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Immune infiltrates in papillary thyroid carcinomas
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Immune infiltrates in papillary thyroid carcinomas
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IMMUNE INFILTRATES IN PAPILLARY THYROID CARCINOMAS by Kate Yuri Kang A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (PHYSIOLOGY AND BIOPHYSICS) December 2012 Copyright 2012 Kate Yuri Kang ii ACKNOWLEDGEMENTS I am profoundly grateful to the following people for their mentorship and for supporting the fruition of my thesis: Dr. Harvey Kaslow Dr. Alan Epstein Dr. Herb Meiselman Dr. Adrian Correa Dr. Trevor Angel Dr. Melissa Lechner Also, much gratitude to my fellow lab members: Connor Church Rikki Bass Julie Jang iii TABLE OF CONTENTS Acknowledgments ii List of Abbreviations v List of Tables vi List of Figures vii Abstract viii Chapter 1: Background Thyroid Gland 1 Thyroid Cancer 4 Second-level: Types of Thyroid Cancer 4 Second-level: Management of Thyroid Cancer 6 Potential Therapies for Thyroid Cancers Not Amenable to Surgical Cure 9 Second-level: Endogenous Thyroid Cell Signaling Pathways 9 Second-level: Kinase Inhibitors 10 Thyroid Cancer and Immunotherapy 13 Second-level: Immune System 13 Second-level: Cancer Immunotherapy 14 Chapter 2: Rationale 18 Why Study Thyroid Cancer? 18 Is Immunotherapy a Viable Treatment Option? 20 Role of Adaptive Immune System in AITD 23 Immunotherapy and Thyroid Cancer 24 Chapter 3: Gaps in Knowledge 25 Tumor Associated Lymphocytes 25 Presence of Lymphocytes and Prognostic Factors 27 T Regulatory and NK Cells in Thyroid Papillary Carcinomas 32 Chapter 4: Study Objectives 34 Chapter 5: Materials and Methods 35 Second-level: Tissue Samples 35 Second-level: Immunohistochemistry Techniques 37 Second-level: Scoring 41 Second-level: Screening for BRAF V600E Mutation 43 iv Chapter 6: Results 44 Chapter 7: Conclusions 50 Chapter 8: Future Studies 51 Bibliography 54 Appendices Appendix A: Immunohistochemistry Protocol-ABC Method 62 Appendix B: Scoring Schematic 64 Appendix C: TNM Staging for Thyroid Cancer 65 v LIST OF ABBREVIATIONS AITD Autoimmune Thyroid Disease cAMP Cyclic Adenosine Monophosphate CTL Cytotoxic T- Lymphocytes DTC Differentiated Thyroid Carcinoma FNAB Fine Needle Aspiration Biopsy LI Lymphocytic Infiltration MHC Majorhistocompatibility Complex MTC Medullary Thyroid Carcinoma NIS Sodium Iodine Symporter NK Natural Killer PTC Papillary Thyroid Carcinoma RAIA Radioactive Iodine Ablation TAL Tumor Associated Lymphocytes TH Thyroid Hormone Tg Thyroglobulin TNG Thyroid Nodule Goiter TPO Thyroid Peroxidase TRH Thyrotropin Releasing Hormone TSH Thyroid Stimulating Hormone vi LIST OF TABLES Table 1: Patient Population 38 Table 2: Antibodies 40 Table 3: Patient Characteristics 44 Table 4: CD8 + and FoxP3 + cell counts with patient age and LN status 49 vii LIST OF FIGURES Figure 1: Front and rear view of thyroid gland 1 Figure 2: Regulation of the hypothalamic-pituitary-thyroid axis 2 Figure 3: Histological derivation of thyroid cancers 5 Figure 4: Cancer immnoediting 17 Figure 5: Histology of normal thyroid papillary thyroid carcinomas 36 Figure 6: Intratumoral vs. invading margin 42 Figure 7: Immunohistochemistry of PTC patient specimens 42 Figure 8: Patterns of tumor immune infiltrates and LN metastasis 47 Figure 9: CD8 + infiltration by lymph node metastasis status 47 Figure 10: Patterns of tumor immune infiltrates and age 48 viii ABSTRACT Introduction: Thyroid cancer is the most common endocrine malignancy. Although typically well controlled with surgery and radioactive iodine treatments, some patients suffer from recurrent disease that can be fatal. For some of these patients current therapies fail and thus new treatment modalities are needed. Immunotherapy is a promising approach for treating cancer in general but the way in which the immune system interacts with thyroid cancer is poorly characterized or understood. A better understanding of this interaction should help guide efforts to use the immune system to treat thyroid cancer. Purpose: Characterize immune responses to thyroid tumors in patients experiencing different degrees of disease progression. Approach: Design and initiate a retrospective study analyzing archived papillary thyroid cancer samples regarding lymphocyte subtype infiltration by immunohistochemistry staining and compare to disease progression. Results: Paraffin-preserved samples of thyroid tumors were obtained from 43 patients. The samples were sectioned, stained, and evaluated for the presence of cells expressing lymphocyte markers CD3, CD8, CD16, CD68, and FoxP3. No significant differences were found in the number of CD8 + , CD68 + , CD16 + and FoxP3 + regulatory T cells with respect to both age and lymph node status in papillary thyroid cancer. A decreasing ix number of CD16 + cells significantly correlated with an increase in age. Moreover, there were generally a greater number of effector CD8 + cells relative to FoxP3 regulatory cells across the patient population. Conclusions: The results support the hypothesis that there is an active immune response to papillary thyroid carcinomas that may relate to disease prognosis. The age and lymph node status does not predict characteristics of the immune response. The correlation between NK cells and prognosis may be worth further exploring as NK cells are associated with tumor rejection and limited disease recurrence. 1 CHAPTER 1: BACKGROUND Thyroid Gland The thyroid gland is a bi-lobed gland shaped like a butterfly located anterior to the trachea in the neck. Figure 1: Front and rear view of thyroid gland (National Cancer Institute) Three major components that play a role in the production of thyroid hormone are (Stathatos, 2012): 1. Thyroid gland (functional unit) and the thyroid follicle (location of synthesis and release). 2. Hypothalamus 3. Pituitary gland 2 The thyroid follicle is a cystic structure with a single layer of epithelial cells termed follicular cells or thyrocytes. The follicular cells form a sphere surrounding a substance termed colloid. The colloid consists mainly of thyroglobulin (Tg) and is linked to the precursor of thyroid hormone (TH). The release of TH into the systemic circulation is mediated by the phagocytosis of thyroglobulin by the follicular cells. Thyroid-stimulating hormone (TSH) released from the anterior pituitary gland is a major regulator of TH release. TSH is sensitive to changes in thyroid hormone serum levels via a negative feedback system. Figure 2: Regulation of the hypothalamic-pituitary-thyroid axis (Stathatos, 2012) Thus, measurement of serum TSH is often the most sensitive test available to clinicians for the diagnosis of various states of thyroid dysfunction such as hypothyroidism. Thyrotropin-releasing hormone (TRH) from the hypothalamus also promotes the release of TSH. TH has a direct inhibitory effect on TRH production (Stathatos, 2012). 3 TSH affects thyrocytes via the TSH receptor and results in increased iodine uptake as well as increased synthesis and release of TH. The stimulation of adenyl cyclase mediates the activation of this receptor, which further results in an increase in the concentration of intracellular cyclic adenosine monophosphate (cAMP). There are 2 main types of thyroid hormone: 1. Thyroxine (T4)-carries 4 iodine atoms 2. Triiodothyronine (T3)-carries 3 iodine atoms Normally, 90% of the thyroid hormone released by the thyroid gland is in the T4 form. The sodium/iodine symporter (NIS) concentrates the iodine in the thyrocytes roughly 20- 40x above its serum concentration. Loss of this expression can lead to decreased iodine uptake. Once the iodine is in the thyrocytes, it is organified into tyrosine residues present in Tg (Stathatos, 2012). Thyroid peroxidase (TPO) enzyme is known to play a role in this process. Although the regulation of TPO is complex, iodine plays an important role as an increase in iodine blocks TPO activity. 4 Thyroid Cancer Thyroid cancer is the most common endocrine malignancy and is approximately 1% of all human cancers. According to the National Cancer Institute (NCI), in 2010 there were 44,670 new cases of thyroid cancer and 1,690 related deaths. The survival rate 20 years after diagnosis is 90% (Brenner, 2002). While this survival rate is encouraging it has not improved during the past 30 years and the incidence of thyroid cancers increased more than two fold between 1997-2002 (Davies e Welch, 2006). Recent candidate adjuvant therapies have shown minimal clinical benefit and thus there is a need for new therapeutic concepts and treatment modalities. Types of Thyroid Cancer There are four different types of thyroid cancer: anaplastic, medullary, follicular and papillary (Figure 3). Diagnosis is based primarily on histology, and the different types have different pathophysiologic origins, clinical courses, and treatments. The most common type of thyroid cancer is differentiated cell (95%), medullary cell (5-8%), or anaplastic (1-2%). Differentiated cells are further sub-classified as papillary or follicular. Papillary thyroid cancer (PTC) is the most frequent (80%), whereas follicular thyroid cancer (FTC) (15%), although less common, is more aggressive Differentiated thyroid carcinoma (DTC) is derived from follicular cells and initially exhibit high levels of NIS. Later DTC’s often lose NIS expression. DTC's are further subclassified into papillary, follicular and hurthle cell. Other uncommon follicular cell 5 Figure 3: Histological derivation of thyroid cancers (Harris e Bible, 2011) 6 derived cancers include "tall cell," "follicular variant PTC," and "poorly differentiated"-- these are associated with worse prognoses. Typically, differentiated tumors (papillary and follicular) respond well to treatment and have a high survival rate (Harris e Bible, 2011). DTC's can also rarely give rise to anaplastic thyroid cancer, which is known to be aggressive, hard to control and have poor prognosis. In contrast, medullary thyroid cancers (MTC) are derived from parafollicular cells and can occur as part of inherited syndromes, such as multiple endocrine neoplasia (MEN)-2. Neither parafollicular nor MTC cells express NIS. MTC cells secrete calcitonin and not Tg (Harris e Bible, 2011). Thus, calcitonin can be a useful tumor marker. Management of Thyroid Cancer The various sub-types of thyroid cancer exhibit a wide range of biologic behaviors. Consequently, it is difficult to initially determine at which end of the prognostic spectrum a patient resides. Nevertheless, one may assess a patient’s risk for tumor recurrence and mortality based on a triad of features: the patient’s age at the time of diagnosis, the tumor stage at presentation, and the tumor’s initial response to therapy. Although staging is helpful in the management process, gaps still remain in the treatment plan for a given patient. These gaps can be attributed to the lack of randomized controlled trials, which may be a result of the low incidence and generally favorable prognosis of the disease (Sipos e Mazzaferri, 2008). 7 The Tumor-Node-Metastases (TNM) system by the American Joint Committee on Cancer (AJCC) is the most common method to describe the stages of thyroid cancer. The system summarizes how large the cancer is as well as how far it has spread (Appendix - C). Current guidelines for treatment of progressive differentiated thyroid cancer are as follows (Sipos e Mazzaferri, 2008): 1. Surgery: Pivotal component of management (sub-total/total thyroidectomy, lymph node and lateral dissection). 2. 131 I: It is unlikely that surgery can remove every minute focus of cancerous thyroid tissue. Concentrating 131 I in remaining thyroid tumors can kill those cells. Successful radioiodine remnant ablation requires increased TSH for adequate uptake of iodine by the tissue. 