate a range of variant muc5ac antigen shed into the blood of these colorectal cancer patients . in contrast , pooled ab serum from healthy donors did not yield a significant signal and was similar to the background levels for the elisa . following this , and other preliminary tests , an optimum serum dilution of 1  :  24 a larger number of serum samples were procured to test the utility of the serum - based elisa in detecting the variant muc5ac antigen . a sampling of 41 colorectal or pancreatic cancer patient sera was compared with sera collected from 28 normal healthy blood donors . in this population of cancer patients , blood was collected serially during an approximately 3-month period for several of the patients while they were undergoing various treatment regimens with a medical oncologist . for multiple reasons , thus , there were 41 patients that donated blood at their first evaluation by the medical oncologist , followed by 33 patients that donated their blood at the second visit , and 25 patients who completed all three blood donations at the third visit . the majority of specimens tested in this preliminary study were from patients diagnosed with stage iii or iv disease . figure 4 shows the results of testing this larger panel of colorectal and pancreatic cancer patient serum specimens , compared to a group of normal healthy blood donors . analysis of the results demonstrated approximately a 0.7 log difference between the cancer patients and the healthy donors at each of the three blood draws . the mean and standard error of the mean for each control group for the assays are normals ( 355  60 ) , col / pan ca : 1-month ( 1,757  580 ) , col / pan ca : 2-month ( 1,894  671 ) , col / pan ca : 3-month ( 1,293  390 ) . using the unpaired t - test ( 2-tailed ) method to evaluate the difference between the normal sera group and the cancer sera groups , the differences for each comparison were normal versus 1-month : p = .0511 ; normal versus 2-month : p = .0397 ; normal versus 3-month : p = .0153 . furthermore , using a cutoff value of 355  cells / well derived from the normal sera average , 73% of col / pan ca , 1-month sera were above the cutoff ( 30 of 41 samples ) , and 88% were above the cutoff in each of the 2-month ( 29 of 33 samples ) , and 3-month ( 22 of 25 samples ) in those groups . overall , the samples represent an average of 82% positive above the cutoff established for the assay . these results show that the npc-1c antibody elisa can distinguish differences between serum from normal donors and colorectal or pancreatic cancer patients , with a promising level of confidence . the cancer patient population tested in this study was further stratified by disease type . figure 5 shows that there was no difference distinguished by the mean npc-1c antibody elisa results among those patients diagnosed with colorectal cancer ( n = 36 ) from those patients diagnosed with pancreatic cancer ( n = 5 ) . both groups separately demonstrated approximately 0.7 log units higher variant muc5ac antigen expression levels compared to the group of healthy donors . from patients that donated multiple serum samples , the amount of variant muc5ac biomarker detected in the assay was plotted versus the time of the blood draw . as shown in figure 6 , some patients appeared to express similar amounts of the variant muc5ac antigen during the 2- or 3-month period when blood was drawn ( subjects 5 , 14 , 15 , 19 , 25 , 28 , 29 ) , whereas some patients appeared to experience an increase in this tumor - specific antigen expression ( subjects 1 , 2 , 7 , 33 , 39 ) or a decrease in the biomarker expression ( subjects 18 , 22 , 23 , 28 , 34 , 36 , 40 ) . the significance of these shifts over time are presently unclear , but may be related to the tumor burden of the patient at the time the blood was drawn , which may be directly related to the specific treatment regimen of individual patients . thus , the npc-1c antibody elisa may be a useful tool as a treatment monitoring biomarker assay . it was not possible within the 3-month time period of the blood collections to determine whether a correlation exists between the rate of variant muc5ac increase or decrease and the outcome of the disease in these patients . a number of tumor tissue specimens were procured to examine the level and incidence of variant muc5ac antigen expression in colorectal and pancreatic cancer patients . biotin - labeled npc-1c antibody was used at 10  g / ml , detected with streptavidin - horseradish peroxidase conjugate , and mounted on glass slides . a positive staining scale ranging from + 1 to + 5 representative examples of the staining results to detect the variant muc5ac antigen are shown in figure 7 . tissues from normal pancreas and colon showed no cross - reactive staining with npc-1c antibody ( panels ( a ) and ( c ) resp . ) . in contrast , tissues from pancreatic and colorectal tumor biopsies demonstrated specific staining of both cytoplasmic and membrane associated antigen ( panels ( b ) and ( d ) resp . ) . in the colorectal cancer tissues , frequent staining of secreted variant muc5ac antigen was observed in the luminal spaces of the tissues ( panel ( d ) ) . the tissue specificity of npc-1c binding was demonstrated by the lack