Christina MacMillan

Molecular cytogenetic analysis of head and neck squamous cell carcinoma: By comparative genomic hybridization, spectral karyotyping, and expression array analysis†

A combination of molecular cytogenetic and expression array analysis has been performed on head and neck squamous cell carcinoma (HNSCC) of the oral cavity and supraglottis. These studies were performed to identify consensus regions of chromosomal imbalance and structural rearrangement to determine whether genes located in these genomic regions are subject to alterations in gene expression. Such combinatorial studies may help to identify recurrent patterns of altered gene expression in the context of specific chromosomal changes.

Abnormalities of the ARF-p53 pathway in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is associated with heavy smoking and drinking, but the molecular pathway of tumorigenesis is not understood. Inactivation of the p53 tumor suppressor gene is likely to play an important role since p53 mutation is frequently found. The p14ARF tumor suppressor gene is functionally linked to p53, because it is activated by oncogenes and causes p53-dependent growth arrest and apoptosis. The relationship between p14ARF and p53 inactivation has not been described for OSCC. We studied 25 cases of OSCC to determine if there is an inverse correlation between p53 mutation and p14ARF inactivation by homozygous deletion or mutation. p53 mutation was found in 16 of 25 cases (64%), including nine missense and seven truncating mutations. While all cases with missense mutations showed abnormal accumulation of p53 protein, there were also five carcinomas which showed increased p53 staining in the absence of mutation. p14ARF deletion or mutation was found in eight cases (32%), six of which also demonstrated p53 mutation. Our findings indicate that OSCC often involves loss of both p14ARF and p53 function and suggest that inactivation of these two tumor suppressor genes are not functionally equivalent during tumorigenesis.

Programmed death-ligand 1 overexpression is a prognostic marker for aggressive papillary thyroid cancer and its variants

Programmed death-ligand 1(PD-L1) expression on tumor cells is emerging as a potential predictive biomarker in anti-PD-L1 directed cancer immunotherapy. We analyzed PD-L1 expression in papillary thyroid carcinoma (PTC) and its variants and determined its prognostic potential to predict clinical outcome in these patients. This study was conducted at an academic oncology hospital which is a prime referral centre for thyroid diseases. Immunohistochemical subcellular localization (IHC) analyses of PD-L1 protein was retrospectively performed on 251 archived formalin fixed and paraffin embedded (FFPE) surgical tissues (66 benign thyroid nodules and 185 PTCs) using a rabbit monoclonal anti-PD-L1 antibody (E1L3N, Cell Signaling Technology) and detected using VECTASTAIN rapid protocol with diaminobenzidine (DAB) as the chromogen. The clinical-pathological factors and disease outcome over 190 months were assessed; immunohistochemical subcellular localization of PD-L1 was correlated with disease free survival (DFS) using Kaplan Meier survival and Cox multivariate regression analysis. Increased PD-L1 immunostaining was predominantly localized in cytoplasm and occasionally in plasma membrane of tumor cells. Among all combined stages of PTC, patients with increased PD-L1 membrane or cytoplasmic positivity had significantly shorter median DFS (36 months and 49 months respectively) as compared to those with PD-L1 negative tumors (DFS, both 186 months with p < 0.001 and p < 0.01 respectively). Comparison of PD-L1+ and PD-L1− patients with matched staging showed increased cytoplasmic positivity in all four stages of PTC that correlated with a greater risk of recurrence and a poor prognosis, but increased membrane positivity significantly correlated with a greater risk of metastasis or death only in Stage IV patients. In conclusion, PD-L1 positive expression in PTC correlates with a greater risk of recurrence and shortened disease free survival supporting its potential application as a prognostic marker for PTC.

Telomerase activity is upregulated in laryngeal squamous cell carcinoma.

Objective/Hypothesis The immortalizing enzyme telomerase has been linked to carcinogenesis and is being targeted as a novel molecular marker. This study investigated telomerase expression in patients with laryngeal squamous cell carcinoma and correlated telomerase activity with conventional prognostic parameters.

