Thyroid nodule pathophysiology

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Overview

Thyroid nodules may arise from different cells in the thyroid parenchyma. The pathogenesis of developing a thyroid nodule may differ based on the type of the nodule, and whether it is malignant or benign. Basically thyroid nodules may develop secondary to hyperplasia, mutations and resultant carcinoma, excess colloid accumulation, or frominflammation of thyroid tissue. Genetic mutation is considered as one of the most important mechanisms of developing thyroid nodules, especially neoplastic thyroid nodules. Most of these mutations occur as somatic mutations, while some may exhibit familial inheritance. The most important variety of familial thyroid cancers are caused by genetic mutations, and are called familial non-medullary thyroid cancer (FNMTC). Other important genes related to thyroid nodule formation include, N&H ras, RET, Gsp, C-MET, TRK, EGF / EGF-R, and P53.

Pathogenesis

A summary of thyroid nodule pathophysiology is presented in the slides below:

• Thyroid nodules may arise from different cells in thyroid parenchyma. The pathogenesis of developing a thyroid nodule may differ based on the type of the nodule, and whether it is malignant or benign.
• Basically thyroid nodules may develop secondary to hyperplasia, mutations and resultant carcinoma, excess colloid accumulation, or frominflammation of thyroid tissue.

(a) Hyperplastic nodules

• Hyperplastic nodule pathogenesis seems to start with an increase in thyroid proliferation, which lead to thyroid hyperplasia.
• Rapid thyroid proliferation mainly occur in response to certain stimulants.
• Stimulants mainly act through TSH mediated activity and production. Following the hyperplasia development phase, a new phase may begin, leading to a neoplasia.

1. TSH role in thyroid nodule formation

• Growth signals in thyroid tissue start by a stimulant, that attaches to the thyroid receptors. The following signals can be transmitted through 3 distinct pathways:
  • Adenylate cyclase/protein kinase A system
  • Phospholipase C pathways
  • Phospholipase A2 system (intracellular metabolism of prostaglandins)

• The most important pathway for thyroid growth is the activation of adenylate cyclase/protein kinase A system. Activation of phospholipase C and phospholipase A2 have only a minor effect on thyroid growth.
• TSH acts as a stimulant by binding to the TSH receptor and leads to activation of both the adenylate cyclase and phospholipase C pathways. As mentioned, the phospholipase C pathway has minor effects, and most of the TSH effect on cell growth is generated by adenylate cyclase pathway. The signal generated by the adenylate cyclase cAMP-dependent pathway is then transduced in the nucleus where transcription factors–upon phosphorylation–induce the expression of cAMP-inducible genes. It has been established that TSH has a main mitogenic role, through cAMP, Gs proteins and protein kinase A, which activates the metabolic cascade leading to the stimulation of growth.
• However, to produce hyperplasia, overproduction of cAMP must be continuous, as it occurs in mutations constitutive of the genes which regulate cAMP production.
• Constitutive cAMP overproduction has been demonstrated to be due to point mutation of the TSH receptor or Gs protein.
• Constitutive cAMP overproduction not only stimulates growth but also function.
• Hyperplastic thyroid nodule pathogenesis can be divided into 2 phases:

2. Thyroid overgrowth stimulants:

Thyroid normally has a low proliferative activity, although it can start proliferation rapidly in response to certain stimulants. Stimulants mainly act through TSH mediated activity and production. The following stimulants appear to have the most important role in pathogenesis of hyperplastic nodules:

• Iodine deficiency:
  • Effects directly or indirectly 
  • The most potent stimulator replication of the cells of thyroid gland 
  • Mechanism of action:
    • Acts as an initiator for TSH rise
    • May enhance the effect of other chemicals that induce a rise in TSH by inducing the promotor overactivity
    • The most important reason of high prevalence of thyroid hyperplasia and nodules in iodine-deficient areas
• Industrial chemicals:
  • DDT
  • Polychlorinated biphenyls
  • Pesticides
• Goitrogens:
  • Complex anions and inorganic atoms (iodine, lithium, CLO4–, TcO4–, BF4–)
  • Thiocyanate (SCN–)
  • Goitrin, isolated in plants of the genus Brassica
  • Aniline derivatives (sulfonamides, tolbutamide, sulfaguanidine, sulfamethoxazole, etc.)
  • Phenol derivatives and polyhydroxyphenols
  • Flavonoids:
    • TPO inhibitors
    • Also act on thyroid metabolism by interacting with the nuclear receptor for thyroid hormones
• Antithyroid drugs:
  • Thionamides that are used in the treatment of hyperthyroidism
• Tobacco:
  • May be the reason of high prevalence of thyroid hyperplasia and nodules in iodine-sufficient areas 

