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1.  Most Non-Parotid “Acinic Cell Carcinomas” Represent Mammary Analogue Secretory Carcinomas 
Acinic cell carcinoma (ACC) is a low grade salivary gland malignancy characterized by serous acinar differentiation. Most ACCs arise in the parotid gland, but ACCs have been reported to originate in non-parotid salivary glands where where serous acini are less abundant. Given the recent discovery of mammary analogue secretory carcinoma (MASC) – a salivary malignancy that histologically mimics ACC – a retrospective re-evaluation of non-parotid ACCs is warranted. The surgical pathology archives of The Johns Hopkins Hospital were searched for all ACCs arising outside of the parotid gland. For each case, the histologic slides were reviewed; immunohistochemistry (mammaglobin, S100 protein) was performed; and confirmatory ETV6 break-apart FISH assay was completed. Demographic and clinical data was obtained from the medical records. Fourteen extra-parotid tumors diagnosed as ACC were identified. Eleven of 14 (79%) tumors harbored the ETV6 translocation (oral cavity = 9 of 11; submandibular gland = 2 of 2). The translocation positive tumors occurred in 7 women and 4 men ranging in age from 20–86 years (mean, 56), and usually presented as painless masses. Immunohistochemistry for mammaglobin and S100 was positive in all 11 translocation positive tumors, but negative in the 3 translocation negative tumors. Histologically, the translocation positive tumors exhibited uniform cells with vacuolated cytoplasm, microcystic/cystic and papillary architecture, and intraluminal secretions; but the presence of basophilic cytoplasmic granules was conspicuously absent. Basophilic cytoplasmic granules, indicative of true serous acinar differentiation, were present in the 3 translocation negative tumors. Of the translocation positive tumors, only one locally recurred, and none metastasized. Most alleged ACCs of non-parotid origin actually represent misclassified MASCs. The impact of diagnostic error is mitigated by the low grade nature of MASC that, like ACCs, do not appear to be clinically aggressive.
PMCID: PMC3686973  PMID: 23681074
Acinic cell carcinoma; mammary analogue secretory carcinoma; ETV6-NTRK3 translocation; minor salivary gland carcinoma; mammaglobin; S100
2.  Human Papillomavirus-Related Carcinoma with Adenoid Cystic-Like Features: A Peculiar Variant of Head and Neck Cancer Restricted to the Sinonasal Tract 
HPV-related carcinomas of the head and neck are characterized by a predilection for the oropharynx, a non-keratinizing squamous morphology, and infection with the HPV 16 type; but comprehensive HPV testing across all head and neck sites has shown that the pathologic features of HPV-related carcinoma may be more wide-ranging than initially anticipated. In particular, a subset of sinonasal carcinomas are HPV positive, and these include a variant that is histologically similar to adenoid cystic carcinoma (ACC). Cases were identified by retrospective and prospective analyses of head and neck carcinomas with ACC features. HPV analysis was performed using p16 immunohistochemistry and high risk HPV in-situ hybridization. HPV-positive cases were confirmed and typed using HPV type-specific quantitative PCR, and further characterized regarding their immunohistochemical profile and MYB gene status. HPV was detected in 8 carcinomas of the sinonasal tract, but it was not detected in any ACCs arising outside of the sinonasal tract. The HPV types were 33 (n=6), 35 (n=1) and indeterminate (n=1). Six patients were women and two were men, ranging in age from 40–73 years (mean 55). The carcinomas were characterized by a nested growth, a prominent basaloid component showing myoepithelial differentiation and forming microcystic spaces, and a minor epithelial component with ductal structures. Squamous differentiation, when present, was restricted to the surface epithelium. The carcinomas were not associated with the MYB gene rearrangement that characterizes a subset of ACCs. These cases draw attention to an unusual variant of HPV-related carcinoma that has a predilection for the sinonasal tract. Despite significant morphologic overlap with ACC, it is distinct in several respects including an association with surface squamous dysplasia, absence of the MYB gene rearrangement, and an association with HPV, particularly type 33. As HPV positivity confers distinct clinico-pathologic characteristics when encountered in the oropharynx, a more comprehensive analysis of risk factors, response to therapy and clinical outcomes is warranted for HPV-related carcinomas of the sinonasal tract.
PMCID: PMC3653997  PMID: 23598962
Human papillomavirus; sinonasal carcinoma; adenoid cystic carcinoma; squamous cell carcinoma
3.  Activation of the NOTCH pathway in Head and Neck Cancer 
Cancer research  2013;74(4):1091-1104.
NOTCH1 mutations have been reported to occur in 10 to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation and mutation analyses. Copy number increases were identified in NOTCH pathway genes including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4/37 of the tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptors mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently.
PMCID: PMC3944644  PMID: 24351288
4.  Human papillomavirus status of head and neck cancer as determined in cytologic specimens using the hybrid-capture 2 assay 
Oral oncology  2014;50(6):600-604.
A standardized assay to determine the HPV status of head and neck squamous cell carcinoma (HNSCC) specimens has not yet been established, particularly for cytologic samples. The goal of this study was to determine whether the hybrid capture-2 (HC-2) assay, already widely used for the detection of high risk HPV in cervical brushings, is applicable to cytologic specimens obtained from patients with suspected HNSCCs.
Materials and methods
Fine needle aspirates (FNA) of cervical lymph nodes were pre-operatively obtained from patients with suspected HNSCCs and evaluated for the presence of HPV using the HC-2 assay. HPV analysis was performed on the corresponding resected tissue specimens using p16 immunohistochemistry (IHC) and HR-HPV in situ hybridization (ISH). A cost analysis was performed using the Center for Medicare & Medicaid Services.
