approximately 30,000 new cases of cancer in the oral cavity and oropharynx are diagnosed in the united states each year , corresponding to about 3% of all malignant tumors . although visibly detectable due to the accessibility of the oral cavity , oral cancer has a high morbidity and mortality because it is typically at an advanced stage when it is finally clinically visible . accordingly , the five - year survival rate for all stages combined is only 51% . of the cancers involving the oral cavity and oropharynx , more than 90% are squamous cell carcinomas [ 13 ] . up to 40% of those diagnosed with squamous cell carcinoma will develop a metastatic tumor or a second primary tumor to a nearby organ at a later time . early diagnosis of oral and oropharyngeal squamous cell carcinoma is crucial for a more favorable prognosis . the oral cavity and oropharynx are easily accessible for routine screening for squamous cell carcinoma , but reliable detection in early stages will require robust markers that are easy to assay . methods for testing oral epithelial cells for malignancy traditionally involve an invasive and often painful biopsy . whether as a diagnostic tool or prognostic marker , the potential to develop a simple , less invasive method for oral cancer screening would be of great value . human calmodulin - like protein calml3 ( synonyms : clp , calmodulin - related protein nb-1 ) is a 148-amino - acid - residue calcium sensor protein closely related to the ubiquitous calmodulin [ 4 , 5 ] . however , in contrast to calmodulin , calml3 is tissue specific and seems to be expressed almost exclusively in normally differentiating epithelia such as those of breast , thyroid , prostate , kidney , and skin . in a study comparing normal reduction mammoplasty specimens to archival breast cancer specimens , rogers et al .   found that calml3 was expressed at high levels in normal breast epithelium and that significant calml3 downregulation occurred in 79% to 88% of invasive ductal carcinoma and lobular carcinoma specimens . the authors concluded that calml3 downregulation is common in breast tumorigenesis and that the status of calml3 expression can serve as a marker for the nonmalignant state . we have previously found that calml3 is expressed at the mrna transcript level and can be detected by immunohistochemistry in normal oral mucosa tissue . by contrast , there was a notable reduction in immuno staining in areas of malignant transformation . here we performed immunohistochemical analysis to compare calml3 expression in tissue samples representing the various phases of oral tumor progression to see if there is a statistically significant trend of changes in calml3 expression during disease progression . in addition , direct comparisons in calml3 expression were made between the individual categories of benign , dysplasia , carcinoma in situ , and invasive squamous cell carcinoma . under irb - approved protocol 754 - 04 , the mayo clinic tissue registry was searched for patients that had undergone surgical biopsy of the oral cavity . a database was compiled placing the tissue specimens into 4 categories : benign squamous mucosa ( including mild reactivity and hyperplasia ) , squamous dysplasia , squamous cell carcinoma in - situ ( cis ) , and invasive squamous cell carcinoma . stained slides for the surgical procedures were evaluated and the most appropriate tissue block for each case was processed for calml3 immunostaining . a staff surgical pathologist ( thomas j. sebo ) confirmed the diagnosis of each sample . samples were eliminated if there were any questionable diagnoses or if the quality of the section was poor . a total of 90 tissue specimens were derived from 52 patients with 62 surgical procedures . for the purpose of this study specimens from the same patient were generally from different lesion sites taken during more than one date of surgery . among the 52 patients , 31 ( 60% ) were male and the overall mean age at the time of diagnosis was 64 years ( sd , 15 ; median , 67 ; range , 6 months87 years ) . among the 90 tissue specimens , 30 ( 33% ) were diagnosed as benign , 6 ( 7% ) as dysplastic , 12 ( 13% ) as carcinoma in situ , and 42 ( 47% ) as invasive squamous cell carcinoma . of the 42 samples diagnosed as squamous cell carcinoma , 2 ( 5% ) were grade 1 , 11 ( 26% ) were grade 2 , 27 ( 64% ) were grade 3 , and 2 ( 5% ) were grade 4 . other data recorded included location of specimen ( tongue , floor of mouth , right , left , etc . ) and type of surgery ( excision versus biopsy ) . the generation , affinity purification , and characterization of a rabbit polyclonal antibody ( tg7 ) against human calml3 have been described . reamstown ) recognize