Trevor E. Angell

Immunogenicity of murine solid tumor models as a defining feature of in vivo behavior and response to immunotherapy

Immune profiling has been widely used to probe mechanisms of immune escape in cancer and identify novel targets for therapy. Two emerging uses of immune signatures are to identify likely responders to immunotherapy regimens among individuals with cancer and to understand the variable responses seen among subjects with cancer in immunotherapy trials. Here, the immune profiles of 6 murine solid tumor models (CT26, 4T1, MAD109, RENCA, LLC, and B16) were correlated to tumor regression and survival in response to 2 immunotherapy regimens. Comprehensive profiles for each model were generated using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and flow cytometry techniques, as well as functional studies of suppressor cell populations (regulatory T cells and myeloid-derived suppressor cells), to analyze intratumoral and draining lymphoid tissues. Tumors were stratified as highly or poorly immunogenic, with highly immunogenic tumors showing a significantly greater presence of T-cell costimulatory molecules and immune suppression in the tumor microenvironment. An absence of tumor-infiltrating cytotoxic T lymphocytes and mature dendritic cells was seen across all models. Delayed tumor growth and increased survival with suppressor cell inhibition and tumor-targeted chemokine+/−dendritic cells vaccine immunotherapy were associated with high tumor immunogenicity in these models. Tumor MHC class I expression correlated with the overall tumor immunogenicity level and was a singular marker to predict immunotherapy response with these regimens. By using experimental tumor models as surrogates for human cancers, these studies demonstrate how select features of an immune profile may be utilized to identify patients most likely to respond to immunotherapy regimens.

Noninvasive Follicular Variant of Papillary Thyroid Carcinoma and the Afirma Gene-Expression Classifier

BACKGROUND It is now recognized that noninvasive follicular variant of papillary thyroid carcinoma (NFVPTC) is a distinct subset of FVPTC with an exceedingly indolent clinical course. The Afirma gene-expression classifier (GEC) helps guide clinicians in the management of thyroid nodules with indeterminate fine-needle aspiration (FNA) results. Thyroid surgery is recommended for nodules with a suspicious Afirma result, whereas observation is deemed reasonable for most nodules with a benign result. The aim of this study was to confirm that the Afirma test detects NFVPTCs and to determine how many carcinomas detected by the Afirma GEC represent NFVPTCs. METHODS From a database of 249 FNAs sent for Afirma testing between January 2012 and October 2014, a search was conducted for cases with a preceding FNA diagnosis of atypia/follicular lesion of undetermined significance (AUS/FLUS) or suspicious for a follicular neoplasm (SFN), a suspicious Afirma result, and a corresponding resection specimen reviewed at Brigham and Womens Hospital. The diagnoses of the prior FNAs and subsequent resection specimens were recorded. Slides for all resection specimens with a diagnosis of FVPTC were reviewed to identify NFVPTCs. RESULTS Sixty-three cases met the inclusion criteria. The preceding FNA diagnosis was AUS/FLUS in 34 (54%) cases and SFN in 29 (46%) cases. The surgical resection specimen demonstrated 16 (25%) FVPTCs, five (8%) follicular thyroid carcinomas, one (2%) classical type PTC, and 41 (65%) benign tumors/nodules. Of the 16 FVPTCs, 14 (88%) were NFVPTCs. Thus, NFVPTCs accounted for 64% of the carcinomas in the cohort. CONCLUSION These results indicate that the Afirma GEC detects NFVPTCs and that many of the carcinomas detected by Afirma are NFVPTCs. While all care should be individualized and include clinical and sonographic assessment, these results suggest lobectomy as opposed to total thyroidectomy should be considered for nodules with a preceding AUS/FLUS or SFN on cytology and a suspicious Afirma result.