HPV status of the cervical lymph node metastases was correctly classified using the HC-2 assay in 84% (21/25) of cases. Accuracy was improved to 100% when cytologic evaluation confirmed the presence of cancer cells in the test samples. The estimated cost savings to CMS using the HC-2 assay ranged from $113.74 to $364.63 per patient.
HC-2 is a reliable method for determining the HPV status of HNSCCs. Its application to HNSCCs may reduce costs by helping to localize the primary site during the diagnostic work-up as well as decrease the interval time of determining the HPV status which would be relevant for providing prognostic information to the patient as well as determining eligibility for clinical trials targeting this unique patient population.
PMCID: PMC4318229  PMID: 24630260
HPV; Head and neck cancer; Squamous cell carcinoma; Hybrid capture 2; Fine needle aspiration; Cytology
5.  Aquaporin-1 Promoter Hypermethylation Is Associated with Improved Prognosis in Salivary Gland Adenoid Cystic Carcinoma 
Aquaporin-1 (AQP1) is a candidate oncogene that is epigenetically modified in adenoid cystic carcinoma (ACC). We sought to (1) assess AQP1 promoter methylation and expression in an ACC cohort, (2) identify correlations between AQP1 and clinical outcomes, and (3) explore the role of AQP1 in tumor progression in vitro.
Study design
Laboratory study, retrospective chart review.
Academic medical center.
DNA and RNA were isolated from ACC tumors and control salivary gland tissues. Quantitative methylation-specific polymerase chain reaction (PCR) was performed on bisulfite-treated DNA. Quantitative reverse transcription PCR was performed after cDNA synthesis. Cell lines stably overexpressing an AQP1 plasmid or empty vector were generated. Cell scratch and Matrigel invasion assays were performed. Retrospective chart review was performed for collection of clinical information.
Methylation results from 77 tumors and 30 controls demonstrated that AQP1 was hypomethylated in tumors (P < .0001). Fifty-eight tumors (75.3%) displayed AQP1 hypomethylation compared with controls. AQP1 expression levels assessed in 58 tumors and 23 controls demonstrated a trend toward increased expression in tumors (P = .08). Univariate analysis revealed that AQP1 hypermethylation was associated with increased overall survival. No associations between AQP1 expression level and survival were found. AQP1 overexpression did not affect cell migratory or invasive capacities in vitro.
AQP1 promoter hypomethylation is common in ACC, and AQP1 tends to be overexpressed in these tumors. Increased AQP1 methylation is associated with improved prognosis on univariate analysis, but expression is not associated with outcomes. Further in vitro studies are necessary to clarify the role of AQP1 in ACC.
PMCID: PMC4318231  PMID: 24493792
adenoid cystic carcinoma; epigenetics; promoter methylation
6.  Detection of human papillomavirus (HPV) in clinical samples: Evolving methods and strategies for the accurate determination of HPV status of head and neck carcinomas 
Oral oncology  2014;50(9):771-779.
Much recent attention has highlighted a subset of head and neck squamous cell carcinomas (HNSCCs) related to human papillomavirus (HPV) that has an epidemiologic, demographic, molecular and clinical profile which is distinct from non-HPV-related HNSCC. The clinical significance of detecting HPV in a HNSCC has resulted in a growing expectation for HPV testing of HNSCCs. Although the growing demand for routine testing is understandable and appropriate, it has impelled an undisciplined approach that has been largely unsystematic. The current state of the art has now arrived at a point where a better understanding of HPV-related tumorigenesis and a growing experience with HPV testing can now move wide scale, indiscriminant and non-standardized testing towards a more directed, clinically relevant and standardized approach. This review will address the current state of HPV detection; and will focus on why HPV testing is important, when HPV testing is appropriate, and how to test for the presence of HPV in various clinical samples. As no single test has been universally accepted as a best method, this review will consider the strengths and weaknesses of some of the more commonly used assays, and will emphasize some emerging techniques that may improve the efficiency of HPV testing of clinical samples including cytologic specimens.
PMCID: PMC4318232  PMID: 24932529
Oropharyngeal carcinoma; Head and neck squamous cell carcinoma; In situ hybridization; p16 immunohistochemistry; Hybrid Capture 2 (HC2) HPV DNA Test; Cervista® HPV HR test; cobas® HPV test
7.  Use of p40 and p63 Immunohistochemistry and Human Papillomavirus Testing as Ancillary Tools for the Recognition of Head and Neck Sarcomatoid Carcinoma and Its Distinction From Benign and Malignant Mesenchymal Processes 
Sarcomatoid carcinoma (SC) is a variant of head and neck squamous cell carcinoma characterized by a prominent and sometimes exclusive spindle cell component. Distinction from a sarcoma or reactive stroma can be problematic, particularly in cases in which the conventional component is not obvious. The value of immunohistochemistry is limited because of the loss of cytokeratin expression in a sizable percentage of cases. Staining for p63 can enhance detection of epithelial differentiation, but its usefulness is offset by expression in various soft tissue proliferations. Staining for p40—a squamous-specific isoform of p63—could potentially improve diagnostic accuracy. Immunohistochemistry for pancytokeratin, p63, and p40 was performed on 37 head and neck SCs, 201 soft tissue neoplasms, and 40 reactive stromal proliferations. The SCs were also stained for p16 in the event that some of the tumors were human papillomavirus (HPV) related. HPV in situ hybridization was performed on p16-positive cases. Twenty-three of 37 (62%) SCs were positive for pancytokeratin, 23 of 37 (62%) were positive for p63, and 20 of 37 (54%) were positive for p40. Compared with p63, p40 staining was less likely to be observed in soft tissue tumors (5% vs. 30%) and reactive stromal proliferations (0% vs. 30%). HPV16 was detected in 3 of 10 (30%) SCs of the oropharynx but in none of the nonoropharyngeal SCs. p40 staining does not improve the sensitivity for diagnosing SC, but it does diminish the risk of misdiagnosing a sarcoma or reactive stroma as SC. The presence of a sarcomatoid variant of HPVrelated oropharyngeal cancer points to HPV testing as a useful diagnostic tool for atypical spindle cell proliferations of the oropharynx.
