RESEARCH TITLE: THE SIGNIFICANCE OF PTC GENE IN DETECTING THYROID

CANCER: A POTENTIAL TUMOR MARKER

Introduction

Based on pathological, clinical, and genetic characteristics, thyroid tumors are now

broadly classified as follicle-derived (thyroid epithelial) neoplasms, other epithelial

tumors, non-epithelial tumors, and secondary tumors (Lam, 2017). Depending on their

biological behavior within the body, these tumors can be benign, borderline, or

malignant (Maia, 2012). The major thyroid epithelial neoplasms are follicular adenoma,

hyalinizing trabecular tumor, encapsulated follicular-patterned thyroid tumors, papillary

thyroid carcinoma (PTC), follicular thyroid carcinoma, Hürthle cell tumor, poorly

differentiated thyroid carcinoma, anaplastic thyroid carcinoma, and squamous cell

carcinoma. Other epithelial tumors found in the thyroid gland include medullary

carcinoma, salivary gland-type tumors, mucinous carcinoma, and thymic tumors, while

non-epithelial thyroid tumors include paraganglioma, peripheral nerve sheath, vascular,

smooth muscle, solitary fibrous and histiocytic tumors, lymphoma, and teratoma

(Paricharttanakul et al., 2016).

PTC accounts for roughly 80% of all thyroid cancer cases. It is also the most common

subtype of thyroid cancer in countries with iodine-sufficient or iodine-excess diets (Liu et

al., 2016). The prevalence of PTC is increasing. The reasons are unknown, but they

may reflect advancements that contribute to earlier cancer detection. PTC can develop

at any age and is only rarely diagnosed as a congenital tumor (Zhu et al., 2015). It is
typically discovered in the third to fifth decades of a patient's life, with a mean age of 40

years.

The prevalence of PTC rises with age, and women outnumber men in ratios ranging

from 2:1 to 4:1. The etiology of PTC has sparked considerable interest. A previous

history of radiation exposure is the only notable or well-established environmental factor

linked to the progression of PTC (Al-Salamah et al., 2022). The atomic bombings of

Hiroshima and Nagasaki at the end of World War II in 1945, as well as the explosion of

the Chernobyl nuclear power station in 1986, confirmed the carcinogenic effect of

radiation that led to PTC. Other risk factors include having a family history of PTC or

having pre-existing benign thyroid disease (LiVolsi, 2011).

The risk of PTC malignancy among patients with benign nodular goitre is inconsistently

reported. Previous research comparing patients with multiple nodular goitre to those

with a single thyroid nodule found no difference in cancer risk (Iglesias et al., 2017).

Later research found that patients with a single thyroid nodule are more likely to develop

thyroid cancer than patients with multiple thyroid nodules (Lee et al., 2016). These

beliefs, however, are no longer valid, as numerous subsequent studies have revealed a

significant risk of PTC in patients with multiple thyroid nodules (Wang et al., 2013).

PTC appears to be the most common type of thyroid cancer discovered by chance in

patients with benign thyroid goiter (Smith et al., 2013). The risk of PTC in multinodular

goitre has been reported to range from 6% to 21.2%. (Lim et al., 2017). An earlier study

found that more than half of PTC cases were discovered by chance in elderly patients
undergoing surgery for pre-existing multinodular goitre. There is also variation in the

frequency of thyroid cancer in Graves' disease, with 0.5% to 18.7% of patients having

PTC (Rahman et al., 2015).

The demographics of PTC patients are also fraught with controversy. An

epidemiological survey of 838 patients with multinodular goiter was conducted.

According to a recent study, younger age, male sex, fewer nodules, and smaller nodule

size are all risk factors for incidental thyroid cancer. Similar findings of an increased risk

of cancer in young males with thyroid nodules have been independently reported

(Bombil et al., 2014). On the contrary, Rahman et al. found that the female gender and

multiple lesions were associated with a higher risk of malignancy, while the risk for

smaller nodule size was also high. Bombil et al., data’s on 166 PTC patients with

concomitant multinodular goitre adds to the controversy when older age (mean age was

46 years) and female gender were associated with a higher risk of malignancy.

