Thyroid cancer is the most common endocrine malignancy accounting for >90% of malignancies of endocrine glands [1] and Papillary carcinoma is the most common tumor of all the thyroid malignancies, constituting 80% of all thyroid malignancies [2]. Papillary thyroid carcinoma is defined as a malignant epithelial tumor showing evidence of follicular cell differentiation and is characterized by distinctive nuclear features [3]. Papillary thyroid carcinoma is usually discovered on routine examination as asymptomatic thyroid nodule that appears as a neck mass. In some instances, the mass may produce local symptoms. It is also one of the least aggressive cancer types and is slow-growing [4]. Some tumors are well-circumscribed and even encapsulated; others infiltrate the adjacent parenchyma and have ill-defined margins. Historically, the identification of papillary carcinoma relied on the presence of papillary architecture. The currently accepted diagnosis is based on the nuclear features that include optical clearing, overlapping, nuclear grooves, and pseudo inclusions. The microscopic hallmarks of papillary neoplasm includes branching papillae having fibrovascular stalk covered by single to multiple layers of cuboidal epithelial cells, the nuclei contain finely dispersed chromatin, which imparts an optically clear or empty appearance, giving rise to ground glass or Orphan Annie eye nuclei, invagination of cytoplasm gives intranuclear inclusion appearance (pseudo inclusion), psammoma bodies (concentric calcified bodies). This tumor gains the maximum attention because of its increasing incidence, quick spread effectively through the lymphatic ducts, resulting frequently in recurrences, metastases and even death [5]. However, identification of these features remains, at times controversial and the distinction of papillary carcinoma from its histological mimickers such as follicular adenoma and follicular carcinoma may sometimes be difficult [6]. It is clear that some cases do raise controversy as being PTC or non-PTC. For example, follicular adenoma and follicular variant of PTC, when some of the nuclear diagnostic criteria for PTC are occasionally present. Unfortunately, such controversy exists between expert thyroid pathologists. Interobserver disagreement among pathologists is well documented⁵ and some cases are unjustifiably over called a follicular variant of PTC. Immunohistochemistry (IHC) is the process whereby antibodies are used to detect proteins

of the institutional research committee.

IHC Staining Method: Tissue used for this study was obtained from the patients mentioned above at the Department of Pathology, Government Medical College, Amritsar. The tissues were embedded in paraffin and sliced into 3-4 μm sections, which was mounted on freshly prepared 0.01% poly-L-lysine coated slides. Slides were dried overnight at 37⁰ C, dewaxed in xylene and hydrated. Endogenous peroxidase activity was blocked by adding freshly prepared 0.3% H₂O₂ in methanol for 10minutes, followed by three washings in PBS. Antigen retrieval was done as per the specification of the kit. Slides were then immersed in citrate buffer and put in the microwave oven for 2 cycles of 10 to 15 minutes at 85⁰C and 100⁰C temperature respectively. The slides were bought to room temperature and immersed in PBS. Sections were washed in phosphate buffer saline and 2 drops of 3% H₂O₂, after which incubated for 10-30minutes. Sections were washed in PBS buffer for 5minutes twice. 2 drops of primary mouse monoclonal antibodies included anti-HBME-1 antibody and anti-TPO antibody (DBS, USA) was used and incubated for 60 minutes. Sections were washed with PBS twice. Sections were then incubated with the linked antibody for 30 minutes and again two washings with PBS were given. Enzyme conjugate was added and incubated for 30 minutes. Again, two washings with PBS were given. One tablet of DAB was dissolved in reagent and incubated on the sections for 6-8 minutes. All incubation was done in a moist chamber. Sections were washed in deionized water for 3 minutes. Hematoxylin counterstaining was done for 2-5 minutes and sections washed under tap water and then dehydrated in ascending concentration of alcohol. The clearing was done in xylene and sections mounted with DPX.

IHC Interpretation: Immunoreactive scoring system was used. The immunoreactive score (IRS) gives a range of 0–12 as a product of multiplication between positive cells proportion score (0–4) and staining intensity score (0–3).The staining intensity was graded as 0 (no staining), 1+ (mild staining), 2+(moderate staining), or 3+ (intense staining), and the proportion of stained cells was scored as 0 (no staining), 1+ (1-10% of cells), 2+(10-50% of cell), 3+(50-80% of cells), 4+(>80% of cells). Cases that showed IRS score of >3, were scored as positive for TPO or HBME-1. A positive result for HBME-1 was considered consistent with malignancy, and a negative result for TPO was considered

*Statistical analysis of data was done by using the Chi-square test. P-value >0.05 was considered significant. # statistically not significant.

Table-3: Expression of TPO in both benign and malignant thyroid lesions (n=50)

*Statistical analysis of data was done by using the Chi-square test. P-value >0.05 was considered significant. # statistically not significant.

