Christopher C. Griffith

New Developments in Salivary Gland Pathology: Clinically Useful Ancillary Testing and New Potentially Targetable Molecular Alterations

Accurate diagnosis of salivary gland tumors can be challenging because of the many diagnostic entities, the sometimes extensive morphologic overlap, and the rarity of most tumor types. Ancillary testing is beginning to ameliorate some of these challenges through access to newer immunohistochemical stains and fluorescence in situ hybridization probes, which can limit differential diagnostic considerations in some cases. These ancillary testing strategies are especially useful in small biopsy samples, including aspiration cytology. Molecular techniques are also expanding our understanding of salivary gland tumor pathology and are helping to identify potential targets that may improve treatment for some of these tumors. Here, we summarize the clinical use of new immunohistochemical markers in our practice and review the current understanding of chromosomal rearrangements in salivary gland tumor pathology, emphasizing the prospects for exploiting molecular alterations in salivary gland tumors for diagnosis and targeted therapy. We find that immunohistochemistry and fluorescence in situ hybridization are powerful tools toward the diagnosis of salivary gland tumors, especially when used in a systematic manner based on morphologic differential-diagnostic considerations. As new targeted therapies emerge, it will become increasingly vital to incorporate appropriate molecular testing into the pathologic evaluation of salivary gland cancers.

Deep convolutional neural networks for classifying head and neck cancer using hyperspectral imaging

Abstract. Surgical cancer resection requires an accurate and timely diagnosis of the cancer margins in order to achieve successful patient remission. Hyperspectral imaging (HSI) has emerged as a useful, noncontact technique for acquiring spectral and optical properties of tissue. A convolutional neural network (CNN) classifier is developed to classify excised, squamous-cell carcinoma, thyroid cancer, and normal head and neck tissue samples using HSI. The CNN classification was validated by the manual annotation of a pathologist specialized in head and neck cancer. The preliminary results of 50 patients indicate the potential of HSI and deep learning for automatic tissue-labeling of surgical specimens of head and neck patients.

Clinical Practice Guideline: Evaluation of the Neck Mass in Adults:

Objective Neck masses are common in adults, but often the underlying etiology is not easily identifiable. While infections cause most of the neck masses in children, most persistent neck masses in adults are neoplasms. Malignant neoplasms far exceed any other etiology of adult neck mass. Importantly, an asymptomatic neck mass may be the initial or only clinically apparent manifestation of head and neck cancer, such as squamous cell carcinoma (HNSCC), lymphoma, thyroid, or salivary gland cancer. Evidence suggests that a neck mass in the adult patient should be considered malignant until proven otherwise. Timely diagnosis of a neck mass due to metastatic HNSCC is paramount because delayed diagnosis directly affects tumor stage and worsens prognosis. Unfortunately, despite substantial advances in testing modalities over the last few decades, diagnostic delays are common. Currently, there is only 1 evidence-based clinical practice guideline to assist clinicians in evaluating an adult with a neck mass. Additionally, much of the available information is fragmented, disorganized, or focused on specific etiologies. In addition, although there is literature related to the diagnostic accuracy of individual tests, there is little guidance about rational sequencing of tests in the course of clinical care. This guideline strives to bring a coherent, evidence-based, multidisciplinary perspective to the evaluation of the neck mass with the intention to facilitate prompt diagnosis and enhance patient outcomes. Purpose The primary purpose of this guideline is to promote the efficient, effective, and accurate diagnostic workup of neck masses to ensure that adults with potentially malignant disease receive prompt diagnosis and intervention to optimize outcomes. Specific goals include reducing delays in diagnosis of HNSCC; promoting appropriate testing, including imaging, pathologic evaluation, and empiric medical therapies; reducing inappropriate testing; and promoting appropriate physical examination when cancer is suspected. The target patient for this guideline is anyone ≥18 years old with a neck mass. The target clinician for this guideline is anyone who may be the first clinician whom a patient with a neck mass encounters. This includes clinicians in primary care, dentistry, and emergency medicine, as well as pathologists and radiologists who have a role in diagnosing neck masses. This guideline does not apply to children. This guideline addresses the initial broad differential diagnosis of a neck mass in an adult. However, the intention is only to assist the clinician with a basic understanding of the broad array of possible entities. The intention is not to direct management of a neck mass known to originate from thyroid, salivary gland, mandibular, or dental pathology as management recommendations for these etiologies already exist. This guideline also does not address the subsequent management of specific pathologic entities, as treatment recommendations for benign and malignant neck masses can be found elsewhere. Instead, this guideline is restricted to addressing the appropriate work-up of an adult patient with a neck mass that may be malignant in order to expedite diagnosis and referral to a head and neck cancer specialist. The Guideline Development Group sought to craft a set of actionable statements relevant to diagnostic decisions made by a clinician in the workup of an adult patient with a neck mass. Furthermore, where possible, the Guideline Development Group incorporated evidence to promote high-quality and cost-effective care. Action Statements The development group made a strong recommendation that clinicians should order a neck computed tomography (or magnetic resonance imaging) with contrast for patients with a neck mass deemed at increased risk for malignancy. The development group made the following recommendations: (1) Clinicians should identify patients with a neck mass who are at increased risk for malignancy because the patient lacks a history of infectious etiology and the mass has been present for ≥2 weeks without significant fluctuation or the mass is of uncertain duration. (2) Clinicians should identify patients with a neck mass who are at increased risk for malignancy based on ≥1 of these physical examination characteristics: fixation to adjacent tissues, firm consistency, size >1.5 cm, or ulceration of overlying skin. (3) Clinicians should conduct an initial history and physical examination for patients with a neck mass to identify those with other suspicious findings that represent an increased risk for malignancy. (4) For patients with a neck mass who are not at increased risk for malignancy, clinicians or their designees should advise patients of criteria that would trigger the need for additional evaluation. Clinicians or their designees should also document a plan for follow-up to assess resolution or final diagnosis. (5) For patients with a neck mass who are deemed at increased risk for malignancy, clinicians or their designees should explain to the patient the significance of being at increased risk and explain any recommended diagnostic tests. (6) Clinicians should perform, or refer the patient to a clinician who can perform, a targeted physical examination (including visualizing the mucosa of the larynx, base of tongue, and pharynx) for patients with a neck mass deemed at increased risk for malignancy. (7) Clinicians should perform fine-needle aspiration (FNA) instead of open biopsy, or refer the patient to someone who can perform FNA, for patients with a neck mass deemed at increased risk for malignancy when the diagnosis of the neck mass remains uncertain. (8) For patients with a neck mass deemed at increased risk for malignancy, clinicians should continue evaluation of patients with a cystic neck mass, as determined by FNA or imaging studies, until a diagnosis is obtained and should not assume that the mass is benign. (9) Clinicians should obtain additional ancillary tests based on the patient’s history and physical examination when a patient with a neck mass is deemed at increased risk for malignancy who does not have a diagnosis after FNA and imaging. (10) Clinicians should recommend evaluation of the upper aerodigestive tract under anesthesia, before open biopsy, for patients with a neck mass deemed at increased risk for malignancy and without a diagnosis or primary site identified with FNA, imaging, and/or ancillary tests. The development group recommended against clinicians routinely prescribing antibiotic therapy for patients with a neck mass unless there are signs and symptoms of bacterial infection.

Detection of Head and Neck Cancer in Surgical Specimens Using Quantitative Hyperspectral Imaging

Purpose: This study intends to investigate the feasibility of using hyperspectral imaging (HSI) to detect and delineate cancers in fresh, surgical specimens of patients with head and neck cancers. Experimental Design: A clinical study was conducted in order to collect and image fresh, surgical specimens from patients (N = 36) with head and neck cancers undergoing surgical resection. A set of machine-learning tools were developed to quantify hyperspectral images of the resected tissue in order to detect and delineate cancerous regions which were validated by histopathologic diagnosis. More than two million reflectance spectral signatures were obtained by HSI and analyzed using machine-learning methods. The detection results of HSI were compared with autofluorescence imaging and fluorescence imaging of two vital-dyes of the same specimens. Results: Quantitative HSI differentiated cancerous tissue from normal tissue in ex vivo surgical specimens with a sensitivity and specificity of 91% and 91%, respectively, and which was more accurate than autofluorescence imaging (P < 0.05) or fluorescence imaging of 2-NBDG (P < 0.05) and proflavine (P < 0.05). The proposed quantification tools also generated cancer probability maps with the tumor border demarcated and which could provide real-time guidance for surgeons regarding optimal tumor resection. Conclusions: This study highlights the feasibility of using quantitative HSI as a diagnostic tool to delineate the cancer boundaries in surgical specimens, and which could be translated into the clinic application with the hope of improving clinical outcomes in the future. Clin Cancer Res; 23(18); 5426–36. ©2017 AACR.

