Immunohistochemical and molecular imaging biomarker signature for the prediction of failure site after chemoradiation for head and neck squamous cell carcinoma.

OBJECTIVE: To identify a failure site-specific prognostic model by combining immunohistochemistry (IHC) and molecular imaging information to predict long-term failure type in squamous cell carcinoma of the head and neck. PATIENT AND METHODS: Tissue microarray blocks of 196 head and neck squamous cell carcinoma cases were stained for a panel of biomarkers using IHC. Gross tumor volume (GTV) from the PET/CT radiation treatment planning CT scan, maximal Standard Uptake Value (SUVmax) of fludeoxyglucose (FDG) and clinical information were included in the model building using Cox proportional hazards models, stratified for p16 status in oropharyngeal carcinomas. Separate models were built for time to locoregional failure and time to distant metastasis. RESULTS: Higher than median p53 expression on IHC tended toward a risk factor for locoregional failure but was protective for distant metastasis, χ2 for difference p = .003. The final model for locoregional failure included p53 (HR: 1.9; p: .055), concomitant cisplatin (HR: 0.41; p: .008), ß-tubulin-1 (HR: 1.8; p: .08), ß-tubulin-2 (HR: 0.49; p: .057) and SUVmax (HR: 2.1; p: .046). The final model for distant metastasis included p53 (HR: 0.23; p: .025), Bcl-2 (HR: 2.6; p: .08), SUVmax (HR: 3.5; p: .095) and GTV (HR: 1.7; p: .063). CONCLUSIONS: The models successfully distinguished between risk of locoregional failure and risk of distant metastasis, which is important information for clinical decision-making. High p53 expression has opposite prognostic effects for the two endpoints; increasing risk of locoregional failure, but decreasing the risk of metastatic failure, but external validation of this finding is needed.

Double positivity for HPV DNA/p16 in tonsillar and base of tongue cancer improves prognostication: Insights from a large population-based study.

The aim was to explore the overall survival (OS) for palatine tonsillar squamous cell carcinoma (TSCC), subdivided, according to certainty of tonsillar tumour origin, into specified tonsillar squamous cell carcinomas (STSCCs) and nonspecified tonsillar squamous cell carcinomas (NSTSCCs), and base of tongue squamous cell carcinoma (BSCC) when stratifying for HPV DNA status, p16 expression and combined HPV/p16 status. We included all patients (n = 797) diagnosed with TSCCs and BSCCs in Eastern Denmark as registered in the Danish Head and Neck Cancer Group (DAHANCA) database and the Danish Pathology Databank, 2000-2010. Patients were treated according to national guidelines (radiotherapy +/- concomitant cisplatin). All specimens were analysed using HPV DNA PCR and p16 immunohistochemistry. Clinical information was retrieved from the DAHANCA database and the Danish National Patient Registry. Information on vital status was obtained from the Danish Civil Registration System. We observed improved OS for HPV+/p16+ BSCCs compared to HPV-/p16- (hazard ratio for death [HR], 0.15; 95% CI, 0.09-0.24). Among STSCCs, HPV+/p16+ showed the lowest HR (0.19, 95% CI, 0.13-0.29); whereas, HPV-/p16+ showed an intermediate HR (0.39; 95% CI, 0.22-0.70). For NSTSCCs, HPV+/p16+ and HPV-/p16+ showed similar OS (HRs, 0.39; 95% CI, 0.26-0.59; and 0.48; 95% CI, 0.24-0.95, respectively). Combined HPV+/p16+ was a significantly better prognostic marker in BSCCs and STSCCs than HPV DNA and p16, alone (all p-values < 0.05). Whereas, combined testing in NSTSCC was not better than p16 (p = 0.53), alone. In conclusion, double positivity for HPV/p16 in conjunction with the certainty of tumour site improved prognosis.

Does age affect prognosis in salivary gland carcinoma patients? A national Danish study.

