ADSC has two distinct histologic components. SCC usually predominates, can be in situ or invasive, and can range from well to poorly differentiated. The adenocarcinomatous component can have a tubular, alveolar and/or glandular morphology. Mucin production is typically present, but it is not a requirement for diagnosis in the presence of true gland formation [1, 2]. The gland formation classically should consist of “punched out” spaces with smooth, rather than ragged, edges.

It has been recently shown that HPV is an important etiologic agent in head and neck carcinomas, particularly in the oropharynx and nasal cavity. These tumors may present as different variants distinct from the typical keratinizing-type SCC, including nonkeratinizing [5, 6], basaloid [7, 8], and papillary SCC [9] and also undifferentiated carcinoma [10]. It has also been documented in several studies that HPV-related carcinomas of the upper aerodigestive tract have more favorable prognosis than HPV negative ones [11–13]. Most previous studies investigated HPV in tumors using DNA-based polymerase chain reaction (PCR) [14] while in more recent years DNA-based in situ hybridization (ISH) has been used [5, 13, 15]. However, it is becoming increasingly recognized that HPV can be present without necessarily being biologically active, such that mere presence of the virus (as detected by these methods) may not indicate biological/clinical significance. The presence of virus in transcriptionally active form, as evidenced by expression of high risk HPV proteins E6 and E7, has emerged as the gold standard for biologically significant HPV in SCC [16]. Newer studies have utilized reverse transcriptase (RT) PCR [17], which is cumbersome for routine clinical use due to the complex procedures of RNA extraction from formalin-fixed paraffin-embedded tissues and RT–PCR. Here, we use a recently developed slide-based RNA ISH assay which allows for visual evaluation for E6/E7 expression directly in individual tumor cells (Fay et al. manuscript in preparation).

p16 is a tumor suppressor protein that is considered an excellent surrogate marker of HPV in SCC of the cervix and head and neck [18]. It is overexpressed in HPV-related SCC but is usually deleted or otherwise inactivated in non-HPV-related head and neck SCC [19, 20]. p16 expression by immunohistochemistry approaches 100% in HPV-positive oropharyngeal carcinomas in some studies [5, 11, 21, 22] and is indicative of transcriptionally-active HPV. HPV-negative SCCs tend to harbor p53 mutations leading to nuclear accumulation of p53 protein that is detectable by immunohistochemistry. In HPV-related SCC, p53 is frequently wild-type, being targeted for ubiquination and degradation by the HPV E6 oncoprotein, leading to little or no detectable protein by immunohistochemistry [6, 23].

Clinical and pathologic data, along with HPV status and immunohistochemistry for p16 and p53, are shown for all patients in Table 1. Patients’ ages ranged from 42 to 87 years (mean 59.7 years; median 60.5 years). Sixteen of the eighteen patients (88.9%) were men. For the 15 patients where status could be obtained, 11 (73.3%) were lifetime tobacco users (nine smokers and two oral tobacco). The larynx was the most common site (8/18–44.5%), followed by the oral cavity (4/18–22.2%), nasal cavity (3/18–16.7%) and oropharynx (3/18–16.7%). Tumor size ranged from 0.5 to 6.5 cm. Angiolymphatic invasion was present in five cases and perineural invasion was present in six. Lymph node metastases were present in eight patients (44.5%) at diagnosis/presentation. Two patients (cases 5 and 6) developed local recurrence 5 months and 1 month after surgery, respectively. Two patients (cases 2 and 15) developed regional recurrence 16 and 24 months post surgery, respectively. Distant metastases developed in four patients (case 4—neck skin, 30 months; case 5—lung and liver, 5 months; case 7—lung, 128 months; case 15—lung, 23 months). Most tumors were stage IV (10/18), with one Stage III, two Stage II and three Stage I tumors. Staging information was not available for two patients. Seventeen patients (94.4%) were treated by surgical excision, supplemented in nine patients with postoperative radiation. Chemotherapy was additionally given in three patients. Treatment information for one patient (case 1) was not available. Follow up data was available for 16 patients and ranged from 1.5 to 128 months (median 30 months). Six patients died of disease and one patient died of unrelated causes. Seven patients were alive with no evidence of disease and two patients were alive with disease. For the group of 10 patients with sufficient follow up data, the 3 years overall survival rate was 50%.

HPV status by ISH and immunohistochemistry (Fig. 2) results according to the location of the tumors are shown in Table 2. The majority of cases were p53 positive (14/18, 78%) above the threshold we utilized to indicate likely mutation. Immunoreactivity was diffuse and intense in all of these cases and was observed in both the squamous and glandular components. Five tumors (27.7%) were HPV positive by DNA ISH and 13 (72.3%) were HPV negative. HPV DNA ISH staining ranged from focal to diffuse. Three tumors (20.0%) were positive by RNA ISH for high risk HPV E6 and E7 expression, and the staining was strong and diffuse in all of them. Three tumors could not be evaluated because, due to RNA degradation, there was inadequate control ubiquitin C staining or in one case there was no tumor remaining in the block. Nine tumors (50.0%) were positive for p16 by IHC. However, strong, diffuse staining with p16 is frequently considered necessary for p16 as a surrogate marker of HPV. As such, seven tumors (38.9%) were p16 positive with greater than 50% of cells staining including all cases which were high risk HPV RNA ISH positive.