Endoscopic Resection of Solitary Fibrous Tumors of the Nose and Paranasal Sinuses

Solitary fibrous tumors (SFTs) are uncommon neoplasms of mesenchymal origin that were first described as primary spindle-cell tumors of the pleura in 1931. Since then, infrequent case reports of extrapleural SFTs have been described including various subsites within the head and neck. Based on a review of the literature and a description of the endoscopic treatment of three patients with SFTs of the nasal cavity and ethmoid sinuses, the challenges associated with the management of sinonasal SFTs are discussed. Successful endoscopic resection was performed at a tertiary referral rhinology practice within a university center in three cases of sinonasal SFTs with no evidence of recurrence at 26, 35, and 49 months following resection. Summarized are the common presenting symptoms, appropriate diagnostic workup, and indicative computed tomography and magnetic resonance imaging appearance of SFTs. Further discussed are the challenge associated with accurate histological and immunohistochemical diagnosis, the difficulty in assessing the aggressiveness and malignant potential of these lesions, and the appropriate treatment and follow-up duration that these neoplasms require.

Prediction of recurrence-free survival using a protein expression-based risk classifier for head and neck cancer

Loco-regional recurrence in 50% of oral squamous cell carcinoma (OSCC) patients poses major challenge for oncologists. Lack of biomarkers that can predict disease aggressiveness and recurrence risk makes the scenario more dismal. On the basis of our earlier global proteomic analyses we identified five differentially expressed proteins in OSCC. This study aimed to develop protein biomarkers-based prognostic risk prediction model for OSCC. Sub-cellular expression of five proteins, S100A7, heterogeneous nuclear ribonucleoproteinK (hnRNPK), prothymosin α (PTMA), 14-3-3ζ and 14-3-3σ was analyzed by immunohistochemistry in test set (282 Indian OSCCs and 209 normal tissues), correlated with clinic–pathological parameters and clinical outcome over 12 years to develop a risk model for prediction of recurrence-free survival. This risk classifier was externally validated in 135 Canadian OSCC and 96 normal tissues. Biomarker signature score based on PTMA, S100A7 and hnRNPK was associated with recurrence free survival of OSCC patients (hazard ratio=1.11; 95% confidence interval 1.08, 1.13, P<0.001, optimism-corrected c-statistic=0.69) independent of clinical parameters. Biomarker signature score stratified OSCC patients into high- and low-risk groups with significant difference for disease recurrence. The high-risk group had median survival 14 months, and 3-year survival rate of 30%, whereas low-risk group survival probability did not reach 50%, and had 3-year survival rate of 71%. As a powerful predictor of 3-year recurrence-free survival in OSCC patients, the newly developed biomarkers panel risk classifier will facilitate patient counseling for personalized treatment.

Molecular characterization of salivary gland malignancy using the Smgb-Tag transgenic mouse model

The molecular mechanisms underlying salivary gland tumorigenesis remain unclear. In order to identify genetic changes that occur during the development of invasive adenocarcinoma from normal salivary gland, we used the Smgb-Tag transgenic mouse model. This transgene induces the progressive development of dysplasia to invasive adenocarcinoma in the submandibular salivary gland. Gene expression patterns from 20 submandibular glands (two normal, nine dysplasia and nine adenocarcinoma samples) were assessed using a mouse 15 K cDNA array. Unsupervised hierarchical clustering was used to group gene expression based on 157 differentially expressed genes distinguishing between dysplasias and adenocarcinomas. Further analysis identified 25 significantly overexpressed and 28 underexpressed cDNA sequences in adenocarcinoma as compared to dysplasia. Differential expression of five genes (Lcn2, Ptn, Cd24a, Mapk6 and Rnps1) was validated by quantitative real-time RT-PCR in a total of 48 mouse salivary gland tissues (seven histologically normal, 13 dysplasias and 28 adenocarcinomas), including the 20 samples analyzed by cDNA arrays. Immunohistochemical analysis was used to validate the expression of Ptn and Cd24a at the protein level in a subset of 16 mouse salivary glands (four normal, five dysplasia and seven adenocarcinoma samples), as well as in 23 human submandibular gland tumors (16 pleomorphic adenomas, three adenoid cystic carcinomas, one acinic cell carcinoma, one adenocarcinoma NOS, one myoepithelial and one mucoepidermoid carcinoma). We thus demonstrated that the Smgb-Tag transgenic mouse model is a useful tool for the identification of genes that are deregulated in salivary gland adenocarcinomas. Our data suggest that Ptn and Cd24a may be genetic markers associated with salivary gland tumorigenesis and/or progression.