a peptide corresponding to the c - terminal residues 127 to 148 of calml3 ( the most divergent region between calml3 and calmodulin ) . affinity - purified antibodies showed excellent sensitivity and specificity for calml3 [ 5 , 7 ] . the paraffin - embedded tissue specimens were cut at a thickness of 6  m , mounted on positively charged slides , deparaffinized , and treated with h2o2/methanol to block endogenous peroxidase activity essentially as described [ 10 , 11 ] . the specimens received heat - induced epitope retrieval in 1  mm edta at a ph of 8.0 for 20 minutes . nonspecific protein - binding sites were blocked in 5% normal goat serum in phosphate - buffered saline solution ( pbs)/0.05% tween-20 for 1  h , and the sections were immunostained by sequential incubations ( 1 - 2  h at room temperature each ) in affinity - purified calml3 antibody tg7 ( 40  g / ml in pbs/0.05% tween-20/1% normal goat serum ) , biotinylated goat anti - rabbit igg ( 1  :  200 in pbs/0.05% tween-20/1% normal goat serum , dako , carpinteria , ca ) , and horseradish peroxidase - conjugated streptavidin ( 1  :  300 , dako , carpinteria ) essentially as described . the sections were then incubated in 3-amino-9-ethylcarbazole in the presence of h2o2 and counterstained with hematoxylin and mounted with coverslips . two observers ( a staff surgical pathologist ( thomas j. sebo ) and a maxillofacial prosthodontist ( michael d. brooks ) ) evaluated the specimens for expression of calml3 . staining patterns and intensity were observed and recorded for all categories of samples ( benign , dysplasia / carcinoma in - situ , and invasive squamous cell carcinoma ) . 35 separate fields of view were analyzed in 2 mucosal cell layers : superficial cells and basal cells . analysis of staining in cellular compartments was further subdivided into cytoplasmic membrane ( cm ) , cytoplasm ( c ) , and nucleus ( n ) . staining was graded on a 4-point scale : 0 : absent ( no staining above background of a negative control incubated with anti - calml3 igg preabsorbed with calml3 ) , 1 : weak ( barely above background and no strong staining in specific cellular regions ) , 2 : intermediate ( moderate staining with varying degree of subcellular staining ) , 3 : strong ( intense staining with distinct cellular localization ) . the cytoplasmic membrane , cytoplasm , and nuclei for each mucosal cell region were individually assigned a grade .   assigning a grade to first , the squamous cells were initially evaluated using two low magnification lenses ( 2.5x and 5.0x for a total magnification strength of 25x and 50x ) to obtain a general sense of staining intensity of all the squamous cells on the slide . second , for cytoplasmic membrane staining , cells either exhibited circumferential membrane staining or no membrane staining . third , membrane staining , apart from cytoplasmic staining , was typically most readily apparent in the intermediate layer of the squamous mucosal cells . as the cells reach the actual superficial layer of , for example , benign mucosa and become flattened , the ability to distinguish cytoplasmic staining from cytoplasmic membrane staining was limited . invariably , tissue samples in which no calml3 staining in the squamous cells was detected could very quickly be assigned a grade of 0 ( no staining ) . in the situation of superficial squamous cell staining , conversely , strong ( 3 + ) calml3 immunostaining could be readily ascertained using the same approach in 5%30% of the case material . in evaluating basally positioned squamous cells , no staining could be detected very quickly in 50% to almost 100% of the case material , whereas strong ( 3 + ) staining was very rare ( about 2% ) . the challenge in this study , and all studies in which immunostain intensity is visually graded , came in grading squamous cells as displaying either weak ( 1 + ) or intermediate ( 2 + ) calml3 expression . in these instances , an overall assessment of staining intensity at low magnification showed that the degree of staining could not clearly be categorized as 0 or 3 + , and a close analysis of all squamous cells in the tissue sample was performed using all magnification lenses ( 2.5x , 5x , 10x , 20x , and 40x for overall magnification of 25x , 50x , 100x , 200x , and 400x ) to arrive at a grade reflecting the staining intensity for all squamous cells . thus , a grade of 1 + ( weak ) indicates that the overall expression of the squamous cells was weak , showing only barely visible staining above 0 . for an intermediate grade ( 2 + ) , the degree of calml3 staining was felt to be unquestionably present but not as visually strong as a grade of 3 + .   