Survival Signals and Targets for Therapy in Breast Implant–Associated ALK− Anaplastic Large Cell Lymphoma

Purpose: Anaplastic lymphoma kinase (ALK)–negative, T-cell, anaplastic, non–Hodgkin lymphoma (T-ALCL) in patients with textured saline and silicone breast implants is a recently recognized clinical entity for which the etiology and optimal treatment remain unknown. Experimental Design: Using three newly established model cell lines from patient biopsy specimens, designated T-cell breast lymphoma (TLBR)-1 to -3, we characterized the phenotype and function of these tumors to identify mechanisms of cell survival and potential therapeutic targets. Results: Cytogenetics revealed chromosomal atypia with partial or complete trisomy and absence of the NPM-ALK (2;5) translocation. Phenotypic characterization showed strong positivity for CD30, CD71, T-cell CD2/5/7, and antigen presentation (HLA-DR, CD80, CD86) markers, and interleukin (IL)-2 (CD25, CD122) and IL-6 receptors. Studies of these model cell lines showed strong activation of STAT3 signaling, likely related to autocrine production of IL-6 and decreased SHP-1. STAT3 inhibition, directly or by recovery of SHP-1, and cyclophosphamide–Adriamycin–vincristine–prednisone (CHOP) chemotherapy reagents, effectively kill cells of all three TLBR models in vitro and may be pursued as therapies for patients with breast implant–associated T-ALCLs. Conclusions: The TLBR cell lines closely resemble the primary breast implant–associated lymphomas from which they were derived and as such provide valuable preclinical models to study their unique biology. Clin Cancer Res; 18(17); 4549–59. ©2012 AACR.

MHC Class I Loss is a Frequent Mechanism of Immune Escape in Papillary Thyroid Cancer that is Reversed by Interferon and Selumetinib Treatment in vitro

Purpose: To evaluate MHC class I expression on papillary thyroid cancer (PTC) and analyze changes in MHC expression and associated immune activation with current and experimental treatments for thyroid cancer using in vitro PTC cell lines. Experimental Design: MHC class I expression and assessment of tumor-infiltrating leukocyte populations were evaluated by immunohistochemistry. PTC cell lines were analyzed for HLA-ABC expression by flow cytometry following tyrosine kinase inhibitor, IFNα or IFNγ, or radiation treatment. Functional changes in antigenicity were assessed by coculture of allogeneic donor peripheral blood leukocytes (PBL) with pretreated or untreated PTC cell lines and measurement of T-cell activation and cytokine production. Results: Both MHC class I and β2-microglobulin expression was reduced or absent in 76% of PTC specimens and was associated with reduced tumor-infiltrating immune cells, including effector (CD3+, CD8+, CD16+) and suppressor (FoxP3+) populations. Treatment of PTC cell lines with the MEK1/2 inhibitor selumetinib or IFN increased HLA-ABC expression. This phenotypic change was associated with increased T-cell activation (%CD25+ of CD3+) and IL2 production by PBL cocultured with treated PTC cell lines. Additive effects were seen with combination selumetinib and IFN treatment. Conclusions: MHC class I expression loss is frequent in human PTC specimens and represents a significant mechanism of immune escape. Increased antigenicity following selumetinib and IFN treatment warrants further study for immunotherapy of progressive PTC. Clin Cancer Res; 20(23); 6034–44. ©2014 AACR.

BRAFV600E in Papillary Thyroid Carcinoma Is Associated with Increased Programmed Death Ligand 1 Expression and Suppressive Immune Cell Infiltration

BACKGROUND There remain a small number of patients with papillary thyroid cancer (PTC) who suffer recurrence, metastases, or death. While mutation of the BRAF gene, corresponding to the constitutively active BRAF(V600E) protein, has been associated with worse clinical outcomes in thyroid cancer, the reasons underlying this observation are presently unknown. Disruption of endogenous host immune surveillance and promotion of tumor immune escape is one mechanism by which BRAF(V600E) tumors may achieve more aggressive behavior. This study evaluated the relationship between BRAF(V600E) status and known strategies of tumor-mediated immune suppression. METHODS Tissue sections of PTC tumors from 33 patients were evaluated by immunohistochemistry for tumor-expressed suppressive ligands and enzymes and effector and suppressor populations of tumor-infiltrating immune cells. Presence of BRAF(V600E) was evaluated by direct DNA sequencing of PTC specimens and the results correlated with tumor-expressed molecules and tumor-infiltrating immune cell populations, as well as patient characteristics and pathologic findings. RESULTS BRAF(V600E) tumors more often express high levels of immunosuppressive ligands programmed death ligand 1 (53% vs. 12.5%) and human leukocyte antigen G (41% vs. 12.5%) compared to BRAF wild-type tumors. There was no association between indoleamine 2,3-dioxygenase 1 expression and BRAF(V600E) status. Furthermore, BRAF(V600E) tumors demonstrate both lower CD8(+) effector to FoxP3(+) regulatory T cell, and CD68(+) pan-macrophage to CD163(+) M2 macrophage ratios, indicating relative increases in suppressive T cell and macrophage components, respectively. CONCLUSIONS Overall, BRAF(V600E) PTC tumors display a broadly immunosuppressive profile and evidence of disturbed host tumor immune surveillance that may contribute to the poorer outcomes observed in this subset of patients with thyroid cancer.