3. Thyroid stimulating hormone suppression: TSH may promote growth and survival of remnant thyroid cancer cells. Administration of thyroid hormone sufficient to suppress TSH levels following radioactive iodine therapy has been shown to reduce adverse clinical events. DTC cells express TSH receptors that respond to TSH stimulation by increasing thyroglobulin and sodium–iodine symporters as well as upregulating cell growth. 4. Follow-up: Measurement of serum thyroglobulin is a key part of monitoring patient for persistent and/or recurrent DTC. 8 The American Thyroid Association (ATA) has published several revisions to the recommendations published in the November 2009 issue of Thyroid (Boelaert, 2010). There are two areas of extensive revisions. First, there is now a specific recommendation on the size of thyroid nodules that require fine-needle aspiration biopsy (FNAB). Second, there is now a more comprehensive classification system of FNAB results, which now includes a new category titled “suspicious for malignancy.” The recommendations are (Boelaert, 2010): Patients with tumors >1cm in diameter should initially be treated with near-total or total thyroidectomy Patients with involvement of central and lateral neck nodes should undergo therapeutic neck dissection Patients with advanced tumors (stages T3, T4) should be treated with prophylactic neck dissection Patients with either tumors >4cm, extrathyoidal invasion, or distant metasteses should receive ablative radioiodine therapy Patients with advanced disease refractory to radioiodine therapy should be candidates to receive tyrosine kinase inhibitors. Although the most common forms are typically well controlled with surgery and radioactive iodine treatment, recurrent disease is associated with significant morbidity and mortality. Cytotoxic chemotherapy treatments are limited due to high toxicity 9 profiles and low short-lasting response rates. Thus, chemotherapy is often not used for thyroid cancer management. It is important to note that there is no effective standard of care treatment for thyroid cancers that do not respond to radioactive iodine treatment. Potential Therapies for Thyroid Cancers Not Amenable to Surgical Cure Endogenous Follicular Thyroid Cell Signaling Pathways In the case of DTC and lack of surgical cure, there are two cell signaling pathways that may be looked at: TSH and NIS pathways. Both pathways are known treatment targets of early and advanced stage DTC’s. 1. TSH: known to stimulate follicular cell proliferation in early DTC. Loss of TSH receptor has been associated with more aggressive DTC and reconstitution of TSH receptor has shown to decrease DTC cell line growth (Hoffmann, Maschuw et al., 2006) 2. NIS: most DTC’s initially express NIS and thereby accumulates iodine intracellularly, which is the basis of radioactive iodine ablation (RAIA). In contrast, medullary thyroid cells do not express NIS so perhaps expression can be induced. Pre-clinical studies have shown that re-induction of NIS expression is possible via a NIS-expressing adenovirus (Spitzweg, Baker et al., 2007). 10 Kinase Inhibitors Considerable effort is being devoted to molecular targeted therapies aimed at the various genetic mutations associated with thyroid cancer (Woyach e Shah, 2009). The addition of molecular analysis may allow for qualitative improvements in therapies by improving diagnosis compared to diagnosis based on indeterminate morphology. The diagnostic tool of choice for preoperative cytologic evaluation is fine needle aspiration biopsy (FNAB). Although cytologic FNAB is a useful diagnostic method, it has certain limitations. It fails to distinguish follicular and Hurthle cell (unusual and relatively rare type of differentiated thyroid cancer) carcinomas from their benign counterparts (Theoharis, Roman et al., 2012). The six-tiered classification system for thyroid nodules recommended by the National Cancer Institute (NCI) aims to address the FNAB limitations. However, some have found a significant risk of malignancy in the nodules that were initially classified as “Not definitive for malignancy.” These limitations have led to efforts to classify thyroid cancers by detecting mutations that are part of the underlying cause of the disease (Theoharis, Roman et al., 2012). Activating mutations of the BRAF gene in human cancer were first shown in 2003 (Davies et al). Shortly after, the V600E (valine to gluatamate) point mutation in BRAF was found in 45% of papillary thyroid carcinomas (Xing, Tufano et al., 2004; Giordano, Kuick et al., 2005) and rarely in the follicular variant of papillary thyroid carcinomas. Additionally, thyroid cells expressing BRAF V600E in transgenic mice induced goiter 11 and invasive PTC (Knauf, Ma et al., 2005). Furthermore, a small inhibitory RNA construct targeting expression of both wild-type BRAF and BRAF V600E reduced thyroid cancer cell viability (Nikiforova, Kimura et al., 2003). The BRAF mutation becomes more evident as the process of dedifferentiation progresses, which suggests that it plays an early role in tumorigenesis and the dedifferentiation process (Nikiforov, 2004). The specificity of the BRAF mutation has been evaluated in cytologic preoperative FNAB samples (Xing, Tufano et al., 2004; Nikiforov, Ohori et al., 2011). The rate of malignancy in FNA-BRAF positive nodules has been shown to be 99.8%. Studies to establish the prognostic value of identifying BRAF mutations are ongoing. However, some have suggested that current evidence is sufficient to treat patients who are positive for the BRAF mutation with aggressive management from the onset, such as complete thyroidectomy, central lymph node dissection, higher doses of radioactive iodine, and closer clinical follow-up (Xing, 2010). Furthermore, another study has posited that BRAF may be used to predict lymph node metastasis but should be correlated to tumor size (So, Son et al., 2011). The study divided 100 consecutive patients into 3 groups according to tumor size. They found that although 12 the BRAF mutation was found in all 3 groups, prediction of lymph node metastasis was only seen in one group (tumor size >0.5, <1cm). Thyroid tumors carrying the BRAF mutations are dependent on the oncoprotein for viability. The inhibition of this expression results in tumor regression and restoration of radioiodine uptake in vivo in mice (Chakravarty, Santos et al., 2011). The development of transgenic mice with dox-inducible expression of BRAF V600E in thyroid follicular cells helps support these findings. Murine thyroid tumors induced by the activation of BRAF V600E following dox administration resulted in high-grade papillary tumors, phenotypically. The discontinuation of the BRAF V600E expression then resulted in regression of the tumors and restoration of iodine incorporation thereby allowing for therapeutic doses of radioiodine. BRAF triggers the canonical signaling pathway, which is thought to result in the activation of MEK and ERK. Therefore, BRAF-positive thyroid cell lines are sensitive to MAPK pathway inhibitors and its growth suppressive effects. The oncoproteins encoded by the BRAF, RET, and RAS constitutively activate MAPK signaling, which has been known to play a critical role in the pathogenesis of thyroid cancer. Moreover, gene expression required for thyroid hormone synthesis (NIS, TG, TPO) has been shown to be particularly sensitive to MAPK pathway signaling. Thus, inhibition of the MAPK pathway may reduce thyroid cancer growth. This approach may be effective regardless of the immune response towards a thyroid tumor. 13 Thyroid Cancer and Immunotherapy The Immune System The immune response can be broken into two subcategories: innate and adaptive. Innate responses provide an immediate defense mechanism to infections from pathogens that an organism has never encountered but does not confer enhanced protection from a subsequent encounter with the same pathogen. The innate response recruits immune cells to the sites of infection through the production of cytokines and activates the complement system. It also removes foreign substances via phagocytosis by white blood cells, which can then trigger an adaptive immune response via antigen presentation. The adaptive immune response is directed towards structures specific to various pathogens and confers specific protection from future encounters with those pathogens. Adaptive immune responses distinguish between non-self and self-antigens during the process of antigen presentation. Furthermore, they generate specific responses tailored to the presented antigen, and develops immunological memory in which the pathogen is memorized by a signature antibody. Adaptive immunity can be further divided into humoral and cell mediated responses. Antibodies mediate the humoral response whereas T cells mainly manage the cell- mediated response. The cell-mediated response does not exclusively involve T cells as certain antibodies also play a role. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense whereby an effector cell of the 14 immune system actively lyses a target cell bound by antibodies. B cells are mainly responsible for the humoral response as they transform into plasma cells and secrete antibodies. These antibodies then bind to antigens on invading substances and flag them for destruction. CD4 + T-helper 2 cells aid the process and provide co-stimulation. There are two types of antibodies: one may activate the complement system while the other activates effector cells. By contrast, the cell-mediated response involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines. The primary way the cell mediated immune response protects is by activating antigen-specific cytotoxic T-lymphocytes, which induce cell death by apoptosis, activating macrophages and NK cells, and secreting additional cytokines that affect other cells in both the innate and adaptive immune responses. For cancer immunotherapy, the goal is to harness the cytotoxic mechanisms of the immune system to kill cancer cells. Cancer Immunotherapy There are at least three distinct roles that the immune system plays in tumor prevention (Schreiber, Old et al., 2011). It protects the host against viral infections that lead to viral- induced tumors, it retards development of inflammatory environments conducive to 15 tumorigenesis, and it can eliminate tumor cells in certain tissues if it recognizes epitopes that differentiate cancer cells from non-transformed cells. The term “immunosurveillance” is based on the idea that tumor cells express epitopes that are different from their non-transformed counterparts. In the past decade, various research studies have observed that transplanted tumors grow more robustly in mice with compromised immune systems (i.e treatment with neutralizing monocloncal antibodies specific for interferon-γ) (Dighe, Richards et al., 1994). These results demonstrate the immune system can protect mice from primary and transplantable tumors and support the hypothesis that immunosurveillance can stop the development of at least some cancers. In addition to the control of tumor quantity, the immune system also controls “tumor quality” (immunogenicity). A 2001 study suggested that the immune system may shape tumor quality (Shankaran, Ikeda et al., 2001). Tumors formed in the absence of an intact immune system appeared to be more “immunogenic” than the tumors that arose in immunocompetent hosts. This observation suggests that in the face of an immune response, tumors can evolve under selective pressure to evade the response by either no longer expressing relevant epitopes or producing immunosuppressive factors. Characterizing the ongoing immune response in thyroid tumors may help shed light on the type of immunosuppression and the extent that it leads to more severe cases of thyroid cancer. 