Clinical Diagnosis

In the diagnosis of PTC, fine needle aspiration and cytology (FNAC) is the preferred

method. A narrow gauge needle is used to obtain a sample of a lesion for microscopic

examination during the minimally invasive and quick procedure. Thyroid biopsy

specimens are classified as benign, suspicious (or indeterminate), or malignant based

on their cytological appearance during this procedure (Ullah et al., 2014). Although a

PTC aspiration smear may reveal papillary structure, preoperative diagnosis is primarily

based on the recognition of typical nuclear features such as 'Orphan Annie' nuclei,
intranuclear pseudoinclusions (due to cytoplasmic invaginations), and nuclear grooves

(folds in the nuclear membrane). The presence of psammoma bodies (calcium salt

deposits) in a cervical lymph node indicates PTC (Pazaitou-Panayiotou et al., 2012).

When compared to postoperative diagnosis of surgical specimens, the accuracy rate of

diagnosis with FNAC is around 90%. Ultrasonography is commonly used to improve the

diagnostic yield of FNAC. The technique is extremely useful in identifying appropriate

nodules to aspirate within a multinodular thyroid or in determining a site within a nodule.

It can detect the presence of nodules that are too small to be palpated, multiple

nodules, and lymphadenopathy in the central or lateral neck. In addition,

ultrasonography provides precise dimensions of a nodule for patient monitoring (Luo et

al., 2012). Microcalcifications, hypoechoic appearance, increased vascularity, and

irregular borders on ultrasound can help differentiate malignancy in PTC patients with

benign goiter. Distant metastasis, usually to the lungs and bones, is less common in

PTC compared to other types of thyroid cancer.

DANGERS OF THYROID CANCER

Thyroid cancer, if left untreated, can spread to the airway, esophagus, or other nearby

neurovascular structures. The lung, bone, and other soft tissue structures are the most

commonly affected by distant metastasis. Both thyroid lobectomy and total

thyroidectomy have the potential for neurovascular injuries, with the most common

involving the recurrent laryngeal nerve, resulting in hoarseness of voice and, in the case

of bilateral injuries, respiratory failure (Nikiforov, 2022).

familial occurrence for MTC. Over the last decades, the incidence of papillary thyroid

cancer has increased worldwide, primarily due to early detection and advanced imaging

technology with the risk of overdetection. Mutations and translocations in the genes

coding for the mitogen-activated protein kinase (MAPK) cellular signaling pathway have

been linked to the genetic basis of the majority of thyroid cancers (Moura, 2015).

Female sex, a family history of thyroid cancer, and thyroid gland radiation exposure

during childhood are the major risk factors for DTC. Thyroid cancer affects both genders

equally, according to autopsy reports, but it may be detected more frequently in women

than men. Access to medical care could explain the disparity (Paulson et al., 2017).

Epidemiology

Thyroid cancer is the fifth most common cancer in women in the United States,

accounting for 1% to 4% of all malignancies. It has a roughly 3:1 female to male ratio.

The global incidence of thyroid cancer has been steadily increasing; specifically, PTC

detection has increased by 240% in the last three decades. This increase in incidence

has been observed in both genders and across all races, and is thought to be primarily

due to an increasing trend in diagnostic imaging rates (Brehar et al., 2013). PTC is the

most common endocrine cancer, accounting for 96% of all new cases and 66.8% of all

endocrine cancer deaths. As previously stated, most thyroid cancers arise from the

follicular epithelium, with PTC and FTC far more common than anaplastic thyroid cancer

(Catalanotto, 2016).
Evaluation

In the case of a thyroid nodule, the most appropriate initial evaluation is a thyroid

function panel. A hyperthyroid state is often associated with a lower risk of cancer; in

such cases, a radionuclide uptake scan is recommended. If the nodule or nodules are

found to be hyperfunctioning, fine-needle aspiration biopsy (FNAB) should be avoided.

This is due to the fact that these nodules are rarely malignant, and FNAB results for

hyperfunctioning nodules are frequently incorrect (Londin er al., 2015).

When a thyroid nodule is biochemically euthyroid or hypothyroid, it should be evaluated

with a high-resolution diagnostic thyroid ultrasound. This can assist in assessing the

nodules for high-risk characteristics, detecting additional nodules not felt on physical

examination, evaluating for neck lymph nodes, and guiding FNAB if necessary. A

significant increase in size from prior imaging, hypoechogenicity, irregular margins, a

size taller than wide, microcalcifications, a solid internal structure, extra-thyroidal

extension, and central vascularity are all high-risk ultrasound features (Chung et al.,

2012).

