Palate Lung Nasal Clone (PLUNC) was first described in the nasal epithelium of the mouse embryo and the trachea and bronchi of adult mouse lung (Weston et al, 1999). We have subsequently shown that human PLUNC belongs to a family of 10 genes located on chromosome 20q11 (Bingle and Bingle, 2000; Bingle and Craven, 2002; Bingle et al, 2004) that are expressed in the upper airways, oro- and nasopharynx. Due to structural similarities with a family of lipid binding/transfer proteins that includes bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide binding protein (LBP), we have suggested that these proteins may play a role in host defence (Bingle and Craven, 2002). PLUNC proteins subdivide into short (SPLUNC) and long (LPLUNC) proteins, which contain domains structurally similar to one or both domains of BPI respectively (Bingle and Craven, 2002). Although many PLUNCs remain poorly characterised, our analysis suggests that they are expressed in the major salivary glands, the minor mucosal glands of the oral cavity and the respiratory epithelium of the nasal, tracheal and bronchial passages (Bingle and Bingle, 2000; Bingle and Craven, 2002). Limited studies have been performed at the protein level but some PLUNCs have been identified in saliva, nasal fluid and pulmonary secretions. For example SPLUNC1, SPLUNC2, and LPLUNC1 have been identified in saliva (Vitorino et al, 2004; Ramachandran et al, 2006; Guo et al, 2006) suggesting that these proteins are produced by the salivary glands. Although human PLUNCs have only recently been identified, a number of rodent orthologues including parotid secretory protein (psp) have been studied extensively (Ball et al, 2003). psp was identified as an abundant component of rat and mouse saliva (Owerbach and Hjorth, 1980; Mirels and Ball, 1992) and is highly expressed in salivary gland tissue. A second psp related protein, submandibular gland protein B (smgb) was also identified as being highly expressed in the salivary glands of rats (Mirels and Ball, 1992). Our recent analysis has shown that SPLUNC2 is the orthologue of rodent psp (Bingle et al, 2004) and consistent with this observation SPLUNC2 is predominantly expressed in the major salivary glands (Bingle and Craven, 2002; Geetha et al, 2005).

At the same time as we identified the human SPLUNC1 gene, it was also identified (and named LUNX) as a potential marker for micrometastasis for non-small cell lung cancer (NSCLC) (Iwao et al, 2001). LUNX expression was also detected in colorectal cancer, esophageal cancer, hepatocellular carcinoma and breast cancer (Iwao et al, 2001). The value of SPLUNC1/LUNX as a diagnostic marker in NSCLC has recently been confirmed in two additional studies (Mitas et al, 2003a; Mitas et al, 2003b) and we have also shown that SPLUNC1 protein is highly expressed in lung cancers with a glandular phenotype (Bingle et al, 2005). Differential expression of PLUNC family members has also been demonstrated in head and neck squamous cell carcinoma (Lemaire et al, 2003), nasopharyngeal carcinoma (Zhang et al, 2003) and breast tumours (Egland et al, 2003). Taken together these studies suggest that differential expression of PLUNC genes may be a characteristic of cancers and the study of these genes may prove to be of diagnostic and prognostic value. PLUNC proteins have not been investigated in salivary gland tumours, but their expression in other glandular neoplasms and in normal salivary glands suggests that they should be expressed and may be useful as diagnostic markers.

Having validated the antibodies on normal salivary gland tissue we applied them to the salivary gland TMAs. An oral squamous cell carcinoma (OSCC) was used as an orientation marker for TMA construction and was negative for all three PLUNC proteins.

The vast majority of the salivary gland tumours were negative for all three PLUNC proteins assessed. No immunoreactivity was seen in any cases of ACC, CEPA, PLGA, PA, AcCC or MC.

In mucoepidermoid carcinomas, LPLUNC1 (Figure 2A) displayed strong cytoplasmic positivity in the mucous cells and the mucin plugs (Figure 2B). Intermediate cells were moderately positive for LPLUNC1 (Figure 2C) and SPLUNC2 (Figure 2D). Epidermoid (Figure 2E) and clear cells (Figure 2F) within these samples were completely negative in all cases. No positive staining for SPLUNC1 was found in any case of MEC. All 10 cases exhibited a similar expression pattern. There were no correlations between PLUNC expression and tumour grade, size, age, gender or site.

Surprisingly only 2 of the 8 types of salivary gland tumours on our TMA demonstrated immunopositivity for any of the PLUNC family members studied. The mucin or mucous cells detected in the MECs and our single PC case were positive showing a strong relation between PLUNC expression and mucus-secreting salivary gland tumours. We have made a similar observation for SPLUNC1 in lung cancer (Bingle et al, 2005). The lack of immunoreactivity for PLUNC proteins in ACC, AcCC, CEPA, PA, PLGA and MC might be related to their histogenesis as these tumours do not produce or secrete mucin.

Papillary cystadenocarcinoma is a rare malignant neoplasm of the salivary gland mainly located in the parotid and intra-oral minor salivary glands (Harimaya et al, 2006). They are characterized by cystic structures lined by cuboidal, columnar and mucus-secreting cells and arranged in a papillary architecture (Nakagawa et al, 2002). The differential diagnosis includes papillary cystic variant of AcCC, low-grade MEC, salivary duct carcinoma, and PLGA (Foss et al, 1996). PC can usually be distinguished from these tumours using traditional stains such as H&E and PAS. However, some equivocal PC cases remain as diagnostic challenges even for senior pathologists. Slootweg (1993) reported the importance of correctly differentiating papillary PLGAs from PC in the oral cavity. Klijanienko and Vielh (1998) retrospectively compared fine needle aspiration samples from 12 PLGAs and 5 PC, both tumours showed variable proportions of malignant cells, occasionally arranged in pseudopapillary formations, whilst some PLGAs presented hyaline globules. In our study PC was immunoreactive for some PLUNC family members whereas the PLGA and AcCC cases were uniformly negative. Thus PLUNCs could aid in distinguishing PLGA and the papillary variant of AcCC from PC.

Metastases from prostate, gastrointestinal and thyroid tumors can affect the head and neck region and may also show papillary architecture. Established immunomarkers, such as PSA and thyroglobulin, could be useful in distinguishing PC from metastatic lesions but as expression of PLUNCs is limited to the lung, airways and oral cavity (Bingle and Bingle, 2000; Bingle and Craven, 2002; Bingle et al, 2004) they have the potential to give improved distinction.

MEC is the most common malignant salivary gland tumour (Speight and Barrett, 2002). Histopathologically, they are graded as low, intermediate or high-grade malignancy. H&E and other staining methods, notably, PAS and mucicarmine, are used to make a full diagnosis and grade lesions appropriately. High-grade lesions show a small number of mucous cells, which are difficult to recognize using these traditional staining methods. Mucous cells of the MEC studied were strongly positive for LPLUNC1 indicating a potential diagnostic use for this antibody in equivocal cases.

Another interesting finding was the immunoreactivity of some intermediate cells for LPLUNC1 and SPLUNC2. Based on these findings we can hypothesize that intermediate cells that are positive for PLUNC may have the potential to become mucous cells but further studies are needed to confirm this.