AIM: To compare incidence, histology, treatment modalities, disease stages, and outcome in elderly patients (≥70 years) compared to younger (<70 years). METHODS: From the national Danish salivary gland carcinoma database, 871 patients diagnosed with a primary salivary gland carcinoma from January 1990 to December 2005 were identified. Variables necessary for statistical analyses were extracted from the database. RESULTS: The younger patients have a significantly better crude, disease-specific and recurrence-free survival than the elderly ones. In univariate analysis, significantly more patients in the young group were WHO performance status 0 and in disease stage I + II, and they presented with significantly more histological low grade tumors. In multivariate analysis, chronological age seemed to be of no prognostic significance to salivary gland carcinoma patients as opposed to performance status, disease stage and histological grade. CONCLUSIONS: Salivary gland carcinoma patients over the age of 70 years have a poor prognosis compared to younger patients, which can be explained by higher disease stages, more histological high grade subtypes and a poorer performance status at the time of diagnosis.

Pleomorphic adenoma of the parotid gland 1985-2010: A Danish nationwide study of incidence, recurrence rate, and malignant transformation.

BACKGROUND: Pleomorphic adenoma is the most frequent salivary gland tumor and is known for its tendency to recur and for its ability to transform to carcinoma ex pleomorphic adenoma (Ca-ex-PA). Along with pleomorphic adenoma demographics, we present the first nationwide study with long-term follow-up on these topics. METHODS: The Danish Pathology Data Bank was searched for parotid pleomorphic adenoma and Ca-ex-PA in the period 1985 to 2010 and all pathology descriptions were reviewed. Ca-ex-PA specimens were reviewed by a pathologist. RESULTS: A total of 5.497 patients were identified and 2.86% had at least one recurrence. An incidence of 4.29/100,000/year was found. The rate of malignant transformation in recurrent pleomorphic adenoma was 3.3%. CONCLUSION: We report an up-to-date assessment of the epidemiology of pleomorphic adenoma. We found an increasing incidence and low recurrence rate compared with previous studies. The risk of Ca-ex-PA for patients with recurrent pleomorphic adenoma was low compared to the literature. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1364-E1369, 2016.

Salivary adenoid cystic carcinoma in Denmark 1990-2005: Outcome and independent prognostic factors including the benefit of radiotherapy. Results of the Danish Head and Neck Cancer Group (DAHANCA).

AIM: To describe outcome and prognostic factors, including the effect of radiotherapy, in a consecutive national series of salivary gland adenoid cystic carcinomas. METHODS: From the national Danish salivary gland carcinoma database in the structure of DAHANCA, 201 patients diagnosed with adenoid cystic carcinoma, and treated with a curative intent, were identified in the period between 1990 and 2005. Variables necessary for statistical analyses were extracted from the database. RESULTS: The 10-year crude survival and disease specific survival rates were 58% and 75%, respectively. The 10-year locoregional control rate was 70%, and 36% of patients experienced a recurrence during follow-up (median 7.5 years); 18% developed distant metastases (most commonly to the lungs). In multivariate analysis, stage and margin status were both important factors with regards to survival and locoregional control. Radiotherapy did not improve survival, but it did improve the locoregional control rate. CONCLUSIONS: The treatment of choice is surgery with as wide margins as possible including elective, selective neck dissection. Adjuvant radiotherapy should be considered in patients with incomplete tumor resection, high disease stages, and tumors with a solid growth pattern.

A reverse Warburg metabolism in oral squamous cell carcinoma is not dependent upon myofibroblasts.

BACKGROUND: The reverse Warburg effect describes the phenomenon that epithelial cancer cells take advantage of the metabolic machinery from nearby cancer-associated fibroblast, inducing them to produce lactate and ketones to fuel the high metabolic demands of the epithelial tumour tissues. This is in breast cancer observed as a lack of stromal caveolin-1 (CAV-1) and an increased expression of monocarboxylate transporter 4 (MCT-4) in the tumour stroma, with a concomitant increase in the expression of monocarboxylate transporter 1 (MCT-1) in the epithelial, tumour compartment. The lack of CAV-1 and increased expression of MCT-4 have been shown to have prognostic importance, primarily in patients with breast cancer. However, this phenomenon has only scarcely been described in oral squamous cell carcinoma (OSCC). Given the prognostic importance of myofibroblasts in OSCC, we also examined a potential relationship between the expression of MCT-4 and the presence of myofibroblasts. METHODS: Paraffin-embedded tissues from 30 patients with OSCC were immunostained with antibodies towards MCT-1, MCT-4, Cav-1, GLUT-1, α-SMA, TOMM20 and KI-67, and evaluated for their specific epithelial and stromal expression. RESULTS AND CONCLUSIONS: In patients with OSCC, we find an increased expression of MCT-1 and MCT-4 in both the epithelial and stromal compartment, with almost no overlap in their spatial expression. We found a large spatial overlap between α-SMA and MCT-1 in the stroma compartment, but no relationship between MCT-4 and myofibroblasts. Interestingly, we did not observe any relationship between the absence of CAV-1 and the presence of MCT-4 as has been shown in breast carcinomas.

