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The distinction of adenoid cystic carcinoma from basaloid squamous cell carcinoma can be difficult or impossible. Basaloid squamous cell carcinomas may have areas of obvious squamous differentiation admixed with solid tumor islands that exhibit peripheral palisading and a thick basement membrane. However, particularly in small biopsies, obvious squamous differentiation and palisading may not be identified and it is not uncommon for moderately to poorly differentiated basaloid squamous cell carcinomas to undergo central cystification with accumulation of hyaline granules and to appear very similar to adenoid cystic carcinoma on hematoxylin and eosin stain (Figure 1). Conversely, adenoid cystic carcinomas composed of solid arrays of tumor cells may be difficult to distinguish from basaloid squamous cell carcinoma.1 The optimal treatment regimen is different for each entity. Treatment of adenoid cystic carcinoma involves surgery and postoperative radiotherapy. There is no effective chemotherapeutic agent. Basaloid squamous cells carcinoma is treated with surgery, radiotherapy, or both, with or without chemotherapy (5-fluorouracil and cisplatin).2, 3, 4

Figure 1
figure 1

Basaloid squamous cell carcinoma. Hematoxylin and eosin-stained section showing central accumulation of hyaline granules, mimicking adenoid cystic carcinoma.

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p63 was discovered in a screen for p53-homologous genes. Like p53, p63 codes for proteins with an amino(N)-terminal transcription-activating region, a middle DNA-binding region, and a carboxyl terminal region responsible for oligomerization.5 p63 comprises at least six different protein isoforms with homology to the tumor suppressor protein p53. They are generated from a single gene by two promoters and alternative splicing of the primary RNA transcript.5 Certain isoforms (TAp63) are able to transactivate p53 target genes. Other isoforms (ΔNp63) act as dominant-negative factors that inhibit transcription activation by both p53 and TAp63 isoforms.5 The ΔNp63α isoform appears to be a master regulator of squamous stem-cell differentiation.6 Transgenic mice lacking p63 are born without skin, mammary, lachrymal and salivary gland tissue, reflecting a critical role in the development and/or maintenance of several types of epithelia.7, 8

Previous studies in several laboratories have shown p63 expression in many normal tissues including squamous epithelia, urothelium, bronchial epithelium, and the myoepithelial layers of breast, prostate and submucosal glands.9, 10, 11, 12 Expression has also been established in a variety of neoplasms, including squamous, urothelial, endometrial, and papillary thyroid carcinomas and thymomas.13, 14, 15, 16, 17, 18, 19, 20

p63 expression in salivary gland and salivary neoplasms has also been investigated. Benign myoepithelial cells of salivary gland, like those of normal breast and prostate epithelium, express p63.21 Di Como et al9 reported p63 expression in four pleomorphic adenomas and two of four carcinomas of the salivary glands. Bilal et al found that for all salivary gland-derived tumor types that differentiated towards luminal and myoepithelial lineages (pleomorphic adenomas, basal cell adenomas, and adenoid cystic carcinomas), p63 was expressed in myoepithelial cells, whereas internal luminal cells were always negative. In 11 out of 11 adenoid cystic carcinomas, they found the peripheral cells of myoepithelial differentiation to be diffusely and strongly reactive for p63 while all luminal cells were p63-nonreactive.22

We investigated the possibility that p63 immunostaining could be of value in resolving the often difficult differential diagnosis of basaloid squamous cell carcinoma vs adenoid cystic carcinoma.

Materials and methods

In total, 14 archival head and neck adenoid cystic carcinomas and 16 basaloid squamous cell carcinomas were examined. The adenoid cystic carcinomas were from parotid (n=4), sublingual gland (n=3), nasopharynx (n=1), maxillary sinus (n=1), maxilla (n=1), submandibular gland (n=1), mandible (n=1), larynx (n=1) and hard palate (n=1). The basaloid squamous cell carcinomas arose in the tonsil (n=7), tongue (n=4), sinonasal area (n=2), larynx (n=2) and pharynx (n=1). Two of the adenoid cystic carcinomas and three of the basaloid squamous cell carcinomas were small biopsies and the remainder were excision specimens.