16 Thus, one of the primary challenges facing cancer immunotherapy is tumor evasion. The dysfunction of the host immune system often allows for tumor evasion mechanisms. Tumors often evade immune destruction by combining several mechanisms. Tumors may evade immune-surveillance by exhibiting partial or complete loss of human leukocyte antigen (HLA) class I, which normally increases susceptibility to natural killer cells lysis, but also produces factors that depress NK cytotoxicity. Tumor cells may also express ligands inducing T cell death, blunt dendritic cell response to activating factors, and recruit T regulatory cells to block immune-surveillance (Rescigno, Avogadri et al., 2007). In order for these situations to be successful, an immunotherapy must recognize other tumor-associated epitopes or overcome tumor-induced immune suppression (Lizée, Cantu et al., 2007; Sadun, Sachsman et al., 2007). The unifying conceptual framework that takes into account the various factors mentioned above is referred to as immunoediting (Fig 4). This hypothesis involves three sequential phases: elimination, equilibrium, and escape (Schreiber, Old et al., 2011). During the elimination phase, the innate and adaptive immune systems work together to sense the developing tumor and destroy it. In addition to the activation of innate immunity, certain expression of tumor antigens capable of propagating the expansion of CD4 + and CD8 + cells are needed. There is complete destruction of the tumor cell if the elimination phase serves as the endpoint of the immnoediting process. 17 Figure 4: Cancer Immnoediting (Schneider, Kimpfler et al., 2011) 18 CHAPTER 2: RATIONALE Why Study Thyroid Cancer? Although the chance for survival at the diagnosis of thyroid cancer may be 95%, it is not now possible at the time of diagnosis to predict with certainty whether any given patient will survive or suffer from progressive disease and die. Thus, there is a need for better diagnostic tools and therapies. Histologically and phenotypically, homogeneous tumors can exhibit a broad range of clinical behavior, and histopathology alone cannot distinguish relatively benign papillary thyroid cancer from highly aggressive, metastatic papillary thyroid cancer. In addition to saving nearly 1700 lives per year, all 44,000 patients diagnosed each year with thyroid cancer would benefit psychologically if they could receive treatments that led to a 100% cure rate without causing side effects that compromised quality of life. The presence of some mutations in signaling pathways, such as BRAF protein, measured at the time of diagnosis, have been associated with more aggressive thyroid cancer in some studies. However, these mutations are not present in all aggressive thyroid cancers and the precise pathophysiologic basis for the association with aggressiveness is not completely understood. As indicated before, there is currently no effective treatment for thyroid cancer refractory to conventional treatments. Standard therapy for differentiated follicular thyroid cancers (DTC) is near-total thyroid resection followed by radioactive iodine ablation (RAIA) 19 therapy and chronic TSH suppression. While the vast majority of patients with DTC are adequately treated with this regimen, some patients develop radioactive iodine refractory, recurrent or metastatic disease. For these patients, death from thyroid cancer is common within 3 years of diagnosis. A subset of patients with refractory disease can progress to local invasion in the neck, and/or distant metastases. Advanced disease in the neck can lead to tracheal invasion and laryngeal nerve damage, causing significant patient morbidity and mortality. Spread of metastatic disease to the lungs, bone, or brain compromises the function of those organs and is largely untreatable with current therapies. Moreover, new chemotherapies for thyroid cancer are available in only a few clinical trials, and patients are typically eligible or referred to an oncologist late in their disease course. However, premature initiation of ineffective and potentially toxic chemotherapy is also risky (Pfister e Fagin, 2008). Thus, alternative systemic strategies such as empiric therapeutic dosing with RAI are utilized when traditional therapeutic approaches fail, rather than traditional chemotherapies. Numerous adjuvant therapies have been evaluated in pre-clinical models and early phase clinical trials (e.g. tyrosine kinase inhibitors, VEGF neutralization, PPAR-γ inhibitors) but with little benefit yet seen. 20 Recent data suggest that most thyroid cancer metastases occur early in the disease process and remain dormant in “metastatic niches” such as the lungs and lymph nodes. Late-stage progression may be related to growth of these dormant cancer cells rather than new secondary metastases (Ringel, 2011). Immunotherapy may be an acceptable way to rid the body of these metastases after surgical removal of the bulk of the cancer just as the immune system clears infections after other treatments (e.g. antibiotics). Is Immunotherapy a Viable Treatment Option for Thyroid Cancer? The thyroid is a site of organ–specific autoimmune diseases. Autoimmune Thyroid Disease (AITD) is characterized by lymphocytic infiltration in the thyroid gland and the production of thyroid autoantibodies, such as those that are against thyroid antigens, which include thyroglobulin, thyroid peroxidase, pendrin and sodium iodine symporter (Raspé, Costagliola et al., 1995; Yoshida, Hisatome et al., 2009). There are 2 main clinical entities that are noted as AITD: Graves’s disease and Hashimoto’s thyroiditis, both of which are pathologically characterized by lymphocytic infiltration. Graves’ disease is an autoantibody-mediated disease (hyperthyroidism). Antigen presentation by majorhistocompatibility complex II (MHCII) to T cell receptors on CD4 + cells stimulates B cells, which results in the stimulation of the TSH receptor antibody, release of thyroid hormone, and clinical hyperthyroidism. Although removal of the thyroid gland cures the hyperthyroid condition, TSH receptors are expressed on other tissues, and when antibodies bind to them, other clinical problems arise (e.g. 21 exophthalamos). This results in the growth of thyroid follicular cells and thereby leads to hyperthyroidism. Thus, TSH receptor does not seem to be an ideal candidate antigen for thyroid cancer immunotherapy. By contrast, the lymphocytic infiltration in Hashimoto’s thyroiditis is more severe (Caturegli, Kimura et al., 2007). The response is cell mediated and involves both the MHCII and MHCI pathways. Thus, antigen presentation by MHCI to TCR’s on CD8 + generates antigen specific cytotoxic T lymphocytes (CTLs) (Nagayama, Nakahara et al., 2012). Hypothyroidism develops as thyroid follicles are destroyed. Some of the antibodies involved in the development of AITDs include (Stathatos e Daniels, 2012): 1. Thyroid autoantibody (TPO): trans-membrane protein important for the production of thyroid hormone via the organification of inorganic iodide and the coupling of iodinated tyrosines to produce T3/T4. TPO is the target of thyroid antibody. Anti-TPO antibodies are prominently present in both Graves’ and Hashimoto’s patients. Specific TPO epitopes recognized by T cells have yet to be known. 2. Thyroid stimulating hormone receptor antibody: primary cause of Graves’ hyperthyroidism. 3. Thyroglobulin antibody: precursor of thyroid hormone and considered to be very immunoreactive. Found in most patients with Hashimoto’s thyroiditis. 22 4. Sodium/Iodine symporter antibody: Found to be present in about 11% of Graves’ patients and in 20% of Hashimoto’s patients. However, its specific role in thyroid autoimmunity remains unclear. Information on the etiology of AITD has been gleaned from several studies. The presentation of autoantigens by autoimmune target cells themselves is important and has been shown in various autoimmune models (Kawashima, Tanigawa et al., 2011). For example, antigen presentation by epithelial cells in the gastrointestinal tract was shown to play a role in the pathology of Crohn’s disease (Silva, 2009). However, in regards to thyroid autoimmunity, MHCII expression by thyrocytes alone appears to be insufficient. Chronic thyroid expression of MHCII in transgenic mice expressing IFN-γ did not result in spontaneous AITD (Caturegli, Hejazi et al., 2000). Perhaps MHCII expression in thyroid cells alone is insufficient for the induction of a self-tolerance disruption. The destruction of the thyroid may be mediated by the induction of Fas-mediated apoptosis in thyroid follicular cells by cytokines (Giordano, Stassi et al., 1997; Mezosi, Wang et al., 2005; O' Reilly, Tai et al., 2009). The cytotoxic immune response leads to thyroid cell destruction as shown in past mouse models. Further spreading of additional autoantigen epitopes may then contribute to the development of AITD (Lehmann, Forsthuber et al., 1992). Both the autoantigen and MHCII are expressed in the same cell. This is also aligned with a murine model of Graves’ disease in which MHCII and TSHR were co-expressed in the same cell (Shimojo, Kohno et al., 1996). 23 It is possible that the antigens mentioned above will correlate with increased immune cell infiltrate in thyroid cancers and would therefore be highly immunogenic protein targets that could be used in cancer vaccines and immunotherapy. Such studies of the antigen and immune profile of thyroid cancer will aid the translation of cancer vaccine therapies from pre-clinical animal models to treatment of thyroid cancer patients. Thyroid follicular cancers display several unique antigens, thyroglobulin (Tg) and thyroid peroxidase (TPO), which may be targeted by dendritic cell vaccines. Role of Adaptive Immune System in AITD The role of regulatory T cells in association with several autoimmune thyroid diseases has been studied. A lack of and/or defective function of CD4 T cells were noted in some AITDs (Sakaguchi, Setoguchi et al., 2006). Marazuela et al. studied the thyroid infiltrate and the peripheral blood lymphocytes of 12 Graves’ disease patients and 8 Hashimoto’s thyroiditis patients. The number of regulatory T cells increased in both thyroid and peripheral blood. However, their suppressive function decreased. This may suggest that AITD regulatory T cells are unable to down-modulate the autoimmune response (Marazuela, García-López et al., 2006). Furthermore, the depletion of CD4 + CD25 + cells increase the “severity of hyperthyroidism” in mice with induced Graves’ disease (Nagayama, Horie et al., 2007). 24 Immunotherapy and Thyroid Cancer Lymphocytes are often found intra and peritumorally in primary thyroid tumors (Hirabayashi e Lindsay, 1965; Clark, Greenspan et al., 1980). Evidence suggests that the presence of local inflammatory response may predict a more favorable response in PTC patients (Matsubayashi, Kawai et al., 1995; Gupta, Patel et al., 2001; Lundgren, Hall et al., 2006). Lymphocytic infiltration was seen in cases where extrathyroidal invasion was reduced although there was no correlation between infiltration and tumor size and lymph node metastases (Matsubayashi, Kawai et al., 1995). In contrast, patients with LN involvement and tumor invasion but no infiltration had slightly higher rate of recurrence. Furthermore, in a retrospective study from 2006, thyroid cancer patients with lymphocyte infiltration had a higher rate of survival (Gupta, Patel et al., 2001). As age is often an important prognostic factor in thyroid cancer, patients 21 years old or younger had improved disease free survival alongside an increased number of proliferating lymphocytes (Gupta, Patel et al., 2001). A mixture of lymphocytes inclusive of T cells, B cells, and NK cells were found within or near these tumors (Modi, Patel et al., 2003). 