A high and increasing HPV prevalence in tonsillar cancers in Eastern Denmark, 2000-2010: the largest registry-based study to date.

The aim was to explore whether the incidence of tonsillar squamous cell carcinomas (TSCCs) increased in Eastern Denmark, 2000-2010, and whether human papillomavirus (HPV) could explain the increase, and to assess the association of HPV prevalence with gender, age, and origin (i.e., the certainty of tonsillar tumor origin). We applied HPV DNA PCR and p16 immunohistochemistry to all TSCCs registered in the Danish Head and Neck Cancer Group (DAHANCA) and in the Danish Pathology Data Bank (n = 632). Pathologists reviewed and subdivided the tumors into two groups: specified and nonspecified TSCCs. Approximately 10% of HPV-positive tumors was genotyped by amplicon next-generation sequencing. The overall crude incidence of TSCCs increased significantly (2.7% per year) and was explained by an increasing incidence of HPV-positive TSCCs (4.9% per year). The overall HPV prevalence was 58%, with HPV16 being the predominant HPV type. In multivariate analysis, the HPV prevalence was associated with age (<55 vs. >60 years) (OR, 1.72; 95% CI 1.13-2.63) and origin (nonspecified vs. specified TSCCs) (OR, 0.15; 95% CI 0.11-0.22). The association of HPV prevalence with origin increased over time in specified TSCCs (OR per year, 1.10; 95% CI 1.01-1.19), whereas no change over time was observed among nonspecified TSCCs (OR per year, 0.99; 95% CI 0.90-1.08). In conclusion, the observed increase in the number of HPV-positive TSCCs can explain the increasing number of TSCCs in Eastern Denmark, 2000-2010. HPV prevalence was associated with younger age (<55 years) and a high certainty of tonsillar tumor origin.

MiR-21 expression in the tumor stroma of oral squamous cell carcinoma: an independent biomarker of disease free survival.

Oral squamous cell carcinoma (OSCC) patients have a high mortality rate; thus, new clinical biomarkers and therapeutic options are needed. MicroRNAs (miRNAs) are short noncoding RNAs that regulate posttranscriptional gene expression and are commonly deregulated in OSCC and other cancers. MicroRNA-21 (miR-21) is the most consistently overexpressed miRNA in several types of cancer, and it might be a useful clinical biomarker and therapeutic target. To better understand the role of miR-21 in OSCC, paraffin-embedded tumor tissue samples from 86 patients with primary OSCC were analyzed by in situ hybridization. We found that miR-21 was primarily expressed in the tumor stroma and in some tumor-associated blood vessels with no expression in the adjacent normal epithelia or stroma. Using image analysis, we quantitatively estimated miR-21 expression levels specifically in the stroma of a cohort of OSCC samples. These miR-21 levels significantly correlated with disease free survival with the highest levels being located in the stroma. Stromal miR-21 expression was independently associated with a poorer prognosis, even after adjusting for clinical parameters (perineural invasion and N-stage) in a multivariate analysis. In summary, we have shown that miR-21 is located in the carcinoma cells, stroma and blood vessels of tumors, and its expression specifically in the stromal compartment has a negative prognostic value in OSCC.

Lacrimal gland pleomorphic adenoma and carcinoma ex pleomorphic adenoma: genomic profiles, gene fusions, and clinical characteristics.

