Role of Cytomorphometry and BRAFV600E Immunocytochemistry With a Cytologic Diagnosis "Suspicious for Papillary Thyroid Carcinoma"

Thyroid nodules (TNs) are a common finding, detected by ultrasound in about 70% of healthy individuals, with women being four times more affected. While most TNs are benign, the primary clinical goal is to exclude malignancy, which occurs in 4-6.5% of cases. Risk factors for TNs include age, gender, iodine imbalance, family history, and radiation exposure. Thyroid cancer incidence has risen due to improved diagnostic methods such as ultrasound, FNAC, CT, MRI, and PET scans.

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy (90%), followed by follicular, Hurthle cell, medullary, and anaplastic carcinomas. Most thyroid cancers have an excellent prognosis, with a 5-year survival rate of 98.6%. FNAC is the gold standard for diagnosing TNs, and the Bethesda system (TBSRTC) categorizes cytology results into six groups with varying malignancy risks. The "suspicious for malignancy" category (74% risk) includes mostly suspicious papillary thyroid carcinoma (SPTC), but false positives occur, especially in cases of Hashimoto's thyroiditis.

SPTC diagnosis can be challenging, leading to potential overtreatment. To improve diagnostic accuracy, additional techniques like detailed ultrasonographic criteria, intraoperative frozen section, and molecular marker testing are used. Cytomorphometric analysis has been explored for distinguishing benign from malignant thyroid lesions, assessing nuclear size, shape, and chromatin patterns. Most studies have focused on histological sections, but applying morphometry to cytology could enhance automated and accurate diagnoses.

Molecular studies reveal that PTCs primarily arise from MAPK pathway mutations, with BRAFV600E being the most common (60%). This mutation is strongly associated with aggressive tumor behavior and recurrence. BRAFV600E testing combined with FNAC significantly improves diagnostic accuracy, increasing sensitivity from 76.71% (mutation testing alone) to 96.62% when combined with cytology. This integration has reduced false-negative PTC diagnoses and facilitated targeted therapies.

Thyroid nodules are common clinical finding in routine daily medical and surgical practice. They are discovered by ultrasonography in about 70% of clinically healthy individuals. Thyroid nodules are four times more prevalent in women and up to 90% of cases are caused by benign conditions.

The main target of clinical management of thyroid nodules is exclusion of malignancy (4-6.5% of all thyroid nodules), assessment of pressure symptoms and evaluation of thyroid dysfunction .

Many risk factors were identified related to development of thyroid nodules including advanced age, gender, increased or decreased iodine, family history of cancer or polyposis and history of head and neck radiation. The most common thyroid nodules are colloid nodules, Hashimoto's thyroiditis, subacute thyroiditis, cysts, follicular adenoma and thyroid cancers.

Regarding thyroid malignant tumors, the incidence of thyroid cancer increased over the past decades in the United States and internationally. It's the 7th most common cancer in the general population and the 5th most common in women. The incidence of thyroid cancer is about 3 times higher in women than men and it increases from adolescence to middle age with the peak around 55 years in women and 65 years in men then decreasing with older age.

The increase of number of cancer cases is caused by increased diagnosis resulting from the widespread use of diagnostic tools including ultrasonography imaging, fine needle aspiration cytology , computed tomography , magnetic resonance imaging and fluorodeoxyglucose positron emission tomography. In addition to the increase in the exposure to the etiologic factors as ionizing radiation during childhood and obesity.

Papillary thyroid carcinoma (PTC) is the most common histologic type of thyroid cancers representing 90% of cases. The remaining 10% include follicular thyroid carcinoma 4%, Hurthle cell carcinoma 2%, medullary thyroid carcinoma 2% and anaplastic thyroid carcinoma 1% .

The prognosis of thyroid cancer is excellent because most cases are papillary thyroid carcinomas localized to the thyroid gland during diagnosis. The 5-year survival rate is 98.6% overall, 99.9% for localized, 98.3% for regional and 54.9% for distant metastasis .

Fine-needle aspiration cytology (FNAC) of the thyroid gland is a gold standard accurate diagnostic test routinely used in the initial evaluation of thyroid nodules as it is simple, safe, cost-effective and has favorable accuracy. The sensitivity and specificity of thyroid FNAC are 68-98% and 56-100% respectively with failure rates are because of the technique and experience of the performance .

The Bethesda system for reporting thyroid cytopathology (TBSRTC) was first published in 2010 and updated many times to standardize reporting terminology and for better communication between cytopathologists and clinicians.