25 CHAPTER 3: GAPS IN KNOWLEDGE Tumor Associated Lymphocytes Tumor associated lymphocytes linked to disease severity have been extensively studied in various types of cancer but less so in thyroid cancer. Leukocyte infiltrates in and around the tumor have traditionally been studied within the context of protumorigenic inflammation and anticancer immunosurveillance (Fridman, Galon et al., 2011). A comprehensive understanding of the architecture, context and composition of leukocyte infiltrates is a worthwhile goal. In the past, tumor infiltrates were viewed in reference to either the protumorigenic effects of inflammation or the maintenance of small tumors in an equilibrium state preceding immune escape. However, with advanced molecular tools, it is now apparent that perhaps distinct immune infiltrates have distinct prognostic and predictive significance. In a variety of tumors, dendritic cells (DC), M1 macrophages, Th1 CD4 + T cells, cytotoxic CD8 + T cells, and natural killer cells present are associated with reduced cancer growth. In contrast, M2 macrophages, myeloid-derived suppressor cells (MDSC), neutrophils, Th2 and Th17 CD4 + T cells, and FoxP3 + and CD4 + regulatory T cells (Treg) are associated with cancer growth (Fridman, Galon et al., 2011). A goal of this study is to initiate a study to determine if this association holds for thyroid cancer. CD8 + cytotoxic T lymphocytes can directly kill tumor cells. CD4 + T helper lymphocytes are a heterogenous cytokine secreting class of T lymphocytes. T helper type 1 (Th1) 26 lymphoyctes play a role in activating CTL’s whereas T helper type 2 (Th2) lymphocytes stimulate humoral immunity and active eosinophils. Recent research has brought to light various aspects of tumor infiltration lymphocytes. In terms of Th1 vs. Th2 and anti-tumor immunity, Th2 activation has been found to be less effective than Th1 activation (Yu e Fu, 2006). Another study has shown that a CD4 + regulatory T lymphocyte subset suppresses effectors T lymphocytes (Curiel, Coukos et al., 2004). The same study also showed that Treg preferentially traffic tumors due to chemokines produced by tumor cells and microenvironment macrophages. Furthermore, the prognostic value of subset ratios has recently gained significance. By analyzing specific ratios such as CD8 + /FoxP3 + (effector: regulatory) a more comprehensive view of disease site and its events may be provided. The study of ratios in addition to specific solitary subsets is aligned with the notion that the immune system is rather complex and inclusive of various checks and balances. The pooled analysis of the six studies looking at ovarian cancer, cervical cancer, hepatocellular carcinomas, gastric cancer, and adenocarcinomas of the oesophagus showed that reported survival based on CD8/FoxP3 ratios was strongly significant with low heterogeneity (Gooden, De Bock et al., 2011). Two additional studies reported positive effects on high CD8/FoxP3 ratio on disease specific (De Jong, Leffers et al., 2009; Leffers, Gooden et al., 2009) and progression-free survival (Cai, Xu et al., 2009; De Jong, Leffers et al., 2009). 27 Pre- and post-chemotherapy immune infiltrates are worth noting in understanding the prognostic vs. predictive benefits of looking at lymphocytic infilatration. A recent study examined the tumor infiltration of CD8 + and FoxP3 + T lymphocytes before and after neoadjuvant chemotherapy (Ladoire, Mignot et al., 2011). Interestingly, high CD8 + and low FoxP3 + infiltrates post-chemotherapy were associated with improved relapse-free survival as well as overall survival. Thus, chemotherapy-induced immune responses may be worth looking at as it seems to have an impact on the success (or failure) of chemotherapy. Presence of Lymphocytes and Prognostic Factors for Differentiated Thyroid Cancer One of the first studies to investigate lymphocytic infiltration in the papillary thyroid carcinoma was published in 1995 by Matsubayashi et al. They suggested that lymphocytic infiltration peri-tumorally or intra-tumorally in PTC may be effective in predicting a favorable prognosis. Although there have been prior studies that have investigated significant prognostic factors for differentiated thyroid cancer, they relied on existing pathology reports without independent review and did not look at specific lymphocyte subsets (Matsubayashi, Kawai et al., 1995; Lundgren, Hall et al., 2006). These studies reported a correlation between lymphocytic infiltration and reduced invasion. In 2001, Gupta et al. examined 39 childhood PTC, 9 follicular thyroid carcinomas, 2 28 medullary thyroid carcinomas, 11 benign thyroid lesions, and 2 normal thyroid glands for the presence of lymphocytes (leukocyte common antigen) and lymphocyte proliferation (proliferating cell nuclear antigen, Ki-67 via immunohistochemistry. The study reported that patients with PTC and the most numerous proliferative lymphocytes exhibited the best prognosis. One should note that this study focused on a population aged between 6- 21yrs. In 2003, Modi et al. (from the same lab as the above) examined 21 PTC for the presence of CD4 + (helper), CD8 + (killer), CD19 + (B cell), and CD56 + (NK) to further define the types of lymphocytes. They also looked at the presence of proliferating lymphocytes with Ki67. Again, this study was focused on PTC patients of the aforementioned age range of 6-21yrs old. The study reported that close to half of the 21 samples had CD4 + , CD8 + , or CD19 + cells. Only one sample was positive for CD56. In addition, about half of the samples showed Ki67 positivity, however dual staining revealed that none of the CD4 + , CD8 + , CD19 + , CD56 + cells were also positive for Ki67. Due to a small cohort and inadequate follow-up they did not present specific conclusions but did posit that the immune response against PTC is complex and involves several types of lymphocytes. A recent retrospective study analyzing archived PTC samples was published in 2010 in the Journal of Clinical Endocrinology Metabolism claims that their study, for the first time, shows that the presence of lymphocytes within and/or surrounding papillary thyroid tumors correlates with more severe disease (French, Weber et al., 2010). Unlike the 29 previous two studies (Matsubayashi, Kawai et al., 1995; Lundgren, Hall et al., 2006), this particular study differentiated between lymphocytic infiltration and background lymphocytic thyroiditis. Lymphocytic infiltration refers to lymphocytic inflammation within the normal tissue whereas infiltration refers to single cells, lymphocytic aggregates (10 lymphocytes in close proximity) both intratumorally and peritumorally. The goal of the study was to investigate whether tumor-associated lymphocytes (TAL), in the absence of background thyroiditis (LT), correlates to disease severity. They initially analyzed 100 PTC patients for the LT and TAL, and 10 PTC patients with TAL were further assessed for lymphocyte infiltration via immunohistochemistry staining. The patient population consisted of 56 conventional PTC and PTC variants, which included 37 follicular, 4 mixed follicular and tall cell, and 1 solid variant. Patients were categorized according to positive/negative for invasion, LN metastases, and autoantibody production (serum thyroglobulin and thyroid peroxidase). Tumors were noted as positive for invasion if the capsule was disrupted and/or extrathyroidal extension. LN status was determined by pathological examination. To further define the types of lymphocytes found in association with PTC, the study analyzed primary tumors from 10 patients exhibiting tumor associated lymphocytes by immunohistofluorescence. These patients had both peritumoral and intratumoral lymphocytes present as both aggregates and single cells or multicellular foci. CD20 + B 30 cells, CD4 + CD3 + T cells and CD8 + CD3 + T cells were present both within and surrounding tumors. Patients with relatively high levels of tumor associated CD4 + T cells presented with larger tumors when relative lymphocyte subset frequency was compared with individual disease parameters. Thus, Treg frequency may be a diagnostic marker in determining a treatment regimen for PTC patients. The study reports the following: Presence of generalized LI has had no effect on disease stage Disease severity is similar between patients without LI and with LT, patients with TAL had significantly higher –stage disease Patients with TAL had higher incidence of invasive tumors and LN metastases in comparison to those with concurrent thyroiditis or no lymphocytic infiltration Patients with relatively high levels of tumor-associated CD4 + cells presented with larger tumors Accumulation of Tregs may explain the negative association of LI and disease severity Given the data, PTC appears to suppress and evade the immune response. A successful vaccine would take these barriers into consideration. The seeming presence of Tregs suggests that immune-based therapies for thyroid cancer may be beneficial (French, Weber et al., 2010). French et al. posited that despite the presence of a tumor-directed immune response, the immune system appears to not be able to eliminate the tumor. As 31 indicated above, increasing frequencies of CD25 + FoxP3 T cells appeared to correlate with a higher degree of LN metastasis in PTC patients. In addition, naïve T cells may differentiate into Th1, Th2, Th17 (Jinfang Zhu, 2012) depending on specific transcription factors and cytokines. Th1 polarization is known to promote tumor elimination whereas Th2 is known to encourage tumor progression. Both the pro-tumor and anti-tumor properties of Th17 have been discussed in prior studies. CD4 + T cell polarization has not been studied in PTC patients. T cell exhaustion refers to T cells that lose proliferative potential and the ability to produce IL-2, TNFalpha, and INFgamma. French et al. aimed to characterize the T cell phenotype in PTC and investigate “T cell exhaustion” in metastatic LN. They assessed the CD4 + T cell polarization and T cell exhaustion via flow cytometry and immunofluorescence. The study reports the following results: Regulatory CD4 + T cells (Treg) were enriched in tumor involved LN compared with uninvolved LN Regulatory CD4 + T cells were elevated in tumor involved LN from those with recurrent disease Programmed Death-1 T cells were present at high frequency in tumor involved LN and further enriched in tumor involved LN that showed evidence of 32 extranodal invasion Treg frequency correlated with Programmed Death-1 T cell frequencies in tumor involved LN Although Programmed Death-1 T cells produced interferon they failed to fully down-regulate CD27 + and were not actively proliferating T Regulatory and NK Cells in Thyroid Papillary Carcinomas As indicated before, Tregs tightly regulate the function of effector T cells by inhibiting T cell proliferation and maintaining tolerance to self-antigens. The actual mechanism of suppression has yet to be elucidated. However, a number of studies have looked at Tregs within the context of ovarian (Bamias, Koutsoukou et al., 2008), breast (Ghebeh, Barhoush et al., 2008), non-small cell lung (Schneider, Kimpfler et al., 2011), pancreatic cancer (Hinz, Pagerols-Raluy et al., 2007), and malignant melanoma (Miracco, Mourmouras et al., 2007). To further shed light on the role of Tregs in papillary thyroid carcinomas, Gogali et al. aimed to explore the correlation of disease extent with Treg and NK’s infiltration. 65 patients with PTC and 25 patients with thyroid nodular goiter (TNG) who underwent complete thyroidectomy were included. Peripheral blood and fresh tissue samples were collected (Gogali, Paterakis et al., 2012). 