on occasion , rare cells within any given tissue specimen graded as 1 + or 2 + showed strong ( 3 + ) or no ( 0 ) expression . however , no effort was made to further stratify case material on the basis of the percentage of cells showing a range of calml3 expressions . as such , our grading system reflects an overall score of calml3 staining intensity . in those instances in which the two reviewers ( thomas j. sebo and michael d. brooks ) recorded different grades for a given case , rereview of the case material was undertaken to arrive at a consensus grade . this occurred in roughly 10% of the case material .   for analysis purposes , grades 0 and 1 were collapsed and reported together as no / weak staining and grades 2 and 3 were collapsed and reported together as intermediate / strong stain . this is because visually weak staining most closely approximates no ( 0 ) staining and intermediate ( 2 + ) staining most closely approximates strong ( 3 + ) staining . total stain scores were derived by summing the stain grades for the three cellular compartments . the cochran - armitage test for trend was used to evaluate whether the proportion of specimens with intermediate / strong expression significantly changed with increasing disease severity . in addition , the chi - square test ( or the fisher 's exact test , as appropriate ) was used to compare the proportion of specimens with intermediate / strong expression between the benign , dysplasia / cis , and invasive tissue groups . the kruskal - wallis test was used to compare the total stain scores between the three groups . if the p value for the overall test of group differences was < 0.05 , then pair - wise comparisons among the three groups were performed . all calculated p values were two - sided and p values less than 0.05 were considered statistically significant . a bonferroni correction could be applied by assessing the pair - wise comparisons between the three tissue types using an alpha level of 0.0167 to account for the three comparisons . statistical analysis was performed using the sas software package ( sas institute , cary , nc ) . as we expected from previous studies [ 6 , 8 ] , benign mucosa displayed robust immunostaining ( figures 1(a ) and 1(b ) ) . distinct staining was noted in the periphery ( cytoplasmic membrane ) and nuclei of the more superficially located epithelial cells ( figure 1(b ) ) . on the other hand , in samples with dysplasia ( figures 1(c ) and 1(d ) ) and carcinoma in - situ ( not shown ) , a marked decrease was seen in strength of stain . a strong reduction ( or complete lack ) of calml3 immunostaining was seen in invasive squamous cell carcinoma compared to both benign tissue and dysplasia / cis tissue ( figures 2 and 3 ) , although in low - grade invasive squamous cell carcinoma , keratin pearls did illustrate mild immunoreactivity . the basal cell layers in general did not stain as intensely as the more superficial cell layers , and no statistical difference was seen with basal nuclear and cytoplasmic staining among the groups . table 1 summarizes the calml3 expression results at the superficial and basal mucosa levels , separately for the tissue types . because similar results were found for the dysplastic and cis specimens , these two tissue types were combined for the analysis . in addition , we found that aside from the superficial cytoplasmic membranes and nuclei of the benign specimens , only a small percentage of the other tissue regions exhibited strong expression . therefore to evaluate whether there was a trend in the expression level with tissue disease severity , the specimens with weak or no staining were combined , as were those with intermediate or strong staining ( see table 1 ) . however , the detailed summary of all staining results broken down by individual tissue type is available upon request . based on the cochran - armitage test for trend , the calml3 expression level significantly decreased with increasing disease severity for the following sites : superficial cytoplasmic membrane ( p < 0.001 ) , superficial nucleus ( p < 0.001 ) , and basal cytoplasmic membrane ( p = 0.009 ) . on the other hand , a statistically significant trend was not observed for superficial cytoplasm ( p = 0.19 ) and basal cytoplasm ( p = 0.19 ) , in which several dysplastic / cis specimens had stronger expression than the benign tissue . none of the basal nucleus sites had a stronger than  weak  staining score for any of the tissue types . the chi - square test ( or the fisher 's exact test , where appropriate ) was used to compare the proportion of specimens with an intermediate or strong stain between the benign , dysplasia / cis , and invasive tissue groups ( table 1 ) . benign tissue specimens had significantly