A pattern-based risk-stratification scheme for salivary gland cytology: A multi-institutional, interobserver variability study to determine applicability: Salivary FNA Interobserver Variability

Salivary gland aspiration cytology is useful in the preoperative management of patients but remains challenging, because of the extensive morphologic overlap of some tumors limits the ability to always determine the presence of malignancy. In response to this challenge, there has been increasing drive to develop a risk‐based categorization scheme for salivary gland aspirates. Herein, the authors examine the interobserver variability of 1 such pattern and risk‐based system.

Ancillary testing strategies in salivary gland aspiration cytology: A practical pattern-based approach

Fine needle aspiration of salivary gland tumors is a common preoperative triage as it is useful in determining which patients should undergo surgical resection and in guiding the extent of surgery in those cases deemed appropriate for resection. While a specific diagnosis can be achieved on a cytologic specimen in many cases, there is also a considerable amount of morphologic diversity that prevents such confident preoperative classification and in these cases it can be a challenge to confidently determine if a tumor is benign or malignant. Recently, a pattern based risk stratification approach was proposed for salivary gland cytology in which basaloid neoplasms are separated by stromal characteristics and oncocytoid neoplasms are separated primarily by background material such as mucus. In addition to potentially providing a stratification in risk of malignancy for salivary gland tumors, this approach is also useful to narrow differential diagnostic considerations and guide ancillary testing. In this review we use this proposed pattern based approach as a framework to discuss immunostains and fluorescence in situ hybridization studies which we find useful in our practice.

Polymerase Chain Reaction Human Papillomavirus (HPV) Detection and HPV Genotyping in Invasive Cervical Cancers With Prior Negative HC2 Test Results

Objectives Recently, three large Chinese cohort studies showed that 7.5% to 15.5% of patients with cervical carcinoma had negative high-risk human papillomavirus (hrHPV) test results on prior cytology specimens. These studies raise the question as to whether these negative hrHPV results represent truly hrHPV-negative carcinomas or false-negative hrHPV test results due to limitations in the testing of cytology specimens. This is increasingly an important question with increasing push to use hrHPV testing alone to screen for cervical cancer. Methods We investigated the hrHPV status on the surgical specimens from these same patients with cervical carcinoma using three polymerase chain reaction methods and a linear assay genotyping method. Results A variety of HPV genotypes were detected in 28 (45.9%) of 61 cases, all belonging to carcinogenic or possibly carcinogenic categories. HPV 16 was the most common genotype detected in positive cases (66.7%). HPV was detected in 25 (50%) of 50 squamous cell carcinomas and two (66.7%) of three adenosquamous carcinomas and only one (12.5%) of eight endocervical adenocarcinomas. Conclusions Our findings demonstrate that negative hrHPV testing on cervicovaginal cytology specimens in women later diagnosed with cervical carcinoma can be explained by the occurrence of truly HPV-negative carcinomas in more than half of patients. These results should be considered in the development of future cervical cancer screening guidelines.

Application of Strict Criteria for Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features and Encapsulated Follicular Variant Papillary Thyroid Carcinoma: a Retrospective Study of 50 Tumors Previously Diagnosed as Follicular Variant PTC

Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) was recently proposed as a designation for a subset of follicular variant papillary thyroid carcinoma (FVPTC). Encapsulated FVPTC has been shown to be a fairly indolent tumor, and NIFTP are expected to represent the most indolent subset of these tumors. Many of the exclusion criteria for NIFTP related to architecture and a lack of psammoma bodies are designed to preclude the inclusion of more aggressive non-FVPTC tumors in this indolent group and also exclude the diagnosis of FVPTC. In addition to strict application of histologic features to ensure that NIFTP represents a subset of encapsulated FVPTC without invasion, other exclusion criteria including high mitotic activity and necrosis may also lead to a lack of one-to-one correlation between the diagnosis of NIFTP and encapsulated FVPTC without invasion. In this series, 50 cases previously diagnosed as FVPTC over a 2-year period from a large academic center are retrospectively reviewed for reclassification as NIFTP. Additionally, cases not meeting criteria for NIFTP are more accurately classified using the most up to date WHO criteria. Prior BRAF V600E mutation testing was examined for these tumors when available. Seventeen of 50 (34%) tumors met criteria for classification as NIFTP and, 17 (34%) were classified as encapsulated FVPTC with invasion. Strict application of architectural features led to classification of 12 (24%) tumors as non-FVPTC with a variety of more aggressive designations. Tumors classified as NIFTP and encapsulated FVPTC with invasion lacked lymph node metastases (0/4; 0/7, respectively) and BRAF mutations (0/12; 0/13, respectively). In contrast, infiltrative FVPTC, encapsulated PTC with or without invasion, and conventional PTC showed more aggressive features with lymph node metastases and BRAF V600E mutations. One case not meeting criteria for NIFTP maintained the diagnosis of encapsulated FVPTC without invasion but demonstrated significant mitotic activity (three mitoses/ten HPF) and lacked lymph node metastases and BRAF V600E mutation. These findings demonstrate the importance of using strict criteria, especially the lack of true papillary architecture, for the diagnosis of NIFTP and encapsulated FVPTC to ensure that only truly indolent tumors will be included in these diagnoses and to allow tumors with potential for more aggressive behavior to be appropriately treated.