Programmed Death - Ligand 1 Expression Distinguishes Invasive Encapsulated Follicular Variant of Papillary Thyroid Carcinoma from Noninvasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features

Background The noninvasive Encapsulated follicular variant of papillary thyroid cancer (EFVPTC) has been reclassified as Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) without a significant risk for malignant behavior. However the evaluation remains a challenge for clinicians. We sought to determine whether programmed death-ligand 1 (PD-L1) expression may serve as a biomarker to predict invasiveness of EFVPTC and assist to distinguish these neoplasms from NIFTP. Methods Immunohistochemical staining of PD-L1 expression was performed in sections of 174 Formalin-fixed paraffin-embedded (FFPE) tissue blocks from surgery removed thyroid nodules. Results Cytoplasmic PD-L1 expression was significantly increased in invasive EFVPTC (4.76 ± 1.49) as compared to NIFTP (3.06 ± 2.16, p < 0.001). Increased cytoplasmic PD-L1 expression was associated with invasiveness in EFVPTC (p < 0.001); PD-L1 positive EFVPTC cases were at 3.16 folds higher risk in developing invasion than the PD-L1 negative cases. No significant difference in cytoplasmic PD-L1 expression was observed between NIFTP and benign nodules. Conclusion PD-L1 expression may serve as a useful biomarker in predicting invasiveness of EFVPTC and distinguishing NIFTP from invasive EFVPTC. To our knowledge this is the first report suggesting the application of a protein biomarker to confirm NIFTP as benign indolent neoplasms.

Institution‐specific risk of papillary thyroid carcinoma in atypia/follicular lesion of undetermined significance

The Bethesda System for Reporting Thyroid Cytopathology (BSRTC) is used in surgical decision‐making according to malignancy risk in each category. Malignancy risk in atypia/follicular lesion of undetermined significance (AUS/FLUS) is estimated in BSRTC to be 5% to 15%, but institutional data have varied widely.

Activated leukocyte cell adhesion molecule is a marker for thyroid carcinoma aggressiveness and disease-free survival.

BACKGROUND Currently, there are no protein biomarkers for aggressive subtypes of thyroid carcinomas (TC) in clinical use that would allow for early detection and patient management. We hypothesized that activated leukocyte cell adhesion molecule (ALCAM or CD166) expression in thyroid tissues will reveal ALCAM to be a potential diagnostic and/or prognostic marker for TC aggressiveness. METHODS Forty-five benign and 158 malignant thyroid tissues were analyzed for ALCAM expression using immunohistochemistry. ALCAM expression was correlated with different subtypes and clinicopathological features of TC, as well as patient disease-free survival. RESULTS Combined membranous and cytoplasmic (total) expression of ALCAM was significantly reduced in patients with poorly/undifferentiated (aggressive) TC as compared to well-differentiated (nonaggressive) tumors (p<0.001; area-under-curve=0.865, sensitivity=82%, specificity=74%). The decreased ALCAM expression in TC correlated significantly with extrathyroidal extension, distant metastasis, and TC histotype. Notably, Kaplan-Meier survival analysis for follow-up data of 134 patients revealed significantly reduced disease-free survival for patients with TC with decreased ALCAM membranous, cytoplasmic, and total expression. Median survival of patients with decreased cytoplasmic ALCAM expression was 6 years, as compared to 13.7 years for patients with higher ALCAM expression (p<0.001). CONCLUSION ALCAM has the potential to serve as a diagnostic and prognostic biomarker for aggressive TC. This protein can be taken forward for analysis in sera of patients with TC to determine its applicability as a minimally invasive serum biomarker for TC aggressiveness and patient disease-free survival.