The 2023 Bethesda system for reporting thyroid cytopathology is a simple category-based reporting system for fine needle aspiration cytology (FNAC) of thyroid and became one of the most used reporting and diagnostic methods and includes 6 categories.

Category (I) non diagnostic with 13%(5-20) risk of malignancy, Category (II) benign with 4%(2-7) risk of malignancy, Category (III) atypia of undetermined significance with 22%(13-30) risk of malignancy, Category (IV) follicular neoplasm with 30%(23-34) risk of malignancy, Category (V) suspicious for malignancy with 74%(67-83) risk of malignancy and Category (VI) malignant with 97%(97-100) risk of malignancy.

The suspicious for malignancy category is used when cytomorphologic features is inconclusive and raise a strong suspicion of malignancy but are quantitatively or qualitatively insufficient for a conclusive diagnosis and the usual management in this case is molecular testing, lobectomy or near total thyroidectomy.

The suspicious for malignancy (SFM) according to Bethesda system category includes suspicious for papillary thyroid carcinoma (SPTC) which is the most common subtype (more than 90%) and less frequently suspicious for non-papillary carcinoma which consists of suspicious for medullary thyroid carcinoma, suspicious for malignant lymphoma, suspicious for poorly differentiated carcinoma and suspicious for malignancy not otherwise specified.

Suspicious for papillary thyroid carcinoma is diagnosed due to patchy nuclear changes (unremarkable follicular cells admixed with cells showing nuclear enlargement, pallor, grooves and fine chromatin, scant or absent intranuclear pseudoinclusions and absent papillary architecture) , incomplete nuclear changes (generalized nuclear enlargement, pallor and grooves, scant or absent irregular nuclear membrane and intranuclear pseudoinclusions and absent papillary architecture and psammoma bodies), sparsely cellular specimen or cystic degeneration (background of cystic debris and macrophages, nuclear enlargement and pallor, atypical histocytoid cells with cytoplasmic vacuoles and absent papillary architecture and psammoma bodies.

Suspicious for papillary thyroid carcinoma is diagnosed in 2.4-8% of fine needle aspiration results and 60-80% of them are confirmed in resected specimens to be papillary thyroid carcinoma and the rest of cases showed benign conditions as chronic lymphocytic thyroiditis (Hashimoto thyroiditis) and nodular hyperplasia. Hashimoto thyroiditis is one of the most common factors in false positive papillary thyroid carcinoma diagnosis because of the strong overlap of morphological features between them.

Suspicious for papillary thyroid carcinoma diagnosis is confusing for surgeons and patients and makes the optimal surgical decision difficult as the prevalence of malignancy in this diagnostic category is high. Some surgeons proceed for total thyroidectomy and this makes about 25% of patients (false positive cases) may suffer from oversurgery and in need for hormone replacement therapy for life. Many trials were made to increase the accuracy of detection of malignancy in this category and decrease the possibility of oversurgery. Some authors applied detailed ultrasonographic criteria for cases diagnosed cytologically as suspicious for papillary thyroid carcinoma and reported increase of accuracy from 86% to 94% while others performed intraoperative frozen section and ancillary tests as molecular markers.

Cytomorphometric analysis has been used as a clinical diagnostic tool and discussed in the literature for over 30 years. It has been used in many organs like nasopharynx, skin, urinary bladder, colon, breast and thyroid. Morphometry is computerized procedure performed through the quantitative analysis of geometric features of structures with any dimension. The nuclear morphometry of malignant nuclei including the size, shape, and chromatin pattern, are different from those of nonmalignant cell nuclei, which allows nuclear morphometry to distinguish between benign and malignant cases. Using nuclear morphometry, we can quantify a number of parameters such as those related to nuclear size and shape.

Morphometry can help as an adjunct to morphological features in thyroid lesions when correctly applied with appropriate cutoffs. Till now, majority of image analysis studies have been performed on histopathological sections and only few studies performed on cytological smears. It has been suggested that nuclear morphometric parameters such as nuclear area and perimeter and shape factors may allow differentiation between thyroid lesions. Applying morphometry to cytology would be useful for the purpose of automated morphometric screening as well as accurate diagnosis of thyroid tumors.

The computerized morphometric study of thyroid histological slides identified efficient morphometric parameters that distinguish the differences between benign and malignant epithelial cells and the most reliable parameters are area, perimeter, length, width, the degree of roundness and irregularity of the nuclear membranes.