Immunohistochemical staining was performed as follows: 4 μm thick sections were deparaffinized, treated with 3% hydrogen peroxide to block endogenous peroxidase activity, then treated with 0.01 M citric acid (pH 6.0) for 5 min at 100°C for antigen retrieval. Slides were then incubated overnight at room temperature with anti-p63 monoclonal antibody 4A4 (1:600 Santa Cruz Biotechnology, Santa Cruz, CA, USA), stained using a streptavidin–biotin kit (BioGenex, San Ramon, CA, USA, catalog no. QP900-9L) and diaminobenzidine as a chromagen, then counterstained with hematoxylin. p63 expression was considered positive only if distinct nuclear staining was present.

Results

p63 was detected in all 14 adenoid cystic carcinomas and 16 basaloid squamous cell carcinomas. In the series of 16 basaloid squamous cell carcinomas, diffuse p63 staining of between 90 and 100% of tumor cells was consistently found. Some large tumor cell nests of basaloid squamous cell carcinoma contained central necrotic areas. p63 staining was characteristically strong, and occasionally of variable intensity (Figures 2 and 3). Only in a rare perinecrotic focus in an otherwise diffusely p63-positive basaloid squamous cell carcinoma, were isolated p63-negative tumor cells detected (Figure 4).

Figure 2
figure 2

Basaloid squamous cell carcinoma. p63 immunohistochemical staining demonstrating diffuse nuclear staining.

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Figure 3
figure 3

Basaloid squamous cell carcinoma. p63 immunohistochemistry staining demonstrating diffuse nuclear staining. Inset: high power, Ă— 400.

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Figure 4
figure 4

Poorly differentiated basaloid squamous cell carcinoma. Focal p63 negativity in a perinecrotic area. The adjacent viable tumor cells are p63 positive.

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In the series of 14 adenoid cystic carcinomas, the p63 staining pattern was compartmentalized and readily distinguishable from the diffuse staining pattern present in basaloid squamous cell carcinoma. Two patterns were noted:

  1. 1)

    a purely peripheral pattern, noted in seven out of 14 cases, consisting of histomorphologically solid or cribriform nests in which p63-positive cells were exclusively located in a single layer at the periphery of the tumor cell nest and all but the peripheral layer of cells were p63-negative (Figures 5 and 6). When situated at the periphery of nests, the p63-positive cells were either scattered or extensively positive (Figures 5 and 6);

    Figure 5
    figure 5

    Adenoid cystic carcinoma. p63 immunohistochemistry demonstrating the peripheral pattern of p63 positivity. Diffuse peripheral positivity is seen.

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    Figure 6
    figure 6

    Adenoid cystic carcinoma. p63 immunohistochemistry demonstrating the peripheral pattern of p63 positivity. Focal peripheral positivity is seen.

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  2. 2)

    a peripheral/internalized staining pattern, seen in five out of 14 cases, in which p63-positive cells were found internally in tumor nests as well as at the periphery. p63 staining appeared to identify glandular/cribriform-like units of p63-positive cells surrounding or interspersed with small groups of p63-negative cells, which in aggregate comprised larger tumor nests (Figures 7 and 8). In several such cases, the p63-positive cells at either the periphery or scattered within the nests were predominantly smaller in size than the p63-negative cells and unlike the p63-negative cells, appeared to lack nucleoli (Figure 8).

    Figure 7
    figure 7

    Adenoid cystic carcinoma with a cribriform pattern and comedo-like central necrosis (N). The peripheral/internalized p63 staining pattern is present, with a high proportion of p63-positive tumor cells.

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    Figure 8
    figure 8

    Adenoid cystic carcinoma with compartmentalized p63 staining. The p63-positive cells are predominantly smaller than the p63-negative cells, and appear to lack nucleoli (high power view of Figure 7 Ă— 200).

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In two of 14 cases, some nests showed the pure peripheral pattern, and other nests the peripheral/internalized pattern.

The case of adenoid cystic carcinoma with the highest proportion of p63-positive cells was a high-grade case consisting histomorphologically of cells with cribriform patterns in large sheets with comedo-like central necrosis. It consisted of p63-positive cells in the peripheral cell lining and many p63-positive cells (one-third to half of all tumor cells) internally in the cribriform regions (Figures 7 and 8).

The only circumstance in which a p63 staining pattern in adenoid cystic carcinoma in any way resembled the diffuse pattern of basaloid squamous cell carcinoma occurred in rare small solid nests that contained mostly p63-positive cells and rare p63-negative cells. However, in such cases, surrounding tumor nests displaying the characteristic compartmentalized staining predominated, allowing for ready distinction from basaloid squamous cell carcinoma (Figure 9).