33 The study reports the following: Greater Treg thyroid tissue infiltration was seen in PTC vs. TNG FC analysis of blood samples showed no difference between PTC and TNG groups Increased NK cells were seen in PTC vs. TNG via flow cytometry CD8 + and CD4 + T cells did not differ in blood and tissue samples Increased Tregs tissue infiltration was positively correlated with advanced disease In accordance with French et al, the study did confirm Treg infiltration of PTC tissue was proportional to disease stage with or without lymph node metastasis (stage) and intrathyroidal metastasis (“tumor load”). The extent of Treg infiltration in PTC was significantly greater compared to TNG, which suggests that the tumor microenvironment appears to activate the existing Tregs or enhance their infiltration. However, in contrast to the findings by French et al., Gogali et al. noted that there was no difference in Treg infiltration between PTC patients and those with thyroiditis. Thus, Gogali et al suggests that the immune response in thyroiditis patients cannot suppress Tregs and tumor proliferation. Moreover, unlike French et al, they did not find any correlation between Tregs infiltration and disease stage in PTC-Hashimoto’s patients nor did thyroiditis patients have better prognosis. 34 CHAPTER 4: STUDY OBJECTIVES Tumor-immune system interactions, including tumor-infiltrating leukocytes, regulatory cell response, and tumor-associated macrophages, have only recently begun to be explored. The degree and type of immune infiltration in thyroid cancers, as well as immune response differences between local and metastatic disease, are poorly understood at this time. For patients without further therapeutic options, immunotherapy is an exciting possibility due to thyroid tissue’s inherent propensity for auto-immune disease if we are able to understand the role of the immune system in this disease. • Specific Aim 1: Use immunohistochemistry to assess various immune cells subset infiltration, both pertitumorally and intratumorally, with known disease parameters. • Specific Aim 2: From the above retrospective study data, develop hypotheses regarding the relationship between lymphocytic infiltration and disease parameters in order to design a prospective study. 35 CHAPTER 5:MATERIALS AND METHODS Tissue Samples Samples were obtained from the USC Norris Cancer Center Tumor Bank. All cases were histologically reviewed by hematoxylin and eosin staining by pathologist Dr. Sue Ellen Martin to identify areas of cancer and normal tissue and to confirm the histologic diagnosis of PTC. Archival, paraffin-embedded papillary thyroid cancer patient tumor samples and nearest noncancerous margin samples were collected (Fig 5). Analysis of the patient samples was retrospective and conducted following Internal Review Board approved procedures. Samples from 43 patients were categorized by age (younger and older than 45 yrs old), gender, pathologic stage, and the presence or absence of LN metastases. PTC staging was established using the criteria set by the Standard American Joint Committee on Cancer tumor-node-metastases (TMN) (7 th edition, 2010). 36 Figure 5. Histology of normal human thyroid and papillary thyroid carcinoma (PTC) 37 Immunohistochemistry Techniques Patient samples (Table 1) were assessed for lymphocyte subsets and tumor-mediated cytokine production using immunohistochemistry techniques. 4-um sections from formalin-fixed paraffin embedded patient tissue block were cut and placed on positively charged glass slides for staining (Appendix A). Duplicate samples from two areas containing tumor center and nearest noncancerous margin were included in each case when available. The samples were deparaffinized in xylene and rehydrated in two changes of ethanol. Antigen retrieval was carried out by steam heating in citrate buffer (0.01 M citrate, pH 6.0) for 25 minutes. Samples were then cooled for 20 minutes and blocked with serum for 30 minutes. Sections were then incubated with primary antibodies specific for human CD3 (PC3/188a; Santa Cruz), CD8 (C8/144B; Dako), CD16 (0.N.82; Abcam), CD20 (L26; Dako), CD68 (PGM1; Dako), FoxP3 (236A/E7; Novus), HIF1-alpha (Alan Epstein, USC), HLA-A (C6; Santa Cruz), and HLA-G (4H84; Santa Cruz) overnight at 4°C (Table 2). Adaptive immune system markers: CD3 identifies all T cells, CD8 is a lineage specific marker expressed primarily on cytotoxic T cell subsets, FoxP3 is used as a marker of immunosuppressive regulatory T cells, and CD20 is a marker of B cells. Innate compartment: CD68 is a macrophage marker, and CD16 identifies natural killer (NK) cells. 38 SAMPLE DIAGNOSIS AGE GENDER STAGE TMN Stage LN TA 6 PTC 22 F I/II N/A N TA 7 PTC 30 F I/II T4aN1bMX Y TA 8 PTC 34 M I/II N/A Y TA 9 PTC 35 F I/II N/A N TA 10 PTC 35 F I/II N/A Y TA 11 PTC 35 F I/II N/A Y TA 12 PTC 35 F I/II Pt1aN0MX N TA 13 PTC 36 F I/II Pt1bN1bMX Y TA 14 PTC 39 F I/II N/A Y TA 15 PTC 40 F I/II N/A N TA 16 PTC 40 F I/II pT1bN1bMX Y TA 17 PTC 41 M I/II pT1aN0MX N TA 18 PTC 43 F I/II pT1N0MX N TA 19 PTC 46 F I pT1apNXMX N/A TA 20 PTC 46 F I pT1An0MX N TA 21 PTC 46 F I pT1An0MX N TA 22 PTC 52 F I N/A N TA 23 PTC 53 M I N/A N TA 24 PTC 54 F I N/A N TA 25 PTC 55 F I/II N/A N TA 26 PTC 56 F II N/A N TA 27 PTC 57 F II N/A N TA 28 PTC 61 F I N/A N TA 29 PTC 62 F I pT1aN0MX N TA 30 PTC 65 F I N/A N/A TA 31 PTC 68 F I PT1aN0MX N TA 32 PTC 73 F I N/A N TA 33 PTC 75 F I pT1aNXMX N/A TA 34 PTC 48 M IV pT1N1bMX Y TA 35 PTC 48 M III N/A Y TA 36 PTC 48 M III/ IV pT1bN1MX Y TA 37 PTC 49 F IV pT4aN0MX N TA 38 PTC 49 F III pT2pN1aMX Y TA 39 PTC 50 F III pT1aN1MX Y TA 40 PTC 53 F III/ IV N/A N TA 41 PTC 56 F IV pT4aN0MX N TA 42 PTC 59 F IV pT2N1bMX Y TA 43 PTC 61 F III pT3N1aMX Y TA 44 PTC 61 F IV pT3N1bMX Y TA 45 PTC 63 F IV pT4aN0MX N TA 46 PTC 64 M IV N/A N TA 47 PTC 72 F IV pT4apN1a Y TA 48 PTC 45 F IV N/A Y Table 1: Patient samples 39 The optimum titer or dilution is the concentration of the antibody found to give the best antigen specific staining with minimum background as determined on control tissue slides (spleen, LN or normal thyroid). Antibody titers on control tissue section were reviewed with Adrian Correa, an academic head and neck pathologist at the USC University Hospital, to select the best dilutions and confirm appropriate cellular localization of each stain. After incubation with primary antibodies, the slides were washed in PBS-Tween 20 solution (IHC washing buffer) 3x to remove unbound antibody and then incubated with 3% H 2 O 2 for 10 min to block endogenous peroxidase activity. The slides were then incubated with the appropriate second antibody, washed and stained using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) per manufacturer's instructions, followed by detection with 3,3'-diaminobenzidine (DAB). Sections were counterstained with hematoxylin, dehydrated, and mounted. Immunohistochemistry images were obtained using the EVO core microscope (Doheny, USC). Negative control slides without the primary antibodies and positive control tissues for each antigen (e.g. spleen, lymph node) were stained in parallel. 40 Antibody Info Clone Concentration CD8 Dako, monoclonal mouse, anti-human C8/144B 1:100 CD3 Santa Cruz Biotech, mouse monoclonal PC3/188ª 1:200 CD 20 Dako, monoclonal mouse, anti-human L26 1:1500 CD 16 Abcam, ab33515, monoclonal mouse, isotype IgG2a 0.N.82 1:100 CD 68 Dako, monoclonal mouse, anti-human PG-M1 1:100 FoxP3 Novus 236A/E7 1:100 HIF1- α Alan Epstein, USC 564 1:200 C/EBPB Santa Cruz Biotech H-7 1:100 HLA-A Santa Cruz, mouse monoclonal C6 1:1000 HLA-G Santa Cruz, mouse mono clonal 4h-84 1:200 Table 2: Antibodies 41 Scoring To develop an immune scoring system for these studies, we completed a review of the published English literature on PubMed using the following search term "systematic immune scoring" from 1991-2011 and read reviews examining immune infiltrate and tumor prognosis. A draft of the scoring system was then adapted for the study with guidance from Adrian Correa (Appendix B). Investigators were blinded to patient information at the time of quantification as each slide had only a coded number. Immunostained sections were scored for immune cell infiltration in two regions of the tumor: at the invasive margin and in tumor cell nests (Figure 6). For each immune cell stain, five representative high-powered fields (x400 magnification) were assessed in each tumor region to ensure representativeness. At the invasive margin of the tumor, the percentage of positively staining immune cells was estimated. The number of positively stained cells infiltrating the tumor cell nests was counted for each high-powered field (Figure 7). Three independent observers scored each section and an additional observer was consulted if significant discrepancies were seen across the 3 scores. Scores from observers were calculated as an average for homogeneity. 42 Figure 6: Intratumoral vs. Invading margin Figure 7: Immunohistochemistry of PTC patient specimens 43 Screening for BRAF V600E Mutation DNA was isolated from formalin fixed paraffin embedded tissue sections using Qiagen QIAmp FFPE kit per manufacturer instructions. Exon 15 of the human BRAF gene was amplified by polymerase chain reaction using forward primer TCATAATGCTTGCTCTGATAGGA and reverse primer GGCCAAAAATTTAATCAGTGGA, as described previously (Davies, Bignell et al., 2002) to yield a 224bp product. The thermocycling conditions were 94˚C for 2min, 35 cycles of 94˚C for 30sec, 60˚C for 30sec, 72˚C for 45sec, and finally 72˚C for 10min. Samples were electrophoresed on a 1.5% agarose gel at 150mV to separate products and the BRAF amplicon was excised from the gel for purification using Qiagen DNA Gel Extraction kit. Purified BRAF-amplification products for each sample were sequencing by the USC Genomics Core Facility and the sequence for each sample evaluated for mutations in the BRAF gene by comparison to the human BRAF consensus sequence (NC_000007.13) provided by the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). 44 CHAPTER 6: RESULTS There were 7 men and 36 women with a median age of 49 years. TMN stage was not available for 20 patients although LN metastases data was present. 12 patients had T1 category tumors, 2 had T2 and T3, and 5 patients had T4. 23 of the 43 patients have no lymph node metastasis. Information of LN status was not available for 3 patients. Intratumoral immune cell populations were assessed by IHC techniques for T cells (CD3), natural killer cells (CD16), macrophages (CD68), cytotoxic T cells (CD8), and regulatory T (Treg) cells (FoxP3). Table 3. Patient Characteristics Only 24 patients were assessed for CD3, CD68 and CD16 while all 43 patients were assessed for CD8 and FoxP3. This discrepancy was due to the fact that the second set of tissues samples were made available after a considerable amount time following the first 45 set of samples. It is important to note that the results fail to capture data from all 43 patients. Data from some patients are not included for the following reasons: 1. Some slides were not properly stained making appropriate scoring difficult. 2. Some slides had more background staining than others. Thus, discerning the positively stained cells was difficult. 3. LN status was not available for a few patients (Table 1). Patients with LN metastasis showed a trend toward decreased tumor infiltration by macrophages; other immune cell populations were observed at similar intratumoral levels in both patient groups (Figure 8). In addition, within the LN positive patient group, those with 1A LN status appeared to have less intratumoral CD8 + cells compared to those with 1B LN status. 1A indicates that the there has been spread to LN’s around the thyroid whereas 1B denotes that spread to lymph nodes in the neck (called cervical) or to lymph nodes behind the throat (retropharyngeal) or in the upper chest (superior mediastinal) is evident. Although the data is limited as LN status wasn’t provided for each patient, the date seems to suggest that a more robust immune response is evident in those with more advanced disease (Figure 9). Age less than 45 years is associated with improved clinical outcome in PTC. Measurement of tumor immune infiltrate in relation to patient age demonstrated trends toward decreased CD16 + natural killer cell (p<0.0001), CD68 + macrophages (p=0.080), and CD8 + cytotoxic T cells (Figure 10). These data are consistent with the concept of 46 immune senescence and decreasing tumor immune surveillance with age, and should be considered in the design and selection of immunotherapy for patients with thyroid cancer. Moreover, there was a minimal trend of decreased FoxP3 + cells with the increase of age. This may be the case because a robust immune response generally decreases with age and regulatory T cells are known to suppress the immune response. Furthermore, the ratio of cytotoxic T cells to Treg (CD8 + /FoxP3 + cell ratio) has been noted as a potential prognostic tool in previous cancer studies. In our data set, actual ratio values were not calculated, as some of the patients exhibited zero FoxP3 + infiltration. The ratio value would have failed to capture this absence. However, in looking at the paired data set, it is apparent that each patient is very different in terms of the CD8 vs. FoxP3 positive cells counts (Table 4). The data emphasize the importance of the relative ratios of immune infiltrate in tumors, in addition to the quality and quantity of infiltrate. 