Only three cases (16%) were positive for transcriptionally-active HPV (i.e. positive by all three assays). One arose in the nasal cavity (case 14) and two in the oropharynx (cases 6 and 12). Of these three cases, two were from smokers (one oropharynx and one nasal cavity) and one (oropharynx) was a non-smoker. Interestingly, all three of these cases showed overexpression of p53.

Both transcriptionally-active HPV-related oropharyngeal ADSC patients were alive and disease free at 103 and 34 months, respectively, while, after excluding those with minimal clinical follow up information, six of the remaining 14 patients (42.8%) had died of their tumors. All six patients with no evidence of HPV, lack of p16 expression, and positive overexpression of p53, regardless of primary anatomic site, had disease recurrence, with five dying of disease and the sixth having regional disease recurrence at 7 months.

Recent studies have demonstrated that head and neck SCC patients, particularly of the oropharynx, have a much better prognosis when their tumors are associated with transcriptionally-active HPV than when they are not [13]. High risk HPV types, particularly type 16, have been detected in as many as 80% of oropharyngeal SCC [11]. The virus contributes to carcinogenesis by expressing E6 and E7 oncoproteins, which bind to p53 and retinoblastoma (Rb), respectively. These latter proteins are critical for apoptosis and cell cycle regulation and, when disrupted, cause cells to proliferate and fail to undergo apoptosis appropriately. Inactivation of Rb, in particular, leads to marked overexpression of the tumor suppressor protein p16, for which transcription is normally repressed by Rb. This marked overexpression, which is very uncommon in non-HPV-related SCC, makes p16 a good surrogate marker of HPV, but more importantly, a good marker of transcriptionally-active HPV [18], because while HPV DNA can be found in a relatively high numbers of head and neck SCC of all anatomic subsites, the ones with better clinical outcomes have only been those where HPV is present in the tumor and in transcriptionally active form [12]. It is generally accepted that if one finds evidence of high risk HPV in a tumor that coexistent overexpression of p16 and/or significant expression of HPV E6 and E7 transcripts, this indicates biologically and clinically significant role of HPV in the tumor. Occasionally, p16 is overexpressed but no HPV can be detected. These tumors, in the oropharynx, still appear to have the same good prognosis, but their biology is still unknown. It is possible that in most such tumors, HPV is present but the tests don’t detect it. But one can also speculate that tumors can truly overexpress p16 in the absence of HPV. This is something yet to be clearly defined. For non-oropharyngeal SCC, the significance of p16 overexpression is not clear. It does not seem to be a good marker of prognosis or necessarily a good surrogate for HPV. We had a few non-oropharyngeal, p16 overexpressing ADSC cases and these did not harbor HPV. Although they appeared to do well clinically, the number of cases is too small to make any meaningful conclusions.

Several histologic variants of SCC, including nonkeratinizing SCC, basaloid SCC, papillary SCC, and undifferentiated carcinoma [7–10], have been shown to harbor high risk HPV when arising in the oropharynx. Papillary SCC, in particular, can have transcriptionally-active HPV in non-oropharyngeal locations such as the larynx [9]. Where clinical outcomes have been investigated in these variants, HPV-positive patients have had better survival. The relationship of head and neck ADSC to HPV has not been previously investigated. In the uterine cervix [24], as many as 75% of cases harbor HPV [45], and in the single series of lung cases, most harbored HPV as well [46].

In our series of 18 ADSC cases, we utilized a novel, slide-based high risk HPV RNA ISH assay along with the traditional p16 IHC and Ventana DNA ISH assays. This new RNA-based assay for transcriptionally-active HPV allows evaluation for viral E6 and E7 directly in tissue sections. This is considered by many to be the “gold standard” for biologically relevant tumor HPV. Only a small minority of ADSC harbored HPV (2 oropharynx, 1 nasal cavity, 2 larynx) with fewer still (2 oropharynx, 1 nasal cavity) harboring transcriptionally active HPV and showing overexpression of p16. As mentioned earlier, the oropharynx and the nasal cavity are sites with known high-prevalence of HPV-related carcinomas. We have added 18 cases of ADSC to the existing literature. Our cases had a similar prognosis to those in the literature review, supporting the notion that ADSC of the head and neck is more aggressive than typical squamous cell carcinoma. ADSC may be relatively even more aggressive if one excludes good prognosis, HPV-related oropharyngeal cases and evaluates the remaining group.

Two of the five HPV DNA ISH positive cases lacked both p16 overexpression and high risk HPV RNA expression. These are likely false positives due to high, nonspecific background blue staining on the DNA ISH masquerading as true staining. Or, alternatively, this may represent the presence of high risk HPV DNA which does not have enough transcriptional activity to demonstrate E6 and E7 expression above background. As is well known, high risk HPV DNA can be found in a large percentage of non-oropharyngeal SCC from PCR assays, but we know from functional assays that there isn’t evidence of transcriptional activity in most non-oropharyngeal SCC. These ADSC cases could simply represent this phenomenon.

It is generally accepted that the majority of HPV-related tumors harbor an unmutated wild type p53 protein. In contrast, we observed a high rate of p53 protein overexpression as evidenced by p53 immunostaining in the majority of our cases, including the HPV-related ones. Similar findings were also reported by Alos et al. in their series of 12 patients [30]. This observation suggests that p53 mutations may play an important role in tumorigenesis of ADSC. The four patients in our series with p53 negative tumors were alive without disease with follow up ranging from 3.5 to 123 months (cases 7, 8, 9, 13). Interestingly, however, all six patients with p16 and HPV negative tumors also showing overexpression of p53 suffered disease recurrence, with most dying with disease.