The Influence of Patient Age on Thyroid Nodule Formation, Multinodularity, and Thyroid Cancer Risk.

INTRODUCTION Although advancing age is known to influence the formation of thyroid nodules, the precise relationship remains unclear. Furthermore, it is uncertain whether age influences the risk that any thyroid nodule may prove cancerous. AIM The aim was to determine the impact of patient age on nodule formation, multinodularity, and risk of thyroid malignancy. METHOD We conducted a prospective cohort analysis of consecutive adults (ages 20-95 y) who presented for evaluation of nodular disease from 1995 to 2011. A total of 6391 patients underwent ultrasound and fine-needle aspiration of 12 115 nodules ≥ 1 cm. Patients were divided into six age groups and compared using sonographic, cytological, and histological endpoints. RESULT The prevalence of thyroid nodular disease increases with advancing age. The mean number of nodules at presentation increased from 1.5 in the youngest cohort (age, 20-30 y) to 2.2 in the oldest cohort (age, >70 y; P < .001), demonstrating a 1.6% annual increased risk for multinodularity (odds ratio, 1.02; P < .001). In contrast, the risk of malignancy in a newly identified nodule declined with advancing age. Thyroid cancer incidence per patient was 22.9% in the youngest cohort, but 12.6% in the oldest cohort (odds ratio, 0.972; P < .001), demonstrating a 2.2% decrease per year in the relative risk of malignancy between ages 20 and 60 years, which stabilized thereafter. Despite a lower likelihood of malignancy, identified cancers in older patients demonstrated higher risk histological phenotypes. Although nearly all malignancies in younger patients were well-differentiated, older patients were more likely to have higher risk papillary thyroid carcinoma variants, poorly differentiated cancer, or anaplastic carcinoma (P < .001). CONCLUSION With advancing age, the prevalence of clinically relevant thyroid nodules increases, whereas the risk that such nodules are malignant decreases. Nonetheless, when thyroid cancer is detected in older individuals, a higher-risk histological phenotype is more likely. These data provide insight into the clinical paradox that confronts physicians managing this common illness.

Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features Accounts for More Than Half of “Carcinomas” Harboring RAS Mutations

Background: Molecular testing of thyroid nodules is increasingly being utilized to guide clinical management decisions. RAS mutations are the most frequent mutations detected in the context of an indeterminate fine-needle aspiration (FNA) diagnosis. The term “noninvasive follicular thyroid neoplasm with papillary-like nuclear features” (NIFTP) was recently introduced to promote conservative management of tumors previously classified as noninvasive follicular variant of papillary thyroid carcinoma (FVPTC). This change in terminology was based on the indolent clinical behavior of these tumors and their molecular profile, which includes frequent RAS mutations. The aim of this study was to determine the percentage of RAS-mutant “carcinomas” that would now be classified as NIFTPs. Methods: A search was performed for cases with known activating RAS mutations in a database of 199 thyroid carcinomas that underwent molecular characterization as part of Profile:Oncopanel between July 2013 and July 2015. Cases of FV...

Clinical Features and Hospital Outcomes in Thyroid Storm: A Retrospective Cohort Study