more calml3 expression in the superficial mucosa compared to invasive specimens ( p < 0.001 ) and dysplasia / cis specimens ( p = 0.013 ) , as revealed by a comparison of the total stain scores ( table 1 and figure 4 ) . this difference in calml3 staining was most pronounced for the cytoplasmic membrane and the nucleus . dysplasia / cis tissue also had significantly ( p = 0.003 ) more expression than invasive tissue in the superficial mucosa ( table 1 and figure 4 ) , and this difference was most clearly demonstrated in the cytoplasm ( 50% versus 7.1% , p < 0.001 ) and in the nuclei ( 38.9% versus 2.4% , p < 0.001 ) . all of the previously outlined comparisons of expression in the superficial layer between the three tissue groups retained statistical significance if a bonferroni correction was applied and significance is assessed using an alpha level of 0.0167 instead of 0.05 . expression was not strong in any of the sites in the basal layer ( table 1 ) , though there was evidence suggesting a slight increase in expression in the cytoplasmic membrane of benign squamous cells compared to invasive carcinoma ( 13.3% versus 0% , p = 0.027 ) . in analyzing the 42 specimens of squamous cell carcinoma separately by grade , previously , loss of calml3 immunoreactivity has been linked to early breast cancer development . here , we show that while calml3 is strongly expressed in the superficial layers of benign oral mucosa , different oral mucosa tissue types representing the various stages of carcinogenesis exhibit a reduction of calml3 expression as squamous cells progress from benign , to dysplastic , to carcinoma in situ , to invasive squamous cell carcinoma . in general , a trend is seen that as disease severity increases , calml3 expression decreases . many of the benign specimens showed an increase in staining intensity the further the cells moved away from the basal cell layer . as the cells mature , they differentiate and migrate to the upper layers as underlying cells develop . the more intense calml3 staining towards the outer layers of mucosa is consistent with previous reports that noted markedly increased staining in more differentiated layers of stratified epithelia . therefore , calml3 may play a role in terminal differentiation of oral keratinocytes , as has been proposed for the basal and suprabasal keratinocytes of the epidermis during wound healing . calml3 is a calcium - sensor protein related to calmodulin and as such is thought to exert its function by regulating specific target proteins . among these , the unconventional myosin - x   is known to be involved in directional cell migration and cell adhesion , both are important events during terminal keratinocyte differentiation . the staining for calml3 in the cytoplasmic membrane of superficial cells in benign oral mucosa may thus reflect its function in regulating myox concentrated at the cell periphery . like calmodulin , calml3 has multiple targets , one of which ( iq motif containing protein e or iqce ) was recently identified in a yeast two - hybrid screen and may be a protein involved in dna metabolism ( richard d. bennett and emanuel e. strehler , unpublished ) . such a putative nuclear calml3 target protein(s ) could help explain the nuclear staining for calml3 that we observed in the superficial cells of the normal oral mucosa . however , additional studies are needed to determine the role of calml3 in the nucleus and its relevance to normal epithelial differentiation techniques being implemented for diagnostic tests include transepithelial brush biopsy / exfoliative cytology , fine needle aspiration , and toluidine blue [ 1523 ] . these techniques all rely on cytological markers to indicate the presence of malignant or premalignant cells . although these tests are preferred over conventional biopsy confirmation because they are less invasive and more tolerable for the patient , their reliability and efficacy have not been validated [ 2426 ] . because the specific expression and cellular localization of calml3 indicate normal cell function , the potential for development of a simple diagnostic test for oral cancer using calml3 as a marker is promising . currently diagnostic screening tools are being utilized to visualize autofluorescence in normal oral tissue with loss of autofluorescence indicative of tissue change or abnormality . noninvasive screening could be implemented by the use of exfoliative brushing cytology , minibiopsy , or a  swish - and - spit  procedure . this would allow a clinician who observes a suspicious area in the mouth to routinely test for calml3 levels . a change in calml3 expression could indicate the presence or the early developing stage of oral cancer .