PMCID: PMC4313886  PMID: 24418859
head and neck squamous cell carcinoma; spindle cell carcinoma; carcinosarcoma; sarcomatoid carcinoma; human papillomavirus
8.  The Sinonasal Tract: Another Potential “Hot Spot” for Carcinomas with Transcriptionally-Active Human Papillomavirus 
Head and Neck Pathology  2013;8(3):241-249.
While high risk human papillomavirus (HPV) is well established as causative and clinically important for squamous cell carcinoma (SCC) of the oropharynx, its role in non-oropharyngeal head and neck SCC is much less clearly elucidated. In the sinonasal region, in particular, although it is a relatively uncommon site for SCC, as many as 20 % of SCC harbor transcriptionally-active high risk HPV. These tumors almost always have a nonkeratinizing morphology and may have a better prognosis. In addition, specific variants of SCC as well as other rare carcinoma types, when arising in the sinonasal tract, can harbor transcriptionally-active HPV. This article reviews the current literature on HPV in sinonasal carcinomas, attempts to more clearly demonstrate what tumors have it and how this relates to possible precursor lesions like inverted papilloma, and discusses the possible clinical ramifications of the presence of the virus.
PMCID: PMC4126925  PMID: 24338611
Human papillomavirus; Sinonasal; Nonkeratinizing; Squamous cell carcinoma; p16
9.  Differential expression of degradome components in cutaneous squamous cell carcinomas 
Although the cure rate for cutaneous squamous cell carcinoma is high, the diverse spectrum of squamous cell carcinoma has made it difficult for early diagnosis, particularly the aggressive tumors that are highly associated with mortality. Therefore, molecular markers are needed as an adjunct to current staging methods for diagnosing high-risk lesions, and stratifying those patients with aggressive tumors. To identify such biomarkers, we have examined a comprehensive set of 200 histologically defined squamous cell carcinoma and normal skin samples by using a combination of microarray, QRT-PCR and immunohistochemistry analyses. A characteristic and distinguishable profile including matrix metalloproteinase (MMP) as well as other degradome components was differentially expressed in squamous cell carcinoma compared with normal skin samples. The expression levels of some of these genes including matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 10 (MMP10), parathyroid hormone-like hormone (PTHLH), cyclin-dependent kinase inhibitor 2A (CDKN2A), A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), FBJ osteosarcoma oncogene (FOS), interleukin 6 (IL6) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) were significantly differentially expressed (P≤0.02) in squamous cell carcinoma compared with normal skin. Furthermore, based on receiver operating characteristic analyses, the mRNA and protein levels of MMP1 are significantly higher in aggressive tumors compared with non-aggressive tumors. Given that MMPs represent the most prominent family of proteinases associated with tumorigenesis, we believe that they may have an important role in modulating the tumor microenvironment of squamous cell carcinoma.
PMCID: PMC4251465  PMID: 24356192
cutaneous squamous cell carcinoma; degradome; gene expression
10.  Tobacco Smoking and Increased Risk of Death and Progression for Patients With p16-Positive and p16-Negative Oropharyngeal Cancer 
Journal of Clinical Oncology  2012;30(17):2102-2111.
Tobacco smoking is associated with oropharynx cancer survival, but to what extent cancer progression or death increases with increasing tobacco exposure is unknown.
Patients and Methods
Patients with oropharynx cancer enrolled onto a phase III trial of radiotherapy from 1991 to 1997 (Radiation Therapy Oncology Group [RTOG] 9003) or of chemoradiotherapy from 2002 to 2005 (RTOG 0129) were evaluated for tumor human papillomavirus status by a surrogate, p16 immunohistochemistry, and for tobacco exposure by a standardized questionnaire. Associations between tobacco exposure and overall survival (OS) and progression-free survival (PFS) were estimated by Cox proportional hazards models.
Prevalence of p16-positive cancer was 39.5% among patients in RTOG 9003 and 68.0% in RTOG 0129. Median pack-years of tobacco smoking were lower among p16-positive than p16-negative patients in both trials (RTOG 9003: 29 v 45.9 pack-years; P = .02; RTOG 0129: 10 v 40 pack-years; P < .001). After adjustment for p16 and other factors, risk of progression (PFS) or death (OS) increased by 1% per pack-year (for both, hazard ratio [HR], 1.01; 95% CI, 1.00 to 1.01; P = .002) or 2% per year of smoking (for both, HR, 1.02; 95% CI, 1.01 to 1.03; P < .001) in both trials. In RTOG 9003, risk of death doubled (HR, 2.19; 95% CI, 1.46 to 3.28) among those who smoked during radiotherapy after accounting for pack-years and other factors, and risk of second primary tumors increased by 1.5% per pack-year (HR, 1.015; 95% CI, 1.005 to 1.026).