• Thyroid stromal cells interact with thyroid follicular cells by cytokines. Inappropriate cytokine activities also seem to be related to TSH overproduction and thyroid hyperplastic nodule formation. The most important cytokines that may lead to differentiation or inhibition of thyroid growth are:
  • TGFβ
  • IFNγ
  • IL-6
  • Somatostatin

3. Hyperplasia development phase:

• Thyroid cells produce the angiogenic vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) sensitive to TSH stimulation.
• The vascular growth factor induces neovascularization by binding to specific receptors on endothelial cells and stimulating new vessel production.
• In response, endothelial cells produce growth factors that increase thyroid cell proliferation and lead to thyroid hyperplasia.
• Neovascularization in thyroid matrix is accompanied by the production of proteolytic enzymes, which facilitate the expansion of thyroid tissue into the extracellular matrix.

4. Neoplasia development phase:

• Each follicle is composed of different clones of cells (polyclonal), but during nodule formation they replicate in a simultaneous and coordinated manner, so each follicle of the nodule reproduces the same heterogeneity of the mother follicle.
• When a neoplasm arises in the nodule, then the neoplastic follicle shows a monoclonal pattern, suggesting that cancer arises from a single cell. 

• Activation of oncogenes is considered the underlying event leading to uncontrolled cell growth.

(b) Neoplastic nodules

• Neoplastic nodules development mainly involve the activation of proto-oncogenes as the underlying event leading to uncontrolled cell growth.
• Proto-oncogene activation is associated with thyroid adenoma, hyperplasia, and malignancies.
• Thyroid gland is made up of different follicles, and each follicle is composed of different clones of cells (polyclonal). During nodule formation, cells replicate in a coordinated fashion simultanously, so each follicle of the nodule shares the same heterogenity with other cells.
• Hyperplastic thyroid nodules are considered a risk factor for the development of neoplasia, as these cells may express neoplastic potential during their rapid proliferation phase.
• During neoplasm formation in the nodule, the neoplastic follicle mostly shows a monoclonal pattern. These findings may indicate that neoplasia arises from a single cell genetic mutation. The most important oncogenes related to thyroid neoplasia development are mentioned in the genetic table below.

• Environmental factors can play an important role in triggering the oncogene mutation. The most important carcinogens involved in the pathogenesis of neoplastic thyroid nodules are:
  • Thioamide compounds
    • Thiourea
    • Methimazole
    • Ethylenethiourea (ETU)
    • Thiouracil
    • Propylthiouracil
  • Aminotriazole: Herbicide
  • Acetylaminofluorene (AAF)
  • Use: Insecticide
  • Oxydianiline (ODA)
  • Use: Azo-Dye
  • Methylene benzenamine
  • Use: Dye intermediate
  • Nitrosamines
  • Nitrosoureas (NMU), (NBU), (ENU)
  • Use: derivatives (BCNU, CCNU, MeCCNU) are drugs against tumors
  • Streptozocin (naturally occurring nitrosourea) is used in the treatment of islet-cell carcinoma of the pancreas

Papillary thyroid carcinoma

• The most important pathogenic factor involved in developing papillary thyroid cancer is an intracellular signaling pathway called MAPK pathyway (Mitogen-activated protein kinases), also known as ERK pathway (extracellular signal-regulated kinase). After antigen binding to tyrosine receptors, MAPK is translocated into the nucleus. Receptor activation leads to cell division, after phosphorylation by MEK (a serine/threonine kinase).
• Other steps leading to MAPK phosphorylation include phosphorylation of RAS which activates BRAF, a serine/threonine kinase followed by MEK and MAPK phosphorylation.
• In papillary thyroid carcinoma, a somatic mutation may lead to activation of this linear signaling cascade.
• As a result, there will be increased transcription of nuclear proteins, which lead to un-regulated activity and reproduction of cancerous cells. This implies that any single alteration is sufficient to play an early role in tumorigenesis.