of positive staining of lung adenocarcinoma tissue ( panel ( f ) ) , whereas a commercially available anti - muc5ac antibody shows that the lung tumor tissue expresses muc5ac ( panel ( e ) ) . thus , while muc5ac is known to be expressed by lung cells , the npc-1c antibody does not react with the muc5ac expressed by lung tumor tissue . tissues stained with npc-1c antibody were considered positive ( + 1 to + 5 ) for 79% of the tumor samples procured and stained ( 30 of 38 ) . these staining results are similar to results from several other studies completed with npc-1c antibody using tissue array slides , and both frozen and paraffin - embedded surgical specimens . the proof of concept has been established for the value of the npc-1c antibody in the detection of the tumor - associated muc5ac antigen recognized by npc-1c antibody . this development suggests a new effective , scalable serum biomarker elisa for the potential diagnosis and immunoregulatory monitoring of patients with colorectal and pancreatic cancer . these results also support further development and large - scale early noninvasive diagnostic screening of healthy populations for colorectal and pancreatic cancer . the preliminary results described here demonstrate that the npc-1c antibody can distinguish normal / healthy serum from serum derived from patients with colorectal or pancreatic cancer . a better defined cohort of healthy serum donors may permit improved comparisons regarding assay specificity and sensitivity . interestingly , should the assays be predictive in diagnosing colorectal and/or pancreatic cancer , some of the  normal  donors tested in the assays described here may be predisposed to developing cancer , and the potential utility of these elisas may be underestimated , if they could detect cancer in asymptomatic persons . indeed , while the patient population studied in this report was predominantly from stage iii and iv cancer patients , we are currently procuring serum specimens from earlier stage colorectal and pancreatic cancer patients ( stage i and ii ) as well as serum from asymptomatic persons at risk for developing these types of cancer . several research laboratories have demonstrated the association of aberrantly expressed muc5ac in colorectal and pancreatic cancers [ 11 , 12 ] . however , none of these appear as specific as the npc-1c antibody in defining the variant muc5ac antigen expressed in colorectal and pancreatic patient serum as compared with normal healthy donors , or as compared with muc5ac antigen expressed by other tumor types . the reason for this difference may be due , in part , to the specific epitope that the npc-1c antibody recognizes , which is currently an area of active investigation . the assay specificity using the normal serum samples presented in this interim report are 71% ( 8/28 normal samples above the mean cutoff ) . the sensitivity for the npc-1c antibody elisa was 82% ( 18/99 cancer samples below the mean normal cutoff ) . future testing and data analysis with new serum from healthy donors should increase the specificities of both serum assays . importantly , testing earlier stage cancer patient ( stage i - ii ) will shed light on the application of this elisa to detect cancers earlier , which will allow earlier interventions and improve treatment outcomes . reviewing the ihc results for each antibody , the tumor biopsy specimens were collected from patients diagnosed pathologically with stage iii and iv colorectal and pancreatic cancer . the chimeric npc-1c therapeutic antibody ( ensituximab ) is currently being tested in a phase i dose escalation clinical trial . as part of the eligibility criteria for the study , patients with advanced pancreatic or colorectal cancers must have their tumor biopsy stain positive for the npc-1c antigen to be considered eligible for treatment . the preliminary immunohistochemical data presented here suggests that approximately 80% of patients may be treated with the therapeutic biological npc-1c . this truly represents the new frontier of theranostics , where a biomarker can act as a companion to a specific therapeutic product . the serum elisa described here may have utility in monitoring colorectal or pancreatic cancer patients during the course of a treatment regimen . patients with pancreatic cancer are typically treated with gemcitabine , whereas the treatment options for colorectal cancer patients can include chemotherapies ( 5-fluorouracil , folfox , or folfiri ) or biologics such as cetuximab and bevacizumab . the npc-1c antibody elisa may be useful to aid in monitoring the patient responses to such therapies . the results shown in figure 6 demonstrate trends for certain patients that may reflect cancer regression , progression , or stable disease . once these data are coupled with the disease status in patients , the correlation may become apparent . serum - based detection of colorectal and pancreatic cancer biomarkers will improve the chances for early detection of these deadly diseases . the preliminary results with the npc-1c elisa to specifically detect tumor - associated muc5ac may improve the diagnosis of these solid tumors as well as aid in the immune monitoring and prognosis of patients undergoing treatment of their disease .