Risk of oropharyngeal cancer progression and death increases directly as a function of tobacco exposure at diagnosis and during therapy and is independent of tumor p16 status and treatment.
PMCID: PMC3397696  PMID: 22565003
11.  Utility of Mammaglobin Immunohistochemistry as a Proxy Marker for the ETV6-NTRK3 Translocation in the Diagnosis of Salivary Mammary Analogue Secretory Carcinoma 
Human pathology  2013;44(10):1982-1988.
Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland neoplasm defined by ETV6-NTRK3 gene fusion. MASC’s morphologyis not entirely specific and overlaps with other salivary gland tumors. Documenting ETV6 rearrangement is confirmatory, but most laboratories are not equipped to perform this test. As MASCs are positive for mammaglobin, immunohistochemistry could potentially replace molecular testing as a confirmatory test, but the specificity of mammaglobin has not been evaluated across a large and diverse group of salivary gland tumors. One hundred-thirty-one salivary gland neoplasms were evaluated by routine microscopy, mammaglobin immunohistochemistry, and ETV6 break-apart fluorescent in situ hybridization. The cases included 15 MASCs, 44 adenoid cystic carcinomas, 33 pleomorphic adenomas (PAs), 18 mucoepidermoid carcinomas, 10 acinic cell carcinomas, 4 adenocarcinomas not otherwise specified, 3 polymorphous low-grade adenocarcinomas, 3 salivary duct carcinomas, and 1 low-grade cribriform cystadenocarcinoma. All 15 MASCs harbored the ETV6 translocation and were strongly mammaglobin-positive. None of the 116 other tumors carried the ETV6 translocation, however mammaglobin staining was present in 1 of 1 (100%) low-grade cribriform cystadenocarcinoma, 2 of 3 (67%) polymorphous low-grade adenocarcinomas, 2 of 3 (67%) salivary duct carcinomas, 2 of 18 (11%) mucoepidermoid carcinomas, and 2 of 33 (6%) pleomorphic adenomas. Mammaglobin is highly sensitive for MASC, but immunostaining can occur in a variety of tumors that do not harbor the ETV6 translocation. Strategic use of mammaglobin immunostaining has a role in the differential diagnosis of salivary gland neoplasms, but it should not be indiscriminately used as a confirmatory test for MASC.
PMCID: PMC3779501  PMID: 23773480
Mammary analogue secretory carcinoma; mammaglobin; acinic cell carcinoma; ETV6-NTRK3
12.  Long term prognosis and risk factors among HPV-associated oropharyngeal squamous cell carcinoma patients 
Cancer  2013;119(19):3462-3471.
A subset of HPV-associated oropharyngeal squamous cell carcinoma (HPV-OSCC) patients experience poor clinical outcomes. We explored prognostic risk factors on overall survival (OS) and recurrence free survival (RFS).
Patients with incident HPV-OSCC treated at the Johns Hopkins Hospital between 1997– 2008 with available tissue for HPV testing and demographic and clinicopathologic information (N=176) were included. Tissue was tested for HPV by in situ hybridization (ISH) and/or p16 immunohistochemistry (IHC). Demographic and clinicopathologic information was extracted from medical records.
90% (157/176) of the OSCC cases were HPV-associated. In the HPV-OSCC patients, we observed a 3- and 5-year OS rate of 93% (95% CI: 88%–98%) and 89% (95% CI: 81%–97%), respectively. Lower survival was observed with older patient age (HR 2.33 per 10-year increase, CI: 1.05–5.16, p=0.038), advanced clinical T-stage (HR 5.78, 95% CI: 1.60–20.8, p=0.007), and current tobacco use (HR 4.38, 95% CI: 1.07–18.0, p=0.04). Disease recurrence was associated with advanced clinical T stage (HR 8.32, 95% CI: 3.06–23, p<0.0001), current/former alcohol use (HR 13, 95% CI: 1.33–120, p=0.03), and unmarried status (HR 3.28, 95% CI: 1.20–9.00, p=0.02).. Patients who remained recurrence-free for 5 years had an 8.6% chance of recurrence by 10 years (one-sided 95% CI upper bound is 19%, p=0.088).
Prognostic risk factors are identified for HPV-OSCC patients. Observed recurrence rates between 5 and 10 years following definitive therapy needs to be validated in additional studies to determine whether extended cancer surveillance is warranted in this cancer population.
PMCID: PMC3788050  PMID: 23861037
HPV; head and neck cancer; oropharyngeal cancer; gender; risk factors
13.  Application of the hybrid capture 2 assay to squamous cell carcinomas of the head and neck: A convenient liquid phase approach for the reliable determination of HPV status 
Cancer Cytopathology  2011;120(1):18-25.
A growing proportion of head and neck squamous cell carcinoma (HNSCCs) is caused by the human papillomavirus (HPV). In light of the unique natural history and prognosis of HPV-related HNSCCs, routine HPV testing is being incorporated into diagnostic protocols. Accordingly, there is an escalating demand for an optimal detection strategy that is sensitive and specific, transferrable to the diagnostic laboratory, standardized across laboratories, cost effective, and amenable to broad application across specimen types including cytologic preparations.
Cytologic preparations (fine needle aspirates and brushes) were obtained from surgically resected HNSCCs and evaluated for the presence of high risk HPV using the hybrid capture 2 assay. HPV analysis was also performed on the corresponding tissue sections using HPV in-situ hybridization and p16 immunohistochemistry. In cases where the immunohistochemical and in-situ hybridization results were discordant, HPV status was determined by real time PCR detection of E7 expression. HPV status in the tissues and corresponding cytologic samples were compared.