Abbrevaitions:

ERK: extracellular signal-regulated kinase; MAPK: mitogen-activated protein kinase

(c) Colloid and cystic nodules

1. Colloid nodules

• The colloid nodules consist of colloid droplets and thyroglobulin vesicles.
• Thyroid gland keeps a balance between colloid and thyroglobulin production by regulating the secretion of thyroglobulin into colloid and reabsorption of colloid into thyroid follicular cells. This regulation is maintained by macro-pinocytosis (pseudopods) and micro-pinocytosis (microvilli).
• Any imbalance between secretion and reabsorption of thyroglobulin leads to a disruption of the equilibrium, and produces a colloid appeared thyroid nodule. These nodules may also be produced as a defect of intraluminal thyroglobulin reabsorption. 

2. Iodine related nodules pathogenesis:

Iodine excess can lead to colloid nodules in thyroid gland, leading to a colloid goitre:

• Endocytosis inhibition: High dosage of iodine may lead to inhibition of the protease activity of thyroid lysosomes thereby inhibiting endocytosis
• Exocytosis inhibition: Iodine reduces the expression of the TSH receptor on the surface of thyroid cells thereby inhibiting and decreasing colloid reabsorption
• Iodine excess in combination with TSH over activity may lead to colloid goitre

Another mechanism that may lead to colloid goitre formation is loss of thyroglobulin packaging ability, that may lead to an enormous enlargement of the follicles and flattening of the epithelium.

3. Cystic thyroid nodules

Cystic thyroid nodules may be classified into the following types:

• Necrotic cystic nodules:
  • May be due to a relative deficiency of blood supply:
    • Inadequate blood supply for neoplastic growth
    • Imbalance between angiogenesis and cell growth
    • Compression of new vessels due to mass effect, leading to cell damage and necrosis
  • Hyperplastic thyroid nodules may proceed towards necrosis, colliquation, and pseudocyst formation
• Serum-like cystic nodules:
  • May be related to autoimmunity
• Apoptotic cystic nodules:
  • Cysts that may be related to normal cellular apoptosis or neoplastic/infected cellular apoptosis
• Vascular growth factor related cystic nodules:
  • Cyst formation may be the result of an increased concentration of VEGF/VPF inside the cystic area
  • VEGF/VPF lead to stimulation of vascular permeability and promoting the accumulation of fluids in the cysts
  • VEGF/VPF are particularly found in the cystic fluid of rapidly enlarging or recurrent cysts

(d) Thyroiditic nodule

Nodular lymphocytic thyroiditis almost always present in combination with other thyroiditic diseases. They can also present as a part of infection. It has been shown that the ability of super-antigens (SAgs) to activate the immune system may play a role in the course of autoimmune disorders. In most of these cases, the mechanism of nodular lesion is the same as the mechanism of the main disease, implying that the thyroid nodule is a part of normal disease pattern. Many of these nodules are not identifiable based on physical exam, and are detected during thyroid scintigraphy. The most important thyroiditic diseases that may present as lymphocytic nodular thyroid are:

• Local infections:
  • Pyogenic infection
  • Tuberculosis
  • Parasites
• Subacute de Quervain’s thyroiditis
• Fibrosing (Riedel’s) thyroiditis
• Plasma cell granuloma
• Plasmacytoma
• Primary amyloid tumor and amyloidosis
• Thymoma
• Primary thyroid lymphoma
  • Thyroiditic nodule due to diffuse B-cell infiltration into lymphoma presented areas
• Histiocytosis X
• Medullary carcinoma
• Papillary carcinoma
  • Thyroiditic nodule may be due to an immune response to some abnormal thyroid antigen expressed in the tumor