47 Figure 8: Patterns of tumor immune infiltrate in PTC with and without lymph node (LN) metastasis Figure 9: CD8 + infiltration by lymph node metastasis status 48 Figure 10: Differential pattern of tumor immune infiltrate with patient age in PTC 49 Table 4: CD8 + and FoxP3 + cell counts with patient age and LN status 50 CHAPTER 7: CONCLUSIONS Papillary thyroid cancer in some cases represents a mortal disease for which there are limited treatments. Immunotherapy uses the body’s own immune system to recognize and eliminate malignant cells, and has the advantages of systemic trafficking and immunologic memory to control or eradicate metastatic and recurrent disease. Therefore immunotherapy should be explored as a potential therapy, particularly given the thyroid gland’s propensity for auto-immune disease. This study found differences in effector cells (CD8+, CD68+, CD16+) and their relative abundance compared to FoxP3+ regulatory T cells, with respect to both age and lymph node status in early stage papillary thyroid cancer. Though limited by small sample size and clinical information, the results establish the practicality of further studies to identify immune responses associated with either good or poor prognosis. Results from such studies may suggest approaches to enhance immunotherapies to improve outcomes. 51 CHAPTER 8: FUTURE STUDIES A Prospective Study The retrospective study described here has these limitations: 1. Limited tissue samples available 2. Limited patient information available, no follow-up data 3. Certain epitope specificity may be lost in paraffin embedded tissue samples 4. Inability to evaluate blood samples from the evaluated patients To ensure future studies do not have these limitations, the Epstein lab has initiated a prospective study to address additional questions, particularly prognostic implications. The study has a target accrual of 300 thyroid cancer tumor biopsy specimens, whereas the retrospective study of this thesis involved 43 samples. The prospective study will create a sample repository with related clinical information to facilitate translational and clinical thyroid cancer research at USC. This will provide a better understanding of immune escape mechanisms in different types and stages of thyroid cancer, to then guide the development and clinical use of immunotherapy regimens in these patients. Specifically, regarding thyroid cancer, the study will test the following hypotheses: Increased HIF1α and VEGFC expression in the tumor correlates with a higher rate of nodal and/or distant metastasis at baseline and at 2 year follow up and with a higher rate of recurrence with two years after initial treatment Increased HIF1α expression in the tumor correlates with increased suppressor cell infiltration in the tumor 52 Higher cytotoxic T cell, B cell, or natural killer cell infiltrate in the tumor at the time of surgery correlates with better prognosis at 2 year follow up Moreover, unlike the retrospective study described herein, the prospective study will include various types of thyroid cancer rather than just the papillary subset. Participants selected for this study will be patients with Differentiated Thyroid Cancer [i.e. Papillary thyroid cancer (PTC) and Follicular thyroid cancer (FTC)], Anaplastic Thyroid Cancer (ATC), and Medullary Thyroid Cancer (MTC), including those with primary, metastatic, and recurrent disease. Blood and tumor and normal tissue samples will also be collected and processed on the day of surgery. The fresh tumor tissue will facilitate pre-clinical studies of thyroid cancer in relation to immunobiology. Papillary and Follicular/Follicular Variant of PTC thyroid cancer samples that have already been collected (surgical staging or surgical treatment) will be enzymatically digested to create single cell suspensions to be grown in flasks or frozen for later studies, including growth in culture. Stable neoplastic clones will be isolated, characterized and validated using morphologic, immunohistochemistry, cytogenetics, and hetero-transplantation techniques in the Epstein laboratory. (Liebertz, Lechner et al., 2010; Lechner, Lade et al., 2011; Russell, Lechner et al., 2011). A cell line will be evaluated for its ability to undergo multiple transfers, its neoplastic derivation, and its expression of certain surface markers. Derived cell lines will also be characterized for morphology, phenotype, gene and protein expression, growth, and mutation status. 53 Established and characterized cell lines will be sent to the American Tissue Culture Collection for further genetic validation and to be made publicly available for other researchers. 54 BIBLIOGRAPHY BAMIAS, A. et al. Correlation of NK T-like CD3+CD56+ cells and CD4+CD25+(hi) regulatory T cells with VEGF and TNF alpha in ascites from advanced ovarian cancer: Association with platinum resistance and prognosis in patients receiving first-line, platinum-based chemotherapy. Gynecologic Oncology, v. 108, n. 2, p. 421-427, Feb 2008. ISSN 0090-8258. Disponível em: < <Go to ISI>://WOS:000253248700027 >. BOELAERT, K. Thyroid gland: Revised guidelines for the management of thyroid cancer. Nat Rev Endocrinol, v. 6, n. 4, p. 185-6, Apr 2010. ISSN 1759-5037. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/20336160 >. BRENNER, H. Long-term survival rates of cancer patients achieved by the end of the 20th century: a period analysis. Lancet, v. 360, n. 9340, p. 1131-5, Oct 2002. ISSN 0140- 6736. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/12387961 >. CAI, M. Y. et al. Human leukocyte antigen-G protein expression is an unfavorable prognostic predictor of hepatocellular carcinoma following curative resection. Clin Cancer Res, v. 15, n. 14, p. 4686-93, Jul 2009. ISSN 1078-0432. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/19584149 >. CATUREGLI, P. et al. Hypothyroidism in transgenic mice expressing IFN-gamma in the thyroid. Proc Natl Acad Sci U S A, v. 97, n. 4, p. 1719-24, Feb 2000. ISSN 0027-8424. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/10677524 >. CATUREGLI, P. et al. Autoimmune thyroid diseases. Curr Opin Rheumatol, v. 19, n. 1, p. 44-8, Jan 2007. ISSN 1040-8711. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/17143095 >. CHAKRAVARTY, D. et al. Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation. J Clin Invest, v. 121, n. 12, p. 4700-11, Dec 2011. ISSN 1558-8238. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/22105174 >. CLARK, O. H.; GREENSPAN, F. S.; DUNPHY, J. E. Hashimoto's thyroiditis and thyroid cancer: indications for operation. Am J Surg, v. 140, n. 1, p. 65-71, Jul 1980. ISSN 0002-9610. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/6893108 >. CURIEL, T. J. et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med, v. 10, n. 9, p. 942-9, Sep 2004. ISSN 1078-8956. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/15322536 >. 55 DAVIES, H. et al. Mutations of the BRAF gene in human cancer. Nature, v. 417, n. 6892, p. 949-954, Jun 2002. ISSN 0028-0836. Disponível em: < <Go to ISI>://WOS:000176441200039 >. DAVIES, L.; WELCH, H. G. Increasing incidence of thyroid cancer in the United States, 1973-2002. JAMA, v. 295, n. 18, p. 2164-7, May 2006. ISSN 1538-3598. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/16684987 >. DE JONG, R. A. et al. Presence of tumor-infiltrating lymphocytes is an independent prognostic factor in type I and II endometrial cancer. Gynecol Oncol, v. 114, n. 1, p. 105-10, Jul 2009. ISSN 1095-6859. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/19411095 >. DIGHE, A. S. et al. Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma receptors. Immunity, v. 1, n. 6, p. 447-56, Sep 1994. ISSN 1074-7613. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/7895156 >. FRENCH, J. D. et al. Tumor-associated lymphocytes and increased FoxP3+ regulatory T cell frequency correlate with more aggressive papillary thyroid cancer. J Clin Endocrinol Metab, v. 95, n. 5, p. 2325-33, May 2010. ISSN 1945-7197. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/20207826 >. FRIDMAN, W. H. et al. Prognostic and predictive impact of intra- and peritumoral immune infiltrates. Cancer Res, v. 71, n. 17, p. 5601-5, Sep 2011. ISSN 1538-7445. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21846822 >. GHEBEH, H. et al. FOXP3(+) Tregs and B7-HI+/PD-I+T lymphocytes co-infiltrate the tumor tissues of high-risk breast cancer patients: Implication for immunotherapy. Bmc Cancer, v. 8, Feb 2008. ISSN 1471-2407. Disponível em: < <Go to ISI>://WOS:000254617200001 >. GIORDANO, C. et al. Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto's thyroiditis. Science, v. 275, n. 5302, p. 960-3, Feb 1997. ISSN 0036-8075. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/9020075 >. GIORDANO, T. J. et al. Molecular classification of papillary thyroid carcinoma: distinct BRAF, RAS, and RET/PTC mutation-specific gene expression profiles discovered by DNA microarray analysis. Oncogene, v. 24, n. 44, p. 6646-56, Oct 2005. ISSN 0950- 9232. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/16007166 >. 56 GOGALI, F. et al. Phenotypical Analysis of Lymphocytes with Suppressive and Regulatory Properties (Tregs) and NK Cells in the Papillary Carcinoma of Thyroid. Journal of Clinical Endocrinology & Metabolism, v. 97, n. 5, p. 1474-1482, May 2012. ISSN 0021-972X. Disponível em: < <Go to ISI>://WOS:000303915900037 >. GOODEN, M. J. et al. The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis. Br J Cancer, v. 105, n. 1, p. 93-103, Jun 2011. ISSN 1532-1827. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21629244 >. GUPTA, S. et al. Infiltration of differentiated thyroid carcinoma by proliferating lymphocytes is associated with improved disease-free survival for children and young adults. J Clin Endocrinol Metab, v. 86, n. 3, p. 1346-54, Mar 2001. ISSN 0021-972X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/11238531 >. HARRIS, P. J.; BIBLE, K. C. Emerging therapeutics for advanced thyroid malignancies: rationale and targeted approaches. Expert Opin Investig Drugs, v. 20, n. 10, p. 1357-75, Oct 2011. ISSN 1744-7658. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21910667 >. HINZ, S. et al. Foxp3 expression in pancreatic carcinoma cells as a novel mechanism of immune evasion in cancer. Cancer Research, v. 67, n. 17, p. 8344-8350, Sep 2007. ISSN 0008-5472. Disponível em: < <Go to ISI>://WOS:000249406700054 >. HIRABAYASHI, R. N.; LINDSAY, S. THE RELATION OF THYROID CARCINOMA AND CHRONIC THYROIDITIS. Surg Gynecol Obstet, v. 121, p. 243-52, Aug 1965. ISSN 0039-6087. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/14320370 >. HOFFMANN, S. et al. Functional thyrotropin receptor attenuates malignant phenotype of follicular thyroid cancer cells. Endocrine, v. 30, n. 1, p. 129-38, Aug 2006. ISSN 1355-008X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/17185801 >. JINFANG ZHU, W. E. P. CD4 T cells: fates, functions, and faults. Blood, v. 112, n. 5, p. 1557, 1 September 2008 2012. KAWASHIMA, A. et al. Innate immune activation and thyroid autoimmunity. J Clin Endocrinol Metab, v. 96, n. 12, p. 3661-71, Dec 2011. ISSN 1945-7197. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21956420 >. KNAUF, J. A. et al. Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res, v. 65, n. 10, p. 4238-45, May 2005. ISSN 0008-5472. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/15899815 >. 57 LADOIRE, S. et al. In situ immune response after neoadjuvant chemotherapy for breast cancer predicts survival. J Pathol, v. 224, n. 3, p. 389-400, Jul 2011. ISSN 1096-9896. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21437909 >. LECHNER, M. G. et al. Breast implant-associated, ALK-negative, T-cell, anaplastic, large-cell lymphoma: establishment and characterization of a model cell line (TLBR-1) for this newly emerging clinical entity. Cancer, v. 117, n. 7, p. 1478-89, Apr 2011. ISSN 0008-543X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21425149 >. LEFFERS, N. et al. Prognostic significance of tumor-infiltrating T-lymphocytes in primary and metastatic lesions of advanced stage ovarian cancer. Cancer Immunol Immunother, v. 58, n. 3, p. 449-59, Mar 2009. ISSN 1432-0851. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/18791714 >. LEHMANN, P. V. et al. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature, v. 358, n. 6382, p. 155-7, Jul 1992. ISSN 0028-0836. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/1377368 >. LIEBERTZ, D. J. et al. Establishment and characterization of a novel head and neck squamous cell carcinoma cell line USC-HN1. Head Neck Oncol, v. 2, p. 5, 2010. ISSN 1758-3284. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/20175927 >. LIZÉE, G.; CANTU, M. A.; HWU, P. Less yin, more yang: confronting the barriers to cancer immunotherapy. Clin Cancer Res, v. 13, n. 18 Pt 1, p. 5250-5, Sep 2007. ISSN 1078-0432. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/17875752 >. LUNDGREN, C. I. et al. Clinically significant prognostic factors for differentiated thyroid carcinoma: a population-based, nested case-control study. Cancer, v. 106, n. 3, p. 524-31, Feb 2006. ISSN 0008-543X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/16369995 >. MARAZUELA, M. et al. Regulatory T cells in human autoimmune thyroid disease. J Clin Endocrinol Metab, v. 91, n. 9, p. 3639-46, Sep 2006. ISSN 0021-972X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/16804051 >. MATSUBAYASHI, S. et al. The correlation between papillary thyroid carcinoma and lymphocytic infiltration in the thyroid gland. J Clin Endocrinol Metab, v. 80, n. 12, p. 3421-4, Dec 1995. ISSN 0021-972X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/8530576 >. MEZOSI, E. et al. Induction and regulation of Fas-mediated apoptosis in human thyroid epithelial cells. Mol Endocrinol, v. 19, n. 3, p. 804-11, Mar 2005. ISSN 0888-8809. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/15563545 >. 58 MIRACCO, C. et al. Utility of tumour-infiltrating CD25(+)FOXP3(+) regulatory T cell evaluation in predicting local recurrence in vertical growth phase cutaneous melanoma. Oncology Reports, v. 18, n. 5, p. 1115-1122, Nov 2007. ISSN 1021-335X. Disponível em: < <Go to ISI>://WOS:000250570400008 >. MODI, J. et al. Papillary thyroid carcinomas from young adults and children contain a mixture of lymphocytes. J Clin Endocrinol Metab, v. 88, n. 9, p. 4418-25, Sep 2003. ISSN 0021-972X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/12970319 >. NAGAYAMA, Y. et al. CD4+CD25+ naturally occurring regulatory T cells and not lymphopenia play a role in the pathogenesis of iodide-induced autoimmune thyroiditis in NOD-H2h4 mice. J Autoimmun, v. 29, n. 2-3, p. 195-202, 2007 Sep-Nov 2007. ISSN 0896-8411. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/17826032 >. NAGAYAMA, Y. et al. Prophylactic and therapeutic efficacies of a selective inhibitor of the immunoproteasome for Hashimoto's thyroiditis, but not for Graves' hyperthyroidism, in mice. Clin Exp Immunol, v. 168, n. 3, p. 268-73, Jun 2012. ISSN 1365-2249. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/22519588 >. NIKIFOROV, Y. E. Genetic alterations involved in the transition from well- differentiated to poorly differentiated and anaplastic thyroid carcinomas. Endocr Pathol, v. 15, n. 4, p. 319-27, 2004. ISSN 1046-3976. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/15681856 >. NIKIFOROV, Y. E. et al. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab, v. 96, n. 11, p. 3390-7, Nov 2011. ISSN 1945-7197. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21880806 >. NIKIFOROVA, M. N. et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab, v. 88, n. 11, p. 5399-404, Nov 2003. ISSN 0021-972X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/14602780 >. O' REILLY, L. A. et al. Membrane-bound Fas ligand only is essential for Fas-induced apoptosis. Nature, v. 461, n. 7264, p. 659-63, Oct 2009. ISSN 1476-4687. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/19794494 >. PFISTER, D. G.; FAGIN, J. A. Refractory thyroid cancer: a paradigm shift in treatment is not far off. J Clin Oncol, v. 26, n. 29, p. 4701-4, Oct 2008. ISSN 1527-7755. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/18541893 >. 59 RASPÉ, E. et al. Identification of the thyroid Na+/I- cotransporter as a potential autoantigen in thyroid autoimmune disease. Eur J Endocrinol, v. 132, n. 4, p. 399-405, Apr 1995. ISSN 0804-4643. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/7711875 >. RESCIGNO, M.; AVOGADRI, F.; CURIGLIANO, G. Challenges and prospects of immunotherapy as cancer treatment. Biochim Biophys Acta, v. 1776, n. 1, p. 108-23, Sep 2007. ISSN 0006-3002. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/17720322 >. RINGEL, M. D. Metastatic Dormancy and Progression in Thyroid Cancer: Targeting Cells in the Metastatic Frontier. Thyroid, v. 21, n. 5, p. 487-492, May 2011. ISSN 1050- 7256. Disponível em: < <Go to ISI>://WOS:000290783900005 >. RUSSELL, S. M. et al. USC-HN2, a new model cell line for recurrent oral cavity squamous cell carcinoma with immunosuppressive characteristics. Oral Oncol, v. 47, n. 9, p. 810-7, Sep 2011. ISSN 1368-8375. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21719345 >. SADUN, R. E. et al. Immune signatures of murine and human cancers reveal unique mechanisms of tumor escape and new targets for cancer immunotherapy. Clin Cancer Res, v. 13, n. 13, p. 4016-25, Jul 2007. ISSN 1078-0432. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/17606736 >. SAKAGUCHI, S. et al. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in self-tolerance and autoimmune disease. Curr Top Microbiol Immunol, v. 305, p. 51-66, 2006. ISSN 0070-217X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/16724800 >. SCHNEIDER, T. et al. Foxp3(+) Regulatory T Cells and Natural Killer Cells Distinctly Infiltrate Primary Tumors and Draining Lymph Nodes in Pulmonary Adenocarcinoma. Journal of Thoracic Oncology, v. 6, n. 3, p. 432-438, Mar 2011. ISSN 1556-0864. Disponível em: < <Go to ISI>://WOS:000287240100004 >. SCHREIBER, R. D.; OLD, L. J.; SMYTH, M. J. Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science, v. 331, n. 6024, p. 1565- 70, Mar 2011. ISSN 1095-9203. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21436444 >. SHANKARAN, V. et al. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature, v. 410, n. 6832, p. 1107-11, Apr 2001. ISSN 0028-0836. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/11323675 >. 60 SHIMOJO, N. et al. Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc Natl Acad Sci U S A, v. 93, n. 20, p. 11074-9, Oct 1996. ISSN 0027-8424. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/8855311 >. SILVA, M. A. Intestinal dendritic cells and epithelial barrier dysfunction in Crohn's disease. Inflamm Bowel Dis, v. 15, n. 3, p. 436-53, Mar 2009. ISSN 1536-4844. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/18821596 >. SIPOS, J. A.; MAZZAFERRI, E. L. The therapeutic management of differentiated thyroid cancer. Expert Opin Pharmacother, v. 9, n. 15, p. 2627-37, Oct 2008. ISSN 1744-7666. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/18803450 >. SO, Y. K. et al. Preoperative BRAF mutation has different predictive values for lymph node metastasis according to tumor size. Otolaryngol Head Neck Surg, v. 145, n. 3, p. 422-7, Sep 2011. ISSN 1097-6817. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/21750338 >. SPITZWEG, C. et al. Image-guided radioiodide therapy of medullary thyroid cancer after carcinoembryonic antigen promoter-targeted sodium iodide symporter gene expression. Hum Gene Ther, v. 18, n. 10, p. 916-24, Oct 2007. ISSN 1043-0342. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/17931047 >. STATHATOS, N. Thyroid physiology. Med Clin North Am, v. 96, n. 2, p. 165-73, Mar 2012. ISSN 1557-9859. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/22443969 >. STATHATOS, N.; DANIELS, G. H. Autoimmune thyroid disease. Curr Opin Rheumatol, v. 24, n. 1, p. 70-5, Jan 2012. ISSN 1531-6963. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/22157414 >. THEOHARIS, C.; ROMAN, S.; SOSA, J. A. The molecular diagnosis and management of thyroid neoplasms. Curr Opin Oncol, v. 24, n. 1, p. 35-41, Jan 2012. ISSN 1531- 703X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/22123232 >. WOYACH, J. A.; SHAH, M. H. New therapeutic advances in the management of progressive thyroid cancer. Endocr Relat Cancer, v. 16, n. 3, p. 715-31, Sep 2009. ISSN 1479-6821. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/19218279 >. XING, M. Prognostic utility of BRAF mutation in papillary thyroid cancer. Mol Cell Endocrinol, v. 321, n. 1, p. 86-93, May 2010. ISSN 1872-8057. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/19883729 >. 61 XING, M. et al. Detection of BRAF mutation on fine needle aspiration biopsy specimens: a new diagnostic tool for papillary thyroid cancer. J Clin Endocrinol Metab, v. 89, n. 6, p. 2867-72, Jun 2004. ISSN 0021-972X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/15181070 >. YOSHIDA, A. et al. Pendrin is a novel autoantigen recognized by patients with autoimmune thyroid diseases. J Clin Endocrinol Metab, v. 94, n. 2, p. 442-8, Feb 2009. ISSN 0021-972X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/19050049 >. YU, P.; FU, Y. X. Tumor-infiltrating T lymphocytes: friends or foes? Lab Invest, v. 86, n. 3, p. 231-45, Mar 2006. ISSN 0023-6837. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/16446705 >. 62 APPENDIX A: IMMUNOHISTOCHEMISTRY PROTOCOL-ABC METHOD Deparaffinize sections: 1) Baked for 30min at 60 o C 2) 2 changes of xylene, 5 minutes each. (use 3 changes for sections thicker than 25um) 3) Hydrated in 2 changes of 100% ethanol for 3 minutes each 4) 95% ethanol for 1 minute 5) 80% ethanol for 1 minute 6) Rinsed in distilled water for 2 minutes 7) Proceeded to Antigen Retrieval Antigen Retrieval-Steamer Method (Citrate Buffer): 1) Pre-heated steamer (DI water) 2) Added citrate buffer to small beaker (enough to cover bottom), placed one slide in each beaker and added a blank slide to allow for capillary action to draw up buffer 3) Placed beakers in pre-warmed steamer 4) Closed the lid and incubated for 25 min then turned off steamer. 5) Allowed slides to cool in steamer for 20 min. 6) Pulled off blank slide (do not slide off). 