Based on benchmark HPV testing of the tissue sections, 14 HNSCCs were classified as HPV positive and 10 as HPV negative. All corresponding cytologic preparations were correctly classified using the hybrid capture 2 assay.
The hybrid capture 2 strategy, already widely used for the detection of high risk HPV in cervical brushes, is readily transferrable to HNSCCs. Consistent accuracy in cytologic preparation suggests its potential application in FNAs from patients who present with lymph node metastases, and may eliminate the need to obtain tissue solely for the purpose of HPV testing.
PMCID: PMC3197962  PMID: 21751428
14.  HPV Analysis in Distinguishing Second Primary Tumors from Lung Metastases in Patients with Head and Neck Squamous Cell Carcinoma 
For patients with head and neck squamous cell carcinoma (HNSqCC), the development of squamous cell carcinoma (SqCC) in the lung may signal a new primary or the onset of metastatic dissemination. Although the distinction influences prognosis and therapy, it may not be straightforward on histological or clinical grounds. Human papillomavirus (HPV) is an etiologic agent for SqCCs arising from the oropharynx, but not for SqCCs arising from other head and neck sites. For patients with HNSqCC who develop a lung SqCC, HPV analysis could be useful in establishing tumor relationships. High risk HPV in-situ hybridization was performed on 54 lung SqCCs from patients with a prior HNSqCC, and on 166 primary lung carcinomas from patients without prior HNSqCC. HPV was detected in 11 of 220 (5%) cases. All HPV-positive cases were from patients with a prior oropharyngeal SqCC. For the paired oropharyngeal and lung SqCCs, HPV status was concordant in 95% of cases. Time from treatment of the HPV-positive oropharyngeal carcinomas to detection of the lung carcinoma ranged from 1 to 97 months (mean 36 months). Two HPV-positive cancers were detected in the lung 8 years after treatment of the oropharyngeal primary. Despite the long interval, E6 sequencing analysis of one of these paired samples confirmed that the tumors harbored the same HPV-16 variant. HPV does not appear to play a role in the development of primary lung cancer. For patients with oropharyngeal SqCC who develop lung SqCCs, HPV analysis may be helpful in clarifying tumor relationships. These relationships may not be obvious on clinical grounds as HPV-related HNSqCC may metastasize long after treatment of the primary tumor.
PMCID: PMC3273906  PMID: 22173119
Human papillomavirus; lung carcinoma; oropharyngeal squamous cell carcinoma
15.  NUT Midline Carcinomas of the Sinonasal Tract 
NUT midline carcinoma (NMC) is a highly lethal tumor defined by translocations involving the NUT gene on chromosome 15q14. NMC involves midline structures including the sinonasal tract, but its overall incidence at this midline site and its full morphologic profile are largely unknown because sinonasal tumors are not routinely tested for the NUT gene translocation. The recent availability of an immunohistochemical probe for the NUT protein now permits a more complete characterization of sinonasal NMCs. The archival files of The Johns Hopkins Hospital Surgical Pathology were searched for all cases of primary sinonasal carcinomas diagnosed from 1995 to 2011. Tissue microarrays were constructed, and NUT immunohistochemical analysis was performed. All NUT-positive cases underwent a more detailed microscopic and immunohistochemical analysis. Among 151 primary sinonasal carcinomas, only 3 (2%) were NUT positive. NUT positivity was detected in 2 of 13 (15%) carcinomas diagnosed as sinonasal undifferentiated carcinoma and in 1 of 87 (1%) carcinomas diagnosed as squamous cell carcinoma. All occurred in men (26, 33, and 48 y of age). The NMCs grew as nests and sheets of cells with a high mitotic rate and extensive necrosis. Two were entirely undifferentiated, and 1 tumor showed abrupt areas of squamous differentiation. Each case had areas of cell spindling, and 2 were heavily infiltrated by neutrophils. Immunohistochemical staining was observed for cytokeratins (3 of 3), epithelial membrane antigen (3 of 3), p63 (2 of 3), CD34 (1 of 3), and synaptophysin (1 of 3). All patients died of the disease (survival time range, 8 to 16mo; mean, 12mo) despite combined surgery and chemoradiation. NMC represents a rare form of primary sinonasal carcinoma, but its incidence is significantly increased in those carcinomas that exhibit an undifferentiated component. Indiscriminant analysis for evidence of the NUT translocation is unwarranted. Instead, NUT analysis can be restricted to those carcinomas that demonstrate undifferentiated areas. The availability of an immunohistochemical probe has greatly facilitated this analysis and is helping to define the full demographic, morphologic, and immunohistochemical spectrum of sinonasal NMC.