PURPOSE: To study genetic alterations in lacrimal gland pleomorphic adenoma (PA) and carcinoma ex pleomorphic adenoma (Ca-ex-PA) with focus on copy number changes and expression patterns of the translocation target genes PLAG1, HMGA2, and CRTC1-MAML2 in relation to clinical data. DESIGN: Experimental study. PARTICIPANTS: A total of 36 tumors from 32 patients with lacrimal gland PA or Ca-ex-PA were included in the study. METHODS: Genome wide, high-resolution array-based comparative genomic hybridization (arrayCGH) and immunohistochemistry were used to study the genomic profiles and expression patterns of the translocation targets PLAG1, HMGA2, and CRTC1-MAML2. MAIN OUTCOME MEASURES: Copy number alterations (gains/losses) and protein expression of PLAG1, HMGA2, and CRTC1-MAML2. RESULTS: Genome-wide arrayCGH analysis revealed normal genomic profiles in 10 of 17 PA samples. The average number of genomic imbalances per tumor was 3.25 (range, 1-7) in primary and recurrent PAs with alterations compared with 7.7 (range, 4-12) in Ca-ex-PAs. Five recurrent copy number alterations were identified in PAs, including losses of 1pter-p31.3, 6q22.1-q24.3, 8q24.22-q24.3, and 13q21.31-q21.33, and gain of 9p23-p22.3. Gain of 9p23-p22.3 also was seen in a Ca-ex-PA. In Ca-ex-PA, gain of 22q12.3-qter was the only recurrent alteration. Detailed analysis of the array data identified NFIB and PDGFB as the 2 major candidate target oncogenes that may be activated as a result of copy number gains involving 9p and 22q. Both genes have been implicated in the pathogenesis of PA and other types of salivary gland tumors. Immunohistochemical analysis revealed frequent overexpression of the translocation target gene PLAG1 in PAs and in 1 Ca-ex-PA. In contrast, overexpression of HMGA2 was observed in only a small subset of PAs. The CRTC1-MAML2 fusion oncoprotein was overexpressed in 2 mucoepidermoid Ca-ex-PAs. CONCLUSIONS: Lacrimal and salivary gland PAs and Ca-ex-PAs have similar genomic profiles and frequently overexpress the PLAG1 oncoprotein. Copy number gains involving 9p23-p22.3 (NFIB) and 22q12-qter (PDGFB) may be of importance for disease progression in a subset of lacrimal gland PAs.

Nodal yield in selective neck dissection.

CONCLUSION: The total lymph node yield in neck dissection is highly variable and depends on anatomical, surgical and pathological parameters. A minimum yield of six lymph nodes for a selective neck dissection (SND) as recommended in guidelines lies in the lower range of the reported clinical nodal yields. A future application of a lymph node ratio may improve the risk stratification of head and neck cancer patients. However, this will require a higher number of retrieved lymph nodes. OBJECTIVES: To compare the clinical guideline recommendations for nodal yield in SND with the number of lymph nodes obtained from cadavers and the clinical nodal yield reported in the literature. METHODS: Lymph nodes retrieved from SND specimens from nine fresh cadavers were quantified histopathologically. The literature on nodal yields reportedly obtained by clinicians performing neck dissections was reviewed. Finally, the discussion makes reference to the six lymph nodes currently recommended in international clinical guidelines. RESULTS: For clinical SNDs (I-III) the lowest mean nodal yield was 19.4, for SNDs (II-IV) it was 26.4. The cadaver SNDs (I-III and II-IV) yielded 8.8 (range 1-15) and 10.4 nodes (range 1-19), respectively.

Adenoid cystic carcinoma of the lacrimal gland: MYB gene activation, genomic imbalances, and clinical characteristics.