Figure 9
figure 9

Adenoid cystic carcinoma showing solid nest with diffuse distribution of small p63 positive cells (left). The adjacent areas (seen on the right of this image) showed clear compartmentalized staining, consistent with adenoid cystic carcinoma.

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Discussion

We describe a means, via p63 immunostaining, of accurately distinguishing adenoid cystic carcinoma from basaloid squamous carcinoma. We found the p63 staining pattern of basaloid squamous cell carcinoma to differ strikingly from the staining pattern in adenoid cystic carcinoma. In the former, diffuse p63 staining of up to 100% of tumor cells was consistently found, whereas in adenoid cystic carcinoma, staining was compartmentalized.

The existence of a subpopulation of p63-positive cells in tumor cell nests in adenoid cystic carcinoma suggests the possibility that p63-positive cells may comprise a stem-cell compartment that drives the growth of this neoplasm. These p63-positive cells may both proliferate into other p63-positive stem cells, as well as differentiate into the centrally located or contiguous p63-negative tumor cells, much as basaloid p63-positive stem cells in normal squamous epithelia are thought to divide into two daughter cells, one retaining the p63-positive undifferentiated stem-cell-like phenotype, and one that differentiates into a p63-negative, nondividing mature squamous cell. This model would pinpoint the p63-positive cells in adenoid cystic carcinoma as the appropriate target cells for anti-adenoid cystic carcinoma therapeutic strategies.

In an alternative model, in adenoid cystic carcinomas manifesting the purely peripheral staining pattern, the peripheral layer of p63-positive cells may be the residual benign myoepithelial lining of normal ducts into which p63-negative cells extend intraductally by mass action rather than true invasion. This latter model would clearly not satisfactorily explain cases of adenoid cystic carcinoma with a peripheral/internalized pattern, with cribriform-like staining, in which p63-positive cells are also found internally within tumor nests.

To our knowledge, this peripheral/internal pattern of adenoid cystic carcinoma has not been previously described. Bilal et al22 reported that all adenoid cystic carcinomas studied showed selective staining of the peripheral cell layer; staining of internally located cells was not reported. It may be of future interest to compare the aggressiveness of adenoid cystic carcinomas displaying the purely peripheral pattern to those with the peripheral/internal pattern of p63 positivity.

In contrast to adenoid cystic carcinoma, the diffuse pattern of p63 positivity in basaloid squamous carcinomas may represent a complete failure of the normal program of squamous differentiation: a malignantly transformed basaloid stem cell, upon division and driven by unknown oncogenic mechanisms, yields two daughter cells, both of which stay undifferentiated, dividing, and p63-positive.

It appears that p63 expression may be a feature of neoplastic and non-neoplastic cell lineages that are squamous or are capable of undergoing squamous differentiation, including squamous-cell carcinomas of all sites, urothelial, endometrial, papillary thyroid carcinomas and thymomas.13, 14, 15, 16, 17, 18, 19, 20 Conversely, renal and Barrett's metaplastic epithelium and associated adenocarcinomas, which lack the capacity for squamous differentiation, are consistently p63-negative.16, 23 Adenoid cystic carcinomas, which lack the capacity for squamous differentiation but are p63-positive, appear to be one exception to this principle.

p63 positivity in adenoid cystic carcinoma presumably represents a neoplastic recapitulation of the p63-positive basal/myoepithelial cell phenotype present in normal salivary gland.

However, the pattern of p63 expression in adenoid cystic carcinoma differs from staining in adenocarcinomas derived from other types of acinar/glandular tissues with p63-positive myoepithelial cells. For example, normal breast and prostate, like salivary gland, have p63-positive basal or myoepithelial cells, yet most adenocarcinomas of the breast or prostate do not express p63.24, 25

While both adenoid cystic carcinoma and basaloid squamous cell carcinoma staining patterns suggest models that implicate tumor stem-cell function and dysfunction and raise interesting additional long-term questions impacting pathogenesis, therapy and prognosis that merit future investigation, the present findings suggest an immediate clinical benefit, via p63 immunostaining, in resolving the differential diagnosis between basaloid squamous cell carcinoma and adenoid cystic carcinoma.