The cell membrane marker, HBME-1 expression (a marker for malignancy) was found absent in 83.3% cases of Benign thyroid lesions. Positive expression of HBME-1 in Malignant thyroid lesions was 78.95%, 50% and 0% in PTC, FTC, and MTC respectively, whereas there was no expression of the marker in 21%, 50% and 100% cases of PTC, FTC and MTC respectively (Fig.1,2,3). Thus, depicting that overall, neoplastic thyroid lesions showed predominantly positive staining with HBME-1 [Table-2]. Cytoplasmic marker TPO showed positive expression in 91.7% of cases of benign thyroid lesions. In malignant thyroid lesions, negative expression of TPO (taking negative expression of TPO as a marker for malignancy) was shown by 63.16%, 0% and 33.33% cases of PTC, FTC, and MTC respectively. [Table-3] (Figure1, 2, 3)

Fig-1; A-C: Photomicrograph showing classical PTC [A]- H and E stained section (200X). [B]- HBME-1 Immunostaining showing strong membranous positivity (IHC; 400x). [C] TPO showing negative immunostaining (IHC; 200X). D-F: Photomicrograph showing PTC- FOLLICULAR VARIANT [D]- H and E stained section (400X). [E]- HBME-1 Immunostaining

critical to accurately diagnose PTC. When the diagnosis of PTC is difficult to distinguish using histological criteria immunohistochemistry techniques are useful. According to the World Health Organization classification, PTC may be histologically classified into 14 variants based on histological structure, tumor size, cell shape, cell type, and extracellular matrix, including papillary microcarcinoma, encapsulated variant, follicular variant, diffuse sclerosing, tall cell, columnar, cribriform morular, hobnail, solid/trabecular, oncocytic, spindle cell variant, clear cell, warthin like and papillary thyroid carcinoma with fibromatosis/fasciitis like stroma variant. However, due to the paucity of diagnostic nuclear features; it is often difficult to distinguish papillary thyroid carcinoma from its architectural and nuclear mimics in thyroid lesions. In this condition, molecular alterations in thyroid cancer, such as TPO, and HBME-1, may help to differentiate papillary thyroid cancers from other thyroid lesions [7]. For example, one of the most frequent difficulties in thyroid pathology is differentiating the follicular variant of PTC from follicular adenoma. The inter observable variability in this area is well recognized. The differentiation is critical for the treatment and long-term management of the tumors. Despite the universal recognition of this variant, it is clearly lacking the minimal histological definition of the follicular variant of PTC. Unfortunately, the immune profile of follicular derived lesions and neoplasms show some overlap and to the best of our knowledge, no single marker or even panel is 100% sensitive and 100% specific for PTC. Papillary thyroid carcinoma is common in persons of all ages, with a peak age range between 20-60years [10,11,12,13]. In the present study age of the patient diagnosed with papillary thyroid cancer ranged from 21-40 years. Papillary thyroid carcinoma most commonly affects women [13]. Our results on gender distribution showed that the female population predominated comprising about 86% (n=43) of cases in contrast to males with Female to male ratio being 6:1. HBME-1 targets an unknown antigen of mesothelial microvilli and is abnormally expressed in thyroid malignancies, showing cytoplasmic location with membrane accentuation [14]. HBME-1 is highly expressed in PTC including both classical and follicular variant and FTC with little or no expression in MCT and ATC [15, 16]. It has been noticed that HBME-1 is not expressed in benign thyroid lesion but expressed in malignant thyroid cancer, thus making HBME-1 the

staining for diagnosis of malignancy as 100% [27]. The sensitivity in the present study was only 63.3% for PTC and 33.33% for follicular carcinomas. The reasons for these discrepancies are unclear. These results do not reflect nonspecific staining, because there were cases of papillary and follicular carcinomas that clearly showed negative TPO staining. Morphological diagnosis methods such as immunohistochemistry and cytology of fine-needle aspiration biopsy play an important role in providing a definitive diagnosis and therapeutic guidance. Testing for expression of HBME-1 has been shown to improve the diagnostic accuracy for thyroid malignant nodules [28,29]. The combination of HBME-1 and TPO may contribute to an accurate diagnosis of thyroid papillary carcinoma by fine-needle aspiration biopsy.

Limitations: In comparison to other studies, a few limitations in the present study were present. It was observed that sensitivity to both the markers in differentiating papillary thyroid carcinoma from another thyroid neoplasm could be better studied with a greater number of cases. Rather, than using a single marker, a panel of markers will be more helpful in reaching diagnosis e.g. galectin-3, CD56, CK19, along with HBME-1 and TPO. No standard IHC scoring system is available for these markers. Percentage and intensity staining scores were calculated manually so interobserver variations can be the reason for discrepancies.

In conclusion, due to the overlapping histological features, diagnostic problems in thyroid pathology still remains. HBME-1 due to its high sensitivity can significantly be used as a marker of identification for papillary thyroid carcinomas while TPO though helps in differentiating benign from malignant thyroid carcinomas but cannot be used alone. Therefore, a panel of IHC markers is always recommended for the diagnosis of malignant thyroid neoplasms.

Using the combination of HBME-1 and TPO is useful in differentiating benign from malignant thyroid neoplasms. However, a panel of markers more specific for papillary thyroid neoplasm including Galectin 3, CD56, CK19 along with HBME-1 and TPO will be more helpful in accurately diagnosing PTC from another thyroid neoplasm.’