PURPOSE: To investigate genetic alterations in lacrimal gland adenoid cystic carcinomas (ACCs) with emphasis on the MYB-NFIB fusion oncogene and its downstream targets, MYB rearrangements, and copy number alterations in relation to clinical data and survival. DESIGN: Experimental study. PARTICIPANTS AND CONTROLS: Fourteen patients with primary lacrimal gland ACC were included. As a control, we also studied the expression of MYB-NFIB in 19 non-ACC lacrimal gland tumors. METHODS: The expression and identity of MYB-NFIB fusion transcripts were studied using reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequence analyses. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to evaluate the expression of MYB/MYB-NFIB target genes. High-resolution array-based comparative genomic hybridization (arrayCGH) and fluorescence in situ hybridization were used to study copy number alterations and MYB rearrangements. MAIN OUTCOME MEASURES: mRNA or protein expression of MYB-NFIB, MYB, and its down stream targets; copy number alterations; and genomic rearrangements. RESULTS: The median age of the patients was 43 years (equal gender distribution), and the median time of survival was 8.6 years. The MYB-NFIB fusion was expressed in 7 of 14 ACCs. In contrast, all non-ACC tumors were fusion-negative. All 13 ACCs tested stained positive for the MYB protein, and for the MYB targets KIT and BCL2, 12 were positive for MYC and CCNE1, and 9 were positive for CCNB1. Rearrangements of MYB were detected in 8 of 13 cases, including 2 cases with gain of an apparently intact MYB gene. The arrayCGH analysis revealed recurrent copy number alterations with losses involving 6q23-q27, 12q12-q14.1, and 17p13.3-p12, and gains involving 19q12, 19q13.31-qter, 8q24.13-q24.21, 11q12.3-q14.1, and 6q23.3. Neither MYB-NFIB fusion nor any copy number alteration correlated with survival. CONCLUSIONS: Lacrimal gland ACCs are frequently positive for the MYB-NFIB fusion, overexpress MYB and its downstream targets, and have genomic profiles characterized by losses involving 6q, 12q, and 17p, and gains involving 19q, 8q, and 11q. Our findings show that lacrimal gland ACCs are genetically and clinically similar to their salivary gland counterparts and that MYB-NFIB is a clinically useful diagnostic biomarker for ACC. Our data also suggest that MYB and its downstream targets are potential therapeutic targets for these tumors. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Implications of a positive sentinel node in oral squamous cell carcinoma.

BACKGROUND: The role of sentinel node biopsy in head and neck cancer is currently being explored. Patients with positive sentinel nodes were investigated to establish if additional metastases were present in the neck, their distribution, and their impact on outcome. METHODS: In all, 109 patients (n = 109) from 15 European centers, with cT1/2,N0 tumors, and a positive sentinel lymph node were identified. Kaplan-Meier and univariate and multivariate logistic regression analysis were used to identify variables that predicted for additional positive nodes and their position within the neck. RESULTS: A total of 122 neck dissections were performed in 109 patients. Additional positive nodes were found in 34.4% of cases (42/122: 18 same, 21 adjacent, and 3 nonadjacent neck level). Additional nodes, especially if outside the sentinel node basin, had an impact on outcome. CONCLUSIONS: The results are preliminary but suggest that both the number and the position of positive sentinel nodes may identify different prognostic groups that may allow further tailoring of management plans.

The prevalence of occult metastases in nonsentinel lymph nodes after step-serial sectioning and immunohistochemistry in cN0 oral squamous cell carcinoma.

OBJECTIVES: To examine the prevalence of isolated tumor cells (ITC) and micrometastases (MM) in nonsentinel lymph nodes (NSN) using additional step-serial sectioning and immunohistochemistry (IHC) as for sentinel lymph nodes (SN). STUDY DESIGN: Prospective, consecutive, and clinically controlled trial. METHODS: Fifty-one patients with oral cavity squamous cell carcinoma (OSCC) T1-T2 and clinically N0 neck underwent surgical treatment including sentinel-node biopsy (SNB) assisted selective neck dissection (SND). The location of the SN was determined using dynamic and planar lymphoscintigraphy and SPECT-CT. The harvested NSN from the neck dissections underwent the same histopathologic examinations as the SN using step-serial sectioning (SSS) at 150-micron intervals. Two sections from each level were stained with hematoxylin-eosin (H&E) and cytokeratin antibodies (AE1/AE3) and examined for tumor deposits. Results were compared with the previous routine examination of the NSN. RESULTS: A total of 403 NSN were examined with a median of 8 per patient. A total of 1/51 patients (2%) had involvement of an additional NSN not found on routine examination. This was the only lymph node with involvement not detected previously. However, this patient had metastases in SN and in another NSN detected on routine examination. The overall incidence of occult metastasis (SN + NSN) was 21.6% (11/51) as previously reported. CONCLUSIONS: The incidence of occult metastases in NSN after additional SSS and IHC was 2%. The risk of NSN involvement would seem to be extremely low in patients with early OSCC and negative SN. This study further validates SNB as an accurate staging tool for cN0 early OSCC.