Genetics

Genetic mutation is considered as one of the most important mechanisms of developing thyroid nodules, especially neoplastic thyroid nodules. Most of these mutations occur as somatic mutations, while some may occur in a familial order. The most important category of familial thyroid cancers are due to genetic mutations, and are called familial non-medullary thyroid cancer (FNMTC), with the following features:

• Rare group of cancers
• Related to other non-medullary tumors
• Inheritance: Autosomal dominant with incomplete penetrance and variable expressivity
• Affected patients in an earlier age
• Associated with:
  • More benign thyroid nodules
  • Multifocal disease
  • A higher rate of locoregional recurrence

The most important genetic mutations associated with thyroid neoplasia development

Oncogenes and growth factors	Gene mechanism	Mutation effect	Neoplasia
N&H ras	Ras-constitutively bound to GTPase-activating protein (GAP)	Activation of adenylate cyclase and calcium channels	Adenomas Papillary carcinoma Follicular carcinoma Anaplastic carcinoma
RET	Encodes a receptor for glial-derived neurotrophic GF Fusion proteins with constitutive thyrosine kinase activities Dimerization of RET thyrosine kinase receptors (TRK)	Mitogenic through constitutive activation of TKR Increased auto-phosphorylation and alteration of substrate specificity	Papillary carcinoma MEN 2A FMTC MEN 2B
Gsp	Ribosylated GS-α at arginine 201	Impairing of GTPase activity	Hot adenomas
C-MET (α and β subunit)	Increased receptors for HGF/SF	Enhancement of receptor kinase activity	Papillary carcinoma
TRK	Receptor for nerve growth factor	Mitogen activated TK cascade	Papillary carcinoma
EGF / EGF-R	Competence factor in cell cycle	Transition through G0-G1 phase	Anaplastic carcinoma
P53	Lack of activation of p21/Waf l gene expression	Loss of regulation at the critical G1 to S phase	Anaplastic carcinoma Papillary carcinoma Follicular carcinoma

Associated conditions

Preoperative serum TSH is an independent risk factor for predicting malignancy in a thyroid nodule, and is associated with:

• Higher differentiated thyroid cancer stage
• Gross extrathyroidal extension
• Neck node metastases

Gross pathology

• On gross pathology, cystic lesions, multiple or a single nodule, and encapsulated lesions are the most important and prevalent characteristic findings of thyroid nodules.
• On gross pathology, follicular thyroid adenoma may present as a big lesion with thick capsule.

Microscopic pathology

Diagnostic speciemen feature: the presence of at least six follicular cell groups, each containing 10–15 cells derived from at least two aspirates of a nodule