A study revealed that the cytomorphometric image analysis with cytomorphology has the potential for distinguishing not only the benign, follicular neoplasm/suspicious for a follicular neoplasm, malignant thyroid lesions of Bethesda system but also can distinguish nodules with follicular patterns as follicular variant of papillary carcinoma, follicular adenoma and follicular carcinoma.

Many molecular based analytic studies have proved that most papillary thyroid carcinomas are caused by two distinct signaling mechanisms of the MAPK ( Mitogen-activated protein kinase) pathway, either BRAF-like or RAS-like.

The most common gene mutation in papillary thyroid carcinoma (60%) is a missense mutation in the BRAF oncogene, valine (V) substituting glutamic acid (E) at amino acid position 600, which finally drives mitogen-activated protein kinase pathway signaling.

A systematic review and meta-analysis showed that the recurrence rate in BRAFV600E mutated papillary thyroid carcinomas (24.9%) was two times high as wild-type papillary thyroid carcinomas (12.6%). The BRAFV600E mutated PTCs has more risk for lymph node metastasis, extrathyroidal extension and associated with advanced AJCC III/IV stage.

The access to molecular testing has facilitated the use of therapeutic agents targeting specific mutations as BRAFV600E.

The BRAFV600E mutation is very common in papillary thyroid carcinoma (38-90% incidence) and rarely present in benign thyroid lesions so its diagnostic specificity is high. It leads to abnormal activation of the mitogen-activated protein kinase pathway which plays an important role in the development of papillary thyroid carcinoma . It was reported that fine needle aspiration cytology combined with the preoperative BRAFV600E mutation detection can improve the diagnostic performance for thyroid nodules compared to fine needle aspiration cytology alone.

The sensitivity and specificity of the BRAFV600E mutation testing alone for papillary thyroid carcinoma were 76.71% and 100% respectively and increased to 96.62% and 88.03% respectively when combined with cytology. So, the combination between fine needle aspiration cytology and BRAFV600E mutation tests has decreased the false negatives in papillary thyroid carcinoma diagnosis.

Material and methods:

A retrospective descriptive study will be performed on FNA smears of thyroid nodules received in the Department of Pathology at Assiut University Hospital over a six-year period (2018-2025). The study will review Papanicolaou (PAP) and Hematoxylin \& Eosin (H\&E) stained smears from 45 patients with thyroid nodules diagnosed as suspicious for papillary thyroid carcinoma (Bethesda V), combined with the histopathological data of their resected thyroid specimens.

All clinical, radiological and pathological data will be collected from medical records.

Personal data including age and gender, clinical data as number of thyroid nodules and their size and the presence or absence of cervical lymph node enlargement and pressure symptoms and radiological data as thyroid imaging reporting and data system (TI-RADS) which includes composition, echogenicity, shape, margin and punctate echogenic foci will be collected.

Morphometric study:

For each case, 50 nuclei from follicular cells within the suspicious cellular group, with well-preserved morphology, will be selected. Nuclei that will exhibit overlapping, will be obscured by inflammation, blood, or mucin, or will display degenerative changes will be excluded. Preference will be given to isolated nuclei or those just touching each other without overlap with a clean background. Each selected nucleus focused on using a 1000x oil immersion objective lens will be imaged using a digital camera and will be analyzed with Image Analysis Software. The software will assign a specific number to each nucleus in the image and quantify its morphometric parameters.

Immunocytochemical study:

BRAFV600E immunocytochemistry will be applied on the smears after destaining them.

Smears will be stained with BRAFV600E antibodies with immunoperoxidase staining as a visualization method.

Antibody dilution and incubation time will be conducted according to manufacturer's instructions.

Correlation between results of cytomorphometric studies, results of BRAFV600E mutation tests and the results of histopathological slides will be done.

Eligibility Criteria:
Inclusion Criteria:

* Only Papanicolaou (PAP) and Hematoxylin \& Eosin (H\&E) stained FNAC smears diagnosed as suspicious for papillary thyroid carcinoma will be included in the study.
* Only FNAC smears with well-preserved nuclear morphology will be included in the study.
* Smears with histopathological slides.

Exclusion Criteria:

* FNAC smears lacking histopathological confirmation.
* Smears with extensive nuclear overlap and poor morphology.
* Smears obscured by inflammation, blood, or mucoid material.
* Smears exhibiting cellular degenerative changes or drying artifacts.
* Any patient with missing data.