A Correlative Analysis of PD-L1, PD-1, PD-L2, EGFR, HER2, and HER3 Expression in Oropharyngeal Squamous Cell Carcinoma

We explored potential associations of the PD-1/PD-L1/PD-L2 pathway with clinical characteristics, outcome, and expression of EGFR, HER2, HER3 in oropharyngeal squamous cell carcinoma (OPSCC) using an institutional database. Protein expression was assessed by IHC on tissue microarray sections (EGFR, HER2, HER3) or whole tissue sections (PD-1/PD-L1/PD-L2). Expression of EGFR, HER2, HER3, PD-L1, and PD-L2 was quantified on tumor cells. Maximum density of PD-1 positive lymphocytes was measured on a scale of 0 to 4 within the tumor mass and peritumoral stroma. Associations between biomarkers and patient outcomes were tested using descriptive and inferential statistics, logistic regression, and Cox proportional hazards models. We analyzed tissue samples from 97 OPSCC cases: median age 59 years, p16+ (71%), male (83.5%), never smokers (18%), stage 3 to 4 disease (77%). Twenty-five percent of cases were PD-L1 positive. The proportion of PD-L1+ tumors was higher in p16+ (29%) than p16− OPSCC (11%, P = 0.047). There was no correlation between PD-L1, PD-L2, PD-1, EGFR, HER2, or HER3 expression. Positive PD-L1 status correlated with advanced nodal disease on multivariate analysis (OR 5.53; 95% CI, 1.06–28.77; P = 0.042). Negative PD-L2 expression was associated with worse survival (HR 3.99; 95% CI, 1.37–11.58; P = 0.011) in p16− OPSCC. Lower density of PD-1 positive lymphocytes in peritumoral stroma was associated with significantly increased risk of death on multivariate analysis (HR 3.17; 95% CI, 1.03–9.78; P = 0.045) after controlling for prognostic factors such as stage and p16 status. PD-L1 expression on tumor cells correlates with p16 status and advanced nodal status in OPSCC. PD-1 positive lymphocytes in peritumoral stroma serve as an independent prognostic factor for overall survival. Mol Cancer Ther; 17(3); 710–6. ©2018 AACR.

Label-free reflectance hyperspectral imaging for tumor margin assessment: a pilot study on surgical specimens of cancer patients

Abstract. A label-free, hyperspectral imaging (HSI) approach has been proposed for tumor margin assessment. HSI data, i.e., hypercube (x,y,λ), consist of a series of high-resolution images of the same field of view that are acquired at different wavelengths. Every pixel on an HSI image has an optical spectrum. In this pilot clinical study, a pipeline of a machine-learning-based quantification method for HSI data was implemented and evaluated in patient specimens. Spectral features from HSI data were used for the classification of cancer and normal tissue. Surgical tissue specimens were collected from 16 human patients who underwent head and neck (H&N) cancer surgery. HSI, autofluorescence images, and fluorescence images with 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) and proflavine were acquired from each specimen. Digitized histologic slides were examined by an H&N pathologist. The HSI and classification method were able to distinguish between cancer and normal tissue from the oral cavity with an average accuracy of 90%±8%, sensitivity of 89%±9%, and specificity of 91%±6%. For tissue specimens from the thyroid, the method achieved an average accuracy of 94%±6%, sensitivity of 94%±6%, and specificity of 95%±6%. HSI outperformed autofluorescence imaging or fluorescence imaging with vital dye (2-NBDG or proflavine). This study demonstrated the feasibility of label-free, HSI for tumor margin assessment in surgical tissue specimens of H&N cancer patients. Further development of the HSI technology is warranted for its application in image-guided surgery.