PMCID: PMC4124635  PMID: 22534723
NUT midline carcinoma; sinonasal tract; sinonasal undifferentiated carcinoma; t(15;19) translocation; BRD4-NUT
16.  Human Papillomavirus-Related Small Cell Carcinoma of the Oropharynx 
Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSqCC) represents an important subgroup of head and neck cancer that is characterized by a distinct risk factor profile, a relatively consistent microscopic appearance, and a favorable prognosis. A growing experience with HPV testing of OPSqCCs has uncovered variants that deviate from prototypic HPV-related cancer with respect to morphology but not clinical behavior. In effect, HPV positivity confers a favorable prognosis independent of morphologic subtype. We report 5 cases of HPV-related oropharyngeal carcinomas with well developed features of small cell carcinoma (SCC) to define the prognostic impact of HPV positivity in a tumor type universally regarded as highly aggressive. Four of the SCCs arose in association with a conventional HPV-related OPSqCC. All 5 SCCs were HPV positive by in-situ hybridization. By immunohistochemistry, all 5 cases were p16 positive, synaptophysin positive, and CK5/6 negative. Four of the patients were males. The mean age was 61 years (range 49–67). The SCCs were associated with metastatic spread to distant sites (60%) and poor survival outcomes: 3 patients (60%) died as a result of their disease (mean survival time, 10 months; range, 6–15 months). HPV testing has disclosed a previously unrecognized variant of HPV-related oropharyngeal carcinoma that is microscopically characterized by the small cell phenotype. Recognition of this component, even in association with conventional HPV-related OPSqCC, is important as it may indicate an aggressive phenotype that supersedes HPV positivity as a prognostic indicator.
PMCID: PMC3193931  PMID: 21997688
Small cell carcinoma; high grade neuroendocrine carcinoma; human papillomavirus; HPV; HPV-related squamous cell carcinoma; p16; p63; in situ hybridization
17.  GATA3 Immunohistochemical Expression in Salivary Gland Neoplasms 
Head and Neck Pathology  2013;7(4):311-315.
GATA3 is a zinc finger transcription factor that regulates the normal development of many tissues and cell types. Recent studies have shown that immunohistochemical nuclear staining for GATA3 among tumors is highly restricted to carcinomas of breast and urothelial origin; however salivary gland tumors have not been tested. Given that breast and salivary gland tissues are very similar with respect to embryologic development and structure, we performed GATA3 staining on a spectrum of salivary gland neoplasms. GATA3 immunohistochemistry was performed on a diverse collection of 180 benign and malignant salivary gland neoplasms including 10 acinic cell carcinomas, 2 adenocarcinomas not otherwise specified, 41 adenoid cystic carcinomas, 2 epithelial-myoepithelial carcinomas, 1 low grade cribriform cystadenocarcinoma, 15 mammary analogue secretory carcinomas, 7 metastatic squamous cell carcinomas, 27 mucoepidermoid carcinomas, 2 oncocytic carcinomas, 5 oncocytomas, 34 pleomorphic adenomas, 4 polymorphous low grade adenocarcinomas, 25 salivary duct carcinomas, and 5 Warthin tumors. Staining for GATA3 was observed in 92/180 (51 %) of salivary gland tumors. GATA3 staining was observed in most of the tumor types, but diffuse immunolabeling was consistently seen in salivary duct carcinoma (25 of 25) and mammary analogue secretory carcinoma (15 of 15)—the two tumor types that most closely resemble breast neoplasia. Background benign salivary gland tissue was also usually weakly positive in both acini and ducts. GATA3 immunostaining is not restricted to tumors of breast and urothelial origin. Rather, it is expressed across many different types of salivary gland neoplasms. As a result, salivary gland origin should be considered in the differential diagnosis of a GATA3-positive carcinoma, particularly in the head and neck. Although GATA3 immunohistochemistry is not helpful in resolving the differential diagnosis between a primary salivary gland neoplasm and metastatic breast cancer, it may have some utility in subtyping salivary gland tumors, particularly salivary duct carcinoma and mammary analogue secretory carcinoma.
PMCID: PMC3824803  PMID: 23604756
GATA3; Immunohistochemistry; Salivary glands; Salivary duct carcinoma; Mammary analogue secretory carcinoma
18.  Novel Insight into Mutational Landscape of Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2014;9(3):e93102.
Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.
PMCID: PMC3965530  PMID: 24667986
19.  Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma 
Cancer research  2013;73(6):1733-1741.
Human papillomavirus-associated head and neck squamous cell carcinomas (HPV-HNSCC) originate in the tonsils, the major lymphoid organ that orchestrates immunity to oral infections. Despite its location, the virus escapes immune elimination during malignant transformation and progression. Here, we provide evidence for the role of the PD-1:PD-L1 pathway in HPV-HNSCC immune resistance. We demonstrate membranous expression of PD-L1 in the tonsillar crypts, the site of initial HPV infection. In HPV-HNSCCs that are highly infiltrated with lymphocytes, PD-L1 expression on both tumor cells and CD68+ tumor associated macrophages (TAMs) is geographically localized to sites of lymphocyte fronts, while the majority of CD8+ tumor infiltrating lymphocytes (TILs) express high levels of PD-1, the inhibitory PD-L1 receptor. Significant levels of mRNA for interferon-γ (IFN-γ), a major cytokine inducer of PD-L1 expression, were found in HPV+ PD-L1(+) tumors. Our findings support the role of the PD-1:PD-L1 interaction in creating an “immune-privileged” site for initial viral infection and subsequent adaptive immune resistance once tumors are established and suggest a rationale for therapeutic blockade of this pathway in patients with HPV-HNSCC.
PMCID: PMC3602406  PMID: 23288508
PD-1; PD-L1; immune checkpoint; adapative resistance; HPV; head and neck cancers; oropharyngeal cancers; squamous cell carcinomas
20.  Molecular etiology of second primary tumors in contralateral tonsils of human papillomavirus-associatedindextonsillar carcinomas 
Oral oncology  2012;49(3):244-248.