7) Rinsed slides in PBS Tween 20 (0.05% Tween in PBS) for 2x2 min (pour off buffer into sink between washes) 8) Blotted off excess buffer with kim wipe and allow to dry before drawing a complete circle around the tissue with wax pen. 9) Proceeded to staining-serum blocking. Staining: 1) Serum Blocking: Incubated sections for 30 minutes with diluted normal blocking serum which was prepared from the species in which the secondary antibody is made (YELLOW solution from vector kit – about 3-4 drops for tissue) 2) Blotted excess serum from sections—poured off/drained serum onto kim wipe. 3) Primary Antibody: Incubated sections overnight at 4C with primary antibody diluted in buffer (make up about 300uL, need about 200uL to cover tissue sections) placed in slide box in cold room 4) Washed slides for 3x2min in PBS-tween 20 5) Peroxidase Blocking: Incubated for 10 min in 3% H2O2 in PBS (stored at 4C) – 200 uL 6) Washed slides 3x2 min in PBS-tween 20 63 7) Secondary Antibody: Incubated sections for 30 minutes with diluted biotinylated secondary antibody solution (BLUE solution from vector kit) 8) Washed slides 3x2 min in PBS-tween 20 9) Detection: Incubated for 30 minutes with VECTASTAIN ABC Reagent (GREY solution) 10) Washed slides 3x2min in PBS-tween 20 11) Incubated sections in DAB peroxidase substrate solution until desired stain intensity develops (3 min). Three minutes is the suggested antigen retrieval time as less than 3 minutes may leave the antigens under-retrieved, leading to weak staining. More than 3 minutes may lead to non-specific binding. a. Added 5 drops 1% DAB (20x stock in baggie on -20C freezer door) to 5 mL PBS (mix well) b. Added 5 drops 0.3% H202 (4C) (mix well) 12) Rinsed section in distilled water for 2min 13) Rinsed section in tap water for 2min 14) Counterstaining: Immersed slides in Vector Hematoxylin for 1-5 minutes a. Rinsed sections in tap water until rinse water is colorless b. Dipped slides 10times in acid rinse solution for 1min followed by 10 dips in tap water c. Incubated slides in bluing solution for 1min followed by 10 dips in tap water 15) Rinsed section in tap water for 2min 16) Rinsed section in distilled water for 2min 17) Dehydrate: 95% ethanol for 1min a. 100% ethanol for 2x3min b. Clear in xylene for 2x5min 18) Blotted of excess in xylene and allowed to dry 19) Mounted coverslips with toluene. 64 APPENDIX B: SCORING SCHEMATIC Notes: -invading margin refers to the intersection of tumor cells and stromal reaction and/or normal tissue -for the intratumoral count, only cells in the tumour tissue were counted -do not score lymphoid follicles that are in the normal tissue or at the border as these represent factories, not the front lines. -CD8+ staining was noted in relation to circular membranous staining. -Foxp3+ staining was based on distinct nuclear staining. 1. Observed and scanned the H/E slide to locate the tumor and invasive margin area using the EVO core microscope (Doheny) at 100x, saved the jpg of this area. 2. Removed the H/E and placed sample slide on microscope. Started at 100x and found the same tumor area or similiar. 3. Switched magnification to 40x. Focused on an specific intratumoral area that was representative of the slide and saved the image. Repeated 4x to capture 5 distinct intratumoral areas. 4. The saved images were printed and patient information was noted on the back as to not create bias. 5. For CD8: Counted general number of lymphocytes in the area as well as the stained lymphocytes. For FoxP3: counted actual number of stained cells, rather than calculating a percentage. 6. Calculate average number from the 5 sampled area. For CD8 only: Calculated percentage of stained lymphocytes. 7. Re-did process for invasive margin cells. 5 HPF per stain Invasive margin Intratumoral cells % positive cells of immune cells CD3 CD8 CD20 CD16 CD68 HIF1α Total Cell Count FoxP3 65 APPENDIX C: TNM STAGING FOR THYROID CANCER (cancer.org) The TNM staging system A staging system is a standard way for the cancer care team to summarize how large a cancer is and how far it has spread. The most common system used to describe the stages of thyroid cancer is the American Joint Committee on Cancer (AJCC) TNM system. The TNM system describes 3 key pieces of information: T indicates the size of the main (primary) tumor and whether it has grown into nearby areas. N describes the extent of spread to nearby (regional) lymph nodes. Lymph nodes are small bean-shaped collections of immune system cells to which cancers often spread first. Cells from thyroid cancers can travel to lymph nodes in the neck and chest areas. M indicates whether the cancer has spread (metastasized) to other organs of the body. (The most common sites of spread of thyroid cancer are the lungs, the liver, and bones.) Numbers or letters appear after T, N, and M to provide more details about each of these factors. The numbers 0 through 4 indicate increasing severity. The letter X means "cannot be assessed because the information is not available." T categories for thyroid cancer (other than anaplastic thyroid cancer) TX: Primary tumor cannot be assessed. T0: No evidence of primary tumor. T1: The tumor is 2 cm (slightly less than an inch) across or smaller and has not grown out of the thyroid. T1a: The tumor is 1 cm (less than half an inch) across or smaller and has not grown outside the thyroid. T1b: The tumor is larger than 1 cm but not larger than 2 cm across and has not grown outside of the thyroid. T2: The tumor is between 2 cm and 4 cm (slightly less than 2 inches) across and has not grown out of the thyroid. T3: The tumor is larger than 4 cm or it has begun to grow a small amount into nearby tissues outside the thyroid. T4a: The tumor is any size and has grown extensively beyond the thyroid gland into nearby tissues of the neck, such as the larynx (voice box), trachea (windpipe), esophagus (tube connecting the throat to the stomach), or the nerve to the larynx. This is also called moderately advanced disease. T4b: A tumor of any size that has grown either back toward the spine or into nearby large blood vessels. This is also called very advanced disease. N categories for thyroid cancer NX: Regional (nearby) lymph nodes cannot be assessed. 66 N0: No spread to nearby lymph nodes. N1: The cancer has spread to nearby lymph nodes. N1a: Spread to lymph nodes around the thyroid in the neck (called pretracheal, paratracheal, and prelaryngeal lymph nodes). N1b: Spread to other lymph nodes in the neck (called cervical) or to lymph nodes behind the throat (retropharyngeal) or in the upper chest (superior mediastinal). M categories for thyroid cancer M0: No distant metastasis. M1: Spread to other parts of the body, such as distant lymph nodes, internal organs, bones, etc. Stage grouping Once the values for T, N, and M are determined, they are combined into stages, expressed as a Roman numeral from I through IV. Sometimes letters are used to divide a stage into substages. Unlike most other cancers, thyroid cancers are grouped into stages in a way that also considers the subtype of cancer and the patient's age. Papillary or follicular (differentiated) thyroid cancer in patients younger than 45 Younger people have a low likelihood of dying from differentiated (papillary or follicular) thyroid cancer. The TNM stage groupings for these cancers take this fact into account. So, all people younger than 45 years with these cancers are stage I if they have no distant spread and stage II if they have distant spread. Stage I (any T, any N, M0): The tumor can be any size (any T) and may or may not have spread to nearby lymph nodes (any N). It has not spread to distant sites (M0). Stage II (any T, any N, M1): The tumor can be any size (any T) and may or may not have spread to nearby lymph nodes (any N). It has spread to distant sites (M1). Papillary or follicular (differentiated) thyroid cancer in patients 45 years and older Stage I (T1, N0, M0): The tumor is 2 cm or less across and has not grown outside the thyroid (T1). It has not spread to nearby lymph nodes (N0) or distant sites (M0). Stage II (T2, N0, M0): The tumor is more than 2 cm but not larger than 4 cm across and has not grown outside the thyroid (T2). It has not spread to nearby lymph nodes (N0) or distant sites (M0). Stage III: One of the following applies: T3, N0, M0: The tumor is larger than 4 cm or has grown slightly outside the thyroid (T3), but it has not spread to nearby lymph nodes (N0) or distant sites (M0). 67 T1 to T3, N1a, M0: The tumor is any size and may have grown slightly outside the thyroid (T1 to T3). It has spread to lymph nodes around the thyroid in the neck (N1a) but not to distant sites (M0). Stage IVA: One of the following applies: T4a, any N, M0: The tumor is any size and has grown beyond the thyroid gland and into nearby tissues of the neck (T4a). It may or may not have spread to nearby lymph nodes (any N). It has not spread to distant sites (M0). T1 to T3, N1b, M0: The tumor is any size and may have grown slightly outside the thyroid gland (T1 to T3). It has spread to certain lymph nodes in the neck (cervical nodes) or to lymph nodes in the upper chest (superior mediastinal nodes) or behind the throat (retropharyngeal nodes) (N1b) but not to distant sites (M0). Stage IVB (T4b, any N, M0): The tumor is any size and has grown either back to the spine or into nearby large blood vessels (T4b). It may or may not have spread to nearby lymph nodes (any N), but it has not spread to distant sites (M0). Stage IVC (any T, any N, M1): The tumor is any size and may or may not have grown outside the thyroid (any T). It may or may not have spread to nearby lymph nodes (any N). It has spread to distant sites (M1).
Abstract (if available)
Abstract
Introduction: Thyroid cancer is the most common endocrine malignancy. Although typically well controlled with surgery and radioactive iodine treatments, some patients suffer from recurrent disease that can be fatal. For some of these patients current therapies fail and thus new treatment modalities are needed. Immunotherapy is a promising approach for treating cancer in general but the way in which the immune system interacts with thyroid cancer is poorly characterized or understood. A better understanding of this interaction should help guide efforts to use the immune system to treat thyroid cancer. ❧ Purpose: Characterize immune responses to thyroid tumors in patients experiencing different degrees of disease progression. ❧ Approach: Design and initiate a retrospective study analyzing archived papillary thyroid cancer samples regarding lymphocyte subtype infiltration by immunohistochemistry staining and compare to disease progression. ❧ Results: Paraffin-preserved samples of thyroid tumors were obtained from 43 patients. The samples were sectioned, stained, and evaluated for the presence of cells expressing lymphocyte markers CD3, CD8, CD16, CD68, and FoxP3. No significant differences were found in the number of CD8⁺, CD68⁺, CD16⁺ and FoxP3⁺ regulatory T cells with respect to both age and lymph node status in papillary thyroid cancer. A decreasing number of CD16⁺ cells significantly correlated with an increase in age. Moreover, there were generally a greater number of effector CD8⁺ cells relative to FoxP3 regulatory cells across the patient population. ❧ Conclusions: The results support the hypothesis that there is an active immune response to papillary thyroid carcinomas that may relate to disease prognosis. The age and lymph node status does not appear to predict characteristics of the immune response. The correlation between NK cells and prognosis may be worth further exploring as NK cells are associated with tumor rejection and limited disease recurrence.
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Asset Metadata
Creator
Kang, Kate Yuri
(author)
Core Title
Immune infiltrates in papillary thyroid carcinomas
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Physiology and Biophysics
Publication Date
08/22/2012
Defense Date
08/22/2012
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
immunotherapy,OAI-PMH Harvest,papillary thyroid carcinomas,thyroid cancer
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Kaslow, Harvey R. (
committee chair
), Epstein, Alan L. (
committee member
), Meiselman, Herbert J. (
committee member
)
Creator Email
kate.kang@gmail.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-92046
Unique identifier
UC11289451
Identifier
usctheses-c3-92046 (legacy record id)
Legacy Identifier
etd-KangKateYu-1162.pdf
Dmrecord
92046
Document Type
Thesis
Rights
Kang, Kate Yuri
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
immunotherapy
papillary thyroid carcinomas
thyroid cancer