A purely cystic nodule, a spongiform appearance, comet tail shadowing, and peripheral

vascularity are all associated with a lower risk of malignancy. The decision to subject a

nodule to FNAB should be based on these radiological criteria, with assistance from

Thyroid Imaging Reporting and Data System (TIRADS) or American Thyroid

Association (ATA) criteria, but clinical indications should be prioritized regardless of

imaging criteria (Qu, 2016).

Molecular testing is commonly used in Bethesda categories 3 and 4, where cytology is

indeterminate. Single mutations, such as BRAF V600E or RET/PTC translocations,

have recently been tested. While these tests have a high specificity (100%), their

sensitivity (50-60%) is usually low (Zhang, 2017).

Molecular Diagnostic Approaches

A 7-gene panel detects multiple genes, including BRAF V600E, HRAS codon 61, KRAS

codons 12/13, and NRAS codon 61 point mutations, as well as RET/PTC1,

PAX8/PPAR, and RET/PTC3 translocations, which account for approximately 70% of

thyroid cancer. This panel has increased the sensitivity and negative predictive value of

these tests to greater than 90%. As a result, mutation tests are good rule-in tests for

thyroid cancer (Huang, 2013). Furthermore, a 167-Gene expression classifier has a

high negative predictive value but only a 50% positive predictive value.] As a result,

gene expression classifier testing is a good rule-out test for thyroid cancer (Chou,

2017).

However, a large multi-gene panel of mutation markers has recently been introduced,

improving sensitivity and specificity even further. While CT and MRI scans are not

routinely used to evaluate thyroid nodules for malignancy, they may be useful in

assessing local spread in more advanced diseases or in patients with enlarged cervical

nodes in conjunction with a suspicious mass. A CT scan is recommended for patients

ultrasound or plain radiographs (Erler et al., 2014).

Thyroid Cancer in Eastern and Western Countries

Thyroid cancer (TC) is the most common malignant tumor of the endocrine system.

Globally, about 567,000 incident TC cases were estimated in 2018, ranking the ninth for

incidence in all cancers, among which 130,889 were males and 436,344 were females,

with age-standardized incidence rates by world population (ASIRs) of 3.1/100,000 and

10.2/100,000, respectively (Cantara, 2014). However, about 41,000 TC deaths were

estimated in 2018 worldwide, including 15,557 males and 25,514 females, with age-

standardized mortality rates by world population (ASMRs) from 0.4 to 0.5 per 100,000 in

males and females. According to the Global Cancer Statistics 2018, there is an over

ten-fold difference in incidence across different parts of the world in both females and

males, the highest incidence area being in North America (23.1/100,000 in females and

6.9/100,000 in males) and the lowest incidence area in Western Africa (1.5/100,000 in

females and 0.5/100,000 in males) (Li et al., 2015). . Incidence rates of TC have largely

increased in many developed countries in recent decades, but at different magnitudes.

Several possible explanations have been proposed for this increase, including more

sensitive diagnostic procedures and increases in environmental carcinogens. Different

risk factors may account for the temporal trend, including exposure to ionizing radiation

in childhood and among young women; however, their effects on TC development have

still not been fully illuminated (Graham et al., 2015).

shown somewhat divergent findings. Although some studies showed that a rapidly

increased incidence of TC might be associated with wider medical surveillance and

more intensive diagnostic practices, others reported that various environmental factors

may contribute to this increasing trend (Yu, 2016). These studies were mainly carried

out in populations in Europe, the United States, and South America, and thus may

reflect a different situation from that in populations in East Asia, including the levels of

diagnostic technology for TC, differences of environmental factors, and government

health policies (Yoruker, 2016).

Thyroid Cancer Associated Diseases

The link between cancer and inflammation has been well established since 1863, when

Rudolf Virchow hypothesized that chronic inflammation could contribute to

tumorigenesis after observing leukocytes in neoplastic tissues. Several lines of

evidence in the following decades suggested a strong link between chronic

inflammation and increased susceptibility to neoplastic transformation and cancer

development. It is estimated that up to 20% of all tumors are caused by chronic

inflammation, such as chronic infections or autoimmune diseases.