For patients with tobacco-related head and neck squamous cell carcinoma (HNSCC), the occurrence of a second primary tumor (SPT) is an ominous development that is attributed to a field cancerization effect and portends a poor clinical outcome. The goal of this study was to determine whether patients with human papillomavirus (HPV)-related index tonsillar carcinomas can also develop SPTs in the contralateral tonsil, and to discern the molecular etiology of HPV-related tumor multifocality.
Materials and Methods
The surgical pathology archives of The Johns Hopkins Hospital were searched for all patients with primary HPV-related tonsillar squamous cell carcinoma who developed a synchronous or metachronous carcinoma in the contralateral tonsil. The HPV-16 E6 exon was sequenced from each independent cancer site to determine whether the tumor pairs harbored the same or a different HPV-16 variant.
Four patients with bilateral HPV-related tonsillar carcinomas were identified. In every case, the HPV DNA sequences derived from the index tumor and corresponding SPT were 100% concordant, indicating that the index and SPTs were caused by the same HPV-16 variant.
For the small subset of patients with tonsillar carcinomas who develop SPTs in the contralateral tonsil, the index case and the SPT consistently harbored the same HPV variant. This finding suggests that HPV-related tumor multi-focality can be attributed either to independent inoculation events by the same virus, or by migration of HPV-infected cells from a single inoculation site to other regions of Waldeyer’s ring.
PMCID: PMC3548989  PMID: 23062994
Human papillomavirus; head and neck cancer; oropharyngeal cancer; squamous cell carcinoma; second primary tumor
IgG4-related disease has been recently defined as a distinct clinic-pathologic entity, characterized by dense IgG-4 plasmacytic infiltration of diverse organs, fibrosis, and tumefactive lesions. Salivary and lacrimal glands are a target of this disease and, when affected, may clinically resemble Küttner tumor, Mikulicz disease, or orbital inflammatory pseudotumor. In some patients, the disease is systemic, with metachronous involvement of multiple organs, including the pancreas, aorta, kidneys, and biliary tract. We report a 66-year old man who presented with salivary gland enlargement and severe salivary hypofunction and was diagnosed with IgG4-related disease on the basis of a labial salivary gland biopsy. Additional features of his illness included a marked peripheral eosinophilia, obstructive pulmonary disease, and lymphoplasmacytic aortitis. He was evaluated in the context of a research registry for Sjögren syndrome and was the only one of 2594 registrants with minor salivary gland histopathologic findings supportive of this diagnosis.
PMCID: PMC3574629  PMID: 23146570
22.  Human Papillomavirus-Related Carcinomas of the Sinonasal Tract 
High risk human papillomavirus (HPV) is an established cause of head and neck carcinomas arising in the oropharynx. The presence of HPV has also been reported in some carcinomas arising in sinonasal tract, but little is known about their overall incidence or their clinicopathologic profile. The surgical pathology archives of The Johns Hopkins Hospital were searched for all carcinomas arising in the sinonasal tract from 1995 to 2011, and tissue microarrays were constructed. P16 immunohistochemistry and DNA in situ hybridization for high-risk types of HPV were performed. Demographic and clinical outcomes data were extracted from patient medical records. Of 161 sinonasal carcinomas, 34 (21%) were positive for high risk HPV DNA, including type 16 (82%), type 31/33 (12%), and type 18 (6%). HPV-positive carcinomas consisted of 28 squamous cell carcinomas and variants (15 non- or partially-keratinizing, 4 papillary, 5 adenosquamous, 4 basaloid), 1 small cell carcinoma, 1 sinonasal undifferentiated carcinoma, and 4 carcinomas that were difficult to classify but exhibited adenoid cystic carcinoma-like features. Immunohistochemistry for p16 was positive in 59/161 (37%) cases, and p16 expression strongly correlated with the presence of HPV DNA: 33 of 34 (97%) HPV positive tumors exhibited high p16 expression, whereas only 26 of 127 (20%) HPV negative tumors were p16 positive (p < .0001). The HPV-related carcinomas occurred in 19 men and 15 women ranging in age from 33 to 87 years (mean 54). A trend toward improved survival was observed in the HPV-positive group (hazard ratio=0.58, 95% confidence interval [0.26, 1.28]). The presence of high risk HPV in 21% of sinonasal carcinomas confirms HPV as an important oncologic agent of carcinomas arising in the sinonasal tract. While non-keratinizing squamous cell carcinoma is the most common histologic type, there is a wide morphologic spectrum of HPV-related disease that includes a variant that resembles adenoid cystic carcinoma. The distinctiveness of these HPV-related carcinomas of the sinonasal tract with respect to risk factors, clinical behavior, and response to therapy remains to be clarified.
PMCID: PMC3545097  PMID: 23095507
Human papillomavirus; sinonasal carcinoma; squamous cell carcinoma; adenoid cystic carcinoma; sinonasal undifferentiated carcinoma
23.  Cytopathologic Features of Mammary Analogue Secretory Carcinoma 
Cancer cytopathology  2012;121(5):10.1002/cncy.21245.
Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland neoplasm that is defined by ETV6-NTRK3 gene fusion. To the best of the authors’ knowledge, only rare case reports of the cytopathologic features of MASC have been published to date.
A wide variety of archival salivary gland tumors were tested for ETV6 translocation by break-apart fluorescent in situ hybridization. Positive cases with preoperative fine-needle aspiration (FNA) specimens or intraoperative touch preparations were retrieved from the archives of The Johns Hopkins Hospital. All smears were reviewed and the cytologic characteristics were described.