CONTEXT Thyroid storm (TS) is a rare but life-threatening manifestation of thyrotoxicosis. Predictive features and outcomes remain incompletely understood, in part because studies comparing TS with hospitalized thyrotoxic patients have rarely been performed. OBJECTIVES Our objectives were to compare the diagnosis and outcomes in TS versus hospitalized compensated thyrotoxic (CT) patients and to assess differences in diagnostic classification using the Burch-Wartofsky scores (BWSs) or Akamizu (Ak) criteria for identifying TS. DESIGN, SETTING, AND PATIENTS This was a retrospective cohort study of hospitalized patients during a 6-year period at an academic tertiary hospital, with age ≥ 18 years, TSH <0.01 mIU/L, and clinically diagnosed TS or CT. OUTCOME MEASURES In-patient mortality, hospital and intensive care unit length of stay, intubation, and ventilator duration were assessed. RESULTS Twenty-five TS and 125 CT patients were identified and analyzed. All but 1 TS patient received thionamides, β-blockade, glucocorticoids, and iodides within 24 hours of diagnosis. CT patients received thionamides and β-blockade alone. In the acute hospital setting, rates of fever (>100.4 °F), heart rate (>100 beats/min), altered mentation, and a precipitating event were all higher for TS than for CT patients. Altered mentation was the only clinical feature significantly different between TS and the subset of CT patients defined as TS by BWS or Ak criteria (P < .001). TS patients had greater in-patient mortality, hospital and intensive care unit length of stay, and ventilation requirements than CT patients. CONCLUSIONS In acutely hospitalized thyrotoxic patients, the presence of central nervous system dysfunction distinguished clinically diagnosed TS from patients with BWS- or Ak-defined TS. Because TS patients had significantly worse outcomes in this study, thyrotoxic patients with possible TS and central nervous system dysfunction may derive the greatest benefit from aggressive supportive and TS-specific treatments.

HIGH-DOSE BIOTIN TREATMENT FOR SECONDARY PROGRESSIVE MULTIPLE SCLEROSIS MAY INTERFERE WITH THYROID ASSAYS.

OBJECTIVE To review cases and increase awareness in clinicians treating patients who may be taking biotin. METHODS We describe the presentation and workup of a woman with secondary progressive multiple sclerosis on high dose biotin with laboratory studies suggestive of thyrotoxicosis. RESULTS Plasma samples showed laboratory evidence of elevated thyroid hormone levels with elevated free thyroxine >7.8 ng/dl (reference interval (RI) 0.9-1.7 ng/dl) and decreased thyroid stimulating hormone <0.02 uIU/ml (RI 0.50-5.70 uIU/ml). Laboratory values normalized when biotin was withheld prior to repeat testing. CONCLUSIONS Our case report demonstrates that ingestion of high dose biotin in multiple sclerosis patients can cause interference with laboratory assessment of thyroid function. This interference causes laboratory values suggestive of thyrotoxicosis and can lead to unnecessary evaluation. Clinicians should be aware of the risk of laboratory interference in this patient demographic.

Afirma Benign Thyroid Nodules Show Similar Growth to Cytologically Benign Nodules During Follow-Up

CONTEXT The Afirma gene expression classifier (GEC) is a molecular diagnostic test that has a high negative predictive value for ruling out malignancy in thyroid nodules with indeterminate cytology. Many patients with a cytologically indeterminate and GEC benign (Cyto-I/GEC-B) nodule undergo monitoring instead of diagnostic surgery, but few data describe their follow-up. OBJECTIVE The objective of the study was to determine the sonographic changes and clinical outcomes for patients with Cyto-I/GEC-B nodules compared with patients with cytologically benign (Cyto-B) nodules. DESIGN This was a retrospective analysis of consecutive Cyto-I/GEC-B nodules evaluated at Brigham and Womens Hospital compared with Cyto-B nodules. MAIN OUTCOMES Nodule growth of 20% or greater in two dimensions or of 50% or greater in volume, change in sonographic features, and rates of repeat fine-needle aspiration, thyroidectomy, and malignancy. RESULTS Ninety-five Cyto-I/GEC-B nodules in 90 patients were identified. Five patients underwent primary surgical resection. Of the remaining 90 nodules, 58 (64.4%) had sonographic follow-up available at a median of 13 months (range 4-40 mo). Cyto-I/GEC-B nodules showed similar growth compared with 1224 Cyto-B nodules using either of the following criteria: 20% or greater in two dimensions (8.6% vs 8.3%, P = .80) or 50% or greater in volume (17.2% vs 13.8%, P = .44). Thyroidectomies were more frequent in the Cyto-I/GEC-B group (13.8% vs 0.9%, P < .0001), but cancer was found in only one patient, with no evidence of persistent disease after initial treatment. CONCLUSIONS Cyto-I/GEC-B nodules demonstrate similar growth to Cyto-B nodules during follow-up. Although Cyto-I/GEC-B nodules were more frequently resected, only one malignancy was found. These data suggest that reassessment of Cyto-I/GEC-B nodules may be performed similarly to those with benign cytology.