Cytology classification		Also referred to as:	Efficient diagnosis		May be seen in:	FNA cytology
			FNA	Surgical biopsy
Follicular lesions	Benign (macrofollicular)	Adenomatoid adenoma Hyperplastic adenoma Colloid adenoma	+		Normal thyroid tissue Sporadic nodular goiter Monoclonal macrofollicular tumors Hyperplastic nodules Colloid adenomas (most common)	May have areas of cystic degeneration with cellular debris and hemosiderin-laden macrophages Cellular characteristics: Small and flat Uniform in size Non-crowded Smeared colloid is seen in the background Follicle size may vary, with a few microfollicles interspersed among the macrofollicles, especially if the sample was obtained from an area close to the capsule of the lesion Colloid: May smear across the slide or occasionally aggregated into droplets due to disruption of follicles during FNA Stains blue on a Papanicolaou stain May be abundant in the background of macrofollicular lesions
	Follicular neoplasm/microfollicular	Cellular adenoma Indeterminate adenoma Trabecular adenoma		+	Follicular adenomas Follicular carcinomas Follicular variant of papillary cancer Occasionally from autonomously functioning thyroid nodules	Well-developed microfollicles Crowding of cells May form clusters and clumps Scant colloid Varying nuclear atypia Varying cellular pleomorphism Follicular carcinoma: Focal microscopic invasion Cellular or trabecular adenomas: Lesions with less definite or no follicle formation May show vascular or capsule invasion
	Follicular lesion of undetermined significance (FLUS)		+		Commonly, especially in nodular goiters	FLUS: The lesion has approximately equal number of macrofollicular fragments and microfollicles AUS: Cells with mild nuclear atypia Mostly due to compromised speciemens: Poor fixation or obscuring blood (FLUS)
	Atypia of undetermined significance (AUS)
	Hürthle cells	Oncocytes Askanazy cells Oxyphil cells	+		Focal Hürthle-cell change: Degenerating macrofollicular lesions Hashimoto's thyroiditis	Large polyclonal cells Oxyphil cytoplasm Considered benign if there is no evidence of vascular or capsular invasion Considered malignant if invasion is present Hürthle-cell cancer Follicular cancer Oxyphil cell type cancer
Papillary cancer		The follicular variant of papillary cancer		+	Epithelioid giant cells Papillary cancer Degenerating areas of macrofollicular nodules Subacute granulomatous thyroiditis Psammoma bodies Papillary carcinoma Benign thyroid lesions	Large cells and nuclei  Ground glass appearance of cytoplasm  Nuclei appearance: Clefts  Grooves  Holes  Intranuclear cytoplasmic inclusions = Orphan Annie eye Small nucleoli  Psammoma bodies Small laminated calcifications Sticky colloid Colloid "stick" to debris and cell clusters, instead of smearing across the slide Epithelioid giant cells Can also be seen in: Degenerating areas of macrofollicular nodules Subacute granulomatous thyroiditis
Medullary cancer				+	Medullary cancer	Spindle-shaped cells Frequently pleomorphic cells without follicle development Supporting stroma may frequently stains for amyloid Red cytoplasmic granules Eccentrically placed nuclei Slightly granular cytoplasm that may be configured as a tear drop or cytoplasmic tail
Anaplastic thyroid cancer			+		Anaplastic thyroid cancer	Spindle cells Pleomorphic giant cell Squamoid Mitosis Numerous mitotic figures Atypical mitoses Extensive necrosis

• Both polyclonal and monoclonal nodules appear similar on fine needle aspiration (FNA) (macrofollicular) and are benign
• The diagnosis of follicular cancer can not be made based on FNA, because vascular or capsular invasion is required to make the diagnosis of follicular cancer. 8420446

Neoplastic thyroid nodules subclassification microscopic pathology:

Neoplasm	Subclass	Features
Follicular thyroid lesions	Minimally invasive follicular carcinoma	Only invasion of the capsule of the tumor without vascular invasion
	Widely invasive follicular carcinoma	Extensive invasion of the tumor capsule A multinodular tumor without a well-defined capsule invading the normal thyroid surrounding the tumor Extensive vascular invasion (>4 foci of angioinvasion)
	Encapsulated follicular variant of papillary thyroid cancer	Minor vascular invasion (≤4 foci of angioinvasion within the tumor or capsule of the tumor) with or without capsular invasion
	Infiltrative variant of papillary thyroid cancer
Papillary thyroid cancer	Tall cell variant	Tumor cells with eosinophilic cytoplasm that are twice as tall as they are wide The primary tumors tend to be large  Often invasive, that many patients have both local and distant metastases at the time of diagnosis
	Insular varient	Solid nests of tumor, often separated by fibrous bands, but the tumor cell nuclei have the same characteristics as do the nuclei of classical papillary cancers
	Columnar variant	Elongated cells with palisading nuclei
	Hürthle or oxyphilic variant	Cellular features of Hürthle cell carcinomas but cells that are arranged in papillary formations.
	Clear cell variant	Clear cell view with clear cytoplasm Must be distinguished from clear cell carcinomas of other organs such as the kidney or colon that have metastasized to the thyroid
	Diffuse sclerosing variant	Diffuse involvement of the thyroid Stromal fibrosis Prominent lymphocytic infiltration
	Cribriform morular variant	Prominent cribriform pattern with solid and spindle cell areas as well as squamous morules Often associated with familial adenomatous polyposis
	Hobnail variant	Multifocal with variably sized complex papillary structures lined by cells Cells with increased nuclear to cytoplasmatic ratios Apically placed nuclei that lead to a surface bulge (hobnail appearance) 19956062