Cervical cancer and papillomavirus are well known associations, as are gastric cancer

and Helicobacter pylori-induced gastritis, esophageal adenocarcinoma and Barrett's

metaplasia, hepatocellular carcinoma, hepatitis B and C viral infections, and many

others. Some of the mechanisms underlying the relationship between inflammation and
tumor have recently been discovered. The presence of host leukocytes both in the

supporting stroma and among tumor cells characterizes the inflammatory

microenvironment of neoplastic tissues, with macrophages, dendritic cells, mast cells,

and T cells being differentially distributed (Balkwill and Mantovani, 2001). Several

cytokines (TNF, IL-1, IL-6) and chemokines produced by tumor cells, as well as

leukocytes and platelets associated with the tumor, have been found to be capable of

maintaining the invasive phenotype (Coussens and Werb, 2002). Tumor-associated

macrophages (TAMs) are a major component of the leukocyte infiltrate, having been

recruited by inflammatory chemokines (e.g., CCL2) and then maintained by cytokines

found in the tumor microenvironment (e.g., CSFs, VEGF-A). TAMs promote tumor

proliferation, progression, and stroma deposition in response to cytokines such as

TGF-, IL-10, and M-CSF, and their density is increased in advanced thyroid cancers

(Ryder et al., 2008).

Papillary thyroid cancer (PTC) is frequently associated with autoimmune thyroid

diseases such as Graves' disease and Hashimoto's thyroiditis. In the series from

different countries, the frequency of association varies greatly, ranging from 0-9% for

Graves' to 9-58% for Hashimoto's (Figure (Figure1).1). It is still unclear whether the

presence of an autoimmune disorder influences the prognosis of PTC. Few studies

reported a worse prognosis (Ozaki et al., 1990; Pellegriti et al., 1998), whereas the

majority of studies found either a protective effect of thyroid autoimmunity

(Matsubasyashi et al., 1995; Loh et al., 1999; Gupta et al., 2001) or a similar behavior

between cancer with and without associated thyroiditis (Yano et al., 2007). These

disparities can be attributed to the small number of patients studied in those studies, the
absence of a control group, the presence of different genetic and epidemiological

backgrounds, or the use of inappropriate criteria to define remission or

persistence/relapse. We recently published data that expands our understanding of the

close relationship between thyroiditis and thyroid cancer. The clinical and molecular

characteristics, as well as the expression of inflammation-related genes, were studied in

a large series of PTCs divided into two groups based on whether or not the tumor was

associated with thyroiditis (Muzza et al., 2010). Interestingly, no significant differences

were found between the two groups in terms of age at diagnosis, gender distribution,

TNM staging, histological variants, and outcome, implying that the association with an

autoimmune thyroid process has no effect on the presentation or clinical behavior of

PTC. The concept that the inflammatory protumourigenic microenvironment of this

cancer is elicited by the oncogenes responsible for thyroid neoplastic transformation

(such as RET/PTC, BRAFV600E, and RASG12V; Borrello et al., 2005, 2008; Melillo et

al., 2005; Mantovani et al., 2005) has emerged as a critical finding in recent

Summary

The RET/PTC oncogene family, particularly RET/PTC1 and RET/PTC3, is important in

the development of some PTCs, particularly pediatric PTCs and those associated with

ionizing radiation exposure. Furthermore, the relative aggressiveness of a given PTC

may be dependent on the specific RET/PTC family member expressed, implying that

these oncoproteins exhibit significant differences in intracellular behavior. Finally, the

observation of nonclonal expression of RET/PTC oncogenes in some benign thyroid

diseases suggests that the local cellular microenvironment may play a role in
modulating RET/PTC oncogenic activity. These findings highlight significant gaps in our

current understanding of the mechanisms by which RET/PTC mediates thyroid

tumorigenesis, mechanisms that, when characterized, could provide critical insights into

the development of new PTC therapeutics. Both molecular-based diagnostics and

molecule-targeted therapeutics are in their early stages, and, perhaps unsurprisingly,

simple preoperative screening for the presence of RET/PTC fusions in thyroid nodule

aspirates has not proven useful in refining surgical decision making. This finding could

be attributed to poor RET/PTC detection methodology and/or a lack of understanding of

the mechanisms by which RET/PTC mediates tumorigenesis. Improvements in

RET/PTC detection techniques, as well as our understanding of the specific molecular

events that occur when RET/PTC fusions result in carcinoma, may significantly improve

the clinical utility of preoperative RET/PTC oncogene screening. Furthermore,

nonspecific targeting of cellular tyrosine kinases with novel agents has yielded some

promising therapeutic results. Research in this area will undoubtedly continue, resulting

in increasingly efficacious and less toxic therapeutics for otherwise treatment-refractory

PTC.
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