Five cases of MASC with cytopathologic material (4 FNA specimens and 1 touch preparation) were identified. The cases occurred in 3 men and 2 women ranging in age from 21 years to 78 years (mean, 52 years). On the cytologic smears, the MASCs were variably cellular and exhibited 2 different architectural patterns: 1) intact tissue fragments with isomorphic cells arranged in a sheet-like or papillary configuration; and 2) dispersed and dissociated cells with a mostly “histiocyte-like” appearance with large cells containing abundant vacuolated cytoplasm. No matrix tissue or stromal spindled cells were present. The cells did not display acinic differentiation in the form of cytoplasmic zymogen granules. In each case, the preoperative FNA correctly identified a neoplasm, and the most frequent diagnostic considerations were acinic cell carcinoma, mucoepidermoid carcinoma, and pleomorphic adenoma.
MASC is a newly described salivary gland tumor that should be considered in the differential diagnosis of low-grade salivary gland neoplasms. Its cytologic features overlap considerably with those of other tumors, especially acinic cell carcinoma and mucoepidermoid carcinoma.
PMCID: PMC3845462  PMID: 23042752
mammary analogue secretory carcinoma; acinic cell carcinoma; cytopathology; salivary glands; fine-needle aspiration
24.  Tissue Imprint for Molecular Mapping of Deep Surgical Margins in Patients with Head and Neck Squamous Cell Carcinoma 
Head & neck  2012;34(11):1529-1536.
Tissue imprinting can generate molecular marker maps of tumor cells at deep surgical margins. This study evaluates the feasibility of this method for detection of residual head and neck squamous cell carcinoma (HNSCC).
Paired fresh tissue and nitrocellulose membrane imprints of tumor and deep margins were collected from 17 HNSCC resections. DNA was amplified using quantitative methylation-specific PCR (qMSP) for p16, DCC, KIF1A, and EDNRB. Levels of methylation in tumors and deep margins were compared.
DNA from imprints was adequate for qMSP. Hypermethylation of target genes was present in 12/17 tumors and in 8 deep margins. Methylation level was better from margin imprints than tissue. During follow-up (median 13 months), local or regional recurrences occurred in six cases of which five had molecularly positive margins.
Tissue imprinting is feasible for molecular detection of residual tumor at deep surgical margins and may correlate with locoregional recurrence.
PMCID: PMC3377010  PMID: 22223471
Tissue imprint; head and neck squamous cell carcinoma; deep surgical margin; molecular diagnosis; quantitative methylation-specific PCR; Imprint; Deep margin; Surgery; Recurrence
25.  Association Between BRAF V600E Mutation and Mortality in Patients With Papillary Thyroid Cancer 
BRAF V600E is a prominent oncogene in papillary thyroid cancer (PTC), but its role in PTC-related patient mortality has not been established.
To investigate the relationship between BRAF V600E mutation and PTC-related mortality.
Design, Setting, and Participants
Retrospective study of 1849 patients (1411 women and 438 men) with a median age of 46 years (interquartile range, 34–58 years) and an overall median follow-up time of 33 months (interquartile range, 13–67 months) after initial treatment at 13 centers in 7 countries between 1978 and 2011.
Main Outcomes and Measures
Patient deaths specifically caused by PTC.
Overall, mortality was 5.3% (45/845; 95% CI, 3.9%–7.1%) vs 1.1% (11/1004; 95% CI, 0.5%–2.0%) (P<.001) in BRAF V600E–positive vs mutation-negative patients. Deaths per 1000 person-years in the analysis of all PTC were 12.87 (95% CI, 9.61–17.24) vs 2.52 (95% CI, 1.40–4.55) in BRAF V600E–positive vs mutation-negative patients; the hazard ratio (HR) was 2.66 (95% CI, 1.30–5.43) after adjustment for age at diagnosis, sex, and medical center. Deaths per 1000 person-years in the analysis of the conventional variant of PTC were 11.80 (95% CI, 8.39–16.60) vs 2.25 (95% CI, 1.01–5.00) in BRAF V600E–positive vs mutation-negative patients; the adjusted HR was 3.53 (95% CI, 1.25–9.98). When lymph node metastasis, extrathyroidal invasion, and distant metastasis were also included in the model, the association of BRAF V600E with mortality for all PTC was no longer significant (HR, 1.21; 95% CI, 0.53–2.76). A higher BRAF V600E–associated patient mortality was also observed in several clinicopathological subcategories, but statistical significance was lost with adjustment for patient age, sex, and medical center. For example, in patients with lymph node metastasis, the deaths per 1000 person-years were 26.26 (95% CI, 19.18–35.94) vs 5.93 (95% CI, 2.96–11.86) in BRAF V600E–positive vs mutation-negative patients (unadjusted HR, 4.43 [95% CI, 2.06–9.51]; adjusted HR, 1.46 [95% CI, 0.62–3.47]). In patients with distant tumor metastasis, deaths per 1000 person-years were 87.72 (95% CI, 62.68–122.77) vs 32.28 (95% CI, 16.14–64.55) in BRAF V600E–positive vs mutation-negative patients (unadjusted HR, 2.63 [95% CI, 1.21–5.72]; adjusted HR, 0.84 [95% CI, 0.27–2.62]).
Conclusions and Relevance
In this retrospective multicenter study, the presence of the BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC. Because overall mortality in PTC is low and the association was not independent of tumor features, how to use BRAF V600E to manage mortality risk in patients with PTC is unclear. These findings support further investigation of the prognostic and therapeutic implications of BRAF V600E status in PTC.
PMCID: PMC3791140  PMID: 23571588

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