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A Pilot Study of Galectin-3, HBME-1, and p27 Triple Immunostaining Pattern for Diagnosis of Indeterminate Thyroid Nodules in Cytology With Correlation to Histology.

Zhang L, Krausz T, DeMay RM - Appl. Immunohistochem. Mol. Morphol. (2015)

Bottom Line: Sensitivity is increased to 95.5% (0.751, 0.998), but specificity is decreased to 69.0% (0.490, 0.840), if Galectin3 and HBME1 are both used in combination as markers for malignancy.However, the level of specificity is increased to 86.2% (0.674, 0.955) and sensitivity remains high 100% (0.808, 1) if in addition, using the Galectin3/HBME1:p27 ratio (ratio ≥2 indicating malignancy) for 2 or 3 markers positive cases.Further studies and validation in different larger populations are warranted.

View Article: PubMed Central - PubMed

Affiliation: Departments of *Pathology, Cytopathology Division †Pathology, Surgical Pathology Division, The University of Chicago, Chicago, IL.

ABSTRACT
Indeterminate thyroid nodules form a heterogenous group of lesions that constitute 5% to 30% of thyroid cytology diagnoses. We introduce a triple immunostaining protocol for subtyping. Galectin-3, HBME-1, and p27 triple immunostaining, performed on destained cytology slides and formalin-fixed paraffin-embedded tissue, was developed and applied to 51 patients retrospectively with preoperative cytologic diagnoses of follicular lesion of undetermined significance (n=40), atypia of undetermined significance (n=6), and suspicious for follicular neoplasm (n=5). The malignant rate in this series was 43.1% (22/51). A hierarchal evaluation algorithm was generated based on digital image quantitation of triple-stained histologic sections, and applied to both cytology and histology specimens. Fifty of 51 cytology cases have triple staining validated by internal controls. In cytology specimens, the individual sensitivities and specificities of p27, Galectin3, and HBME1 for cancer with 95% confidence interval are: 86.2% (0.674, 0.955)/66.7% (0.431, 0.845); 77.3% (0.542, 0.913)/72.4% (0.525, 0.866); and 72.7% (0.496, 0.884)/93.1% (0.758, 0.988), respectively. Sensitivity is increased to 95.5% (0.751, 0.998), but specificity is decreased to 69.0% (0.490, 0.840), if Galectin3 and HBME1 are both used in combination as markers for malignancy. However, the level of specificity is increased to 86.2% (0.674, 0.955) and sensitivity remains high 100% (0.808, 1) if in addition, using the Galectin3/HBME1:p27 ratio (ratio ≥2 indicating malignancy) for 2 or 3 markers positive cases. Thus, the triple staining method on cytology slides and histology sections shows a similar sensitivity/specificity/positive predictive value/negative predictive value of 100.0%/86.2%/84.0%/100.0% and 95.5%/86.2%/84.0%/96.2%, respectively (P=0.92). Overall, p27 is the most frequent single positive marker (19/50, 38% in cytology), consistent with benign nature of most indeterminate thyroid nodules. Galectin-3 and HBME-1 colocalization (positive in the same cell) was demonstrated in thyroid cancer in 45.5% (10/22) of histology sections, but in none of the normal thyroid tissues and benign thyroid lesions. This supports the notion that synchronous activation of Galectin-3 and HBME-1 occurs in thyroid malignancy and is highly specific for malignancy. We have demonstrated the performance and pattern of triple immunostaining for subtyping indeterminate thyroid nodules. Further studies and validation in different larger populations are warranted.

No MeSH data available.


Related in: MedlinePlus

Illustration of triple-stained cases. Case #16 showed very subtle nuclear clearing and slightly overcrowded nuclei on cytology (Diff Quik). This patient has been aspirated 3 times with 2 AUS and 1 FLUS cytology diagnoses. Triple staining on cytology revealed coexpression of Galectin-3 and HBME-1 in the same cell with no p27 labeling. The histology diagnosis is PTC. Case #4 showed mixed macrofollicles and microfollicles with very scant colloid on cytology (Diff Quik). The cytology diagnosis is FLUS. Triple staining on cytology demonstrated colabeling of p27 and HBME-1 in the same cell. Histology diagnosis is multiple nodular goiter. Control staining from liquid-based preparation and 1 PTC formalin-fixed paraffin-embedded section is shown in the first column. Case #9 showed microfollicles with very rare trabecular architecture on cytology (Pap stain). The cytology diagnosis is FLUS and histology diagnosis is follicular adenoma. Triple staining highlights the nuclei of benign follicular cells in both cytology and histology. The vascular stromal cells are positive for Galectin-3 on histology. AUS indicates atypia of undetermined significance; FLUS, follicular lesion of undetermined significance; PTC, papillary thyroid carcinoma.
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Figure 1: Illustration of triple-stained cases. Case #16 showed very subtle nuclear clearing and slightly overcrowded nuclei on cytology (Diff Quik). This patient has been aspirated 3 times with 2 AUS and 1 FLUS cytology diagnoses. Triple staining on cytology revealed coexpression of Galectin-3 and HBME-1 in the same cell with no p27 labeling. The histology diagnosis is PTC. Case #4 showed mixed macrofollicles and microfollicles with very scant colloid on cytology (Diff Quik). The cytology diagnosis is FLUS. Triple staining on cytology demonstrated colabeling of p27 and HBME-1 in the same cell. Histology diagnosis is multiple nodular goiter. Control staining from liquid-based preparation and 1 PTC formalin-fixed paraffin-embedded section is shown in the first column. Case #9 showed microfollicles with very rare trabecular architecture on cytology (Pap stain). The cytology diagnosis is FLUS and histology diagnosis is follicular adenoma. Triple staining highlights the nuclei of benign follicular cells in both cytology and histology. The vascular stromal cells are positive for Galectin-3 on histology. AUS indicates atypia of undetermined significance; FLUS, follicular lesion of undetermined significance; PTC, papillary thyroid carcinoma.

Mentions: Representative triple Galectin-3/HBME-1/p27 immunocytochemical stains and immunohistochemical stains are illustrated in Figure 1 (see online supplementary Fig. 1, Supplemental Digital Content 1, http://links.lww.com/AIMM/A59, for more pictures and quantification of staining). Positive HBME-1 and Galectin-3 stains are shown in case #16 (AUS in cytology and diagnosis of PTC in histology), and the cells that are simultaneously positive for both markers are better visualized in cytology staining. Case #4 (FLUS in cytology and colloid nodule in histology) shows positive HBME-1 and p27 staining in the same cells, again better demonstrated in cytology preparation. Case #9 (SFN in cytology and FA in histology) is positive for p27 only. HBME-1 and Galectin-3 positive individually or in combination (case #16) indicates malignancy. p27 positive only (case #9) supports diagnosis of benign lesion. An algorithm is required for Galectin-3/HBME-1 and p27 dual-positive or triple-positive cases.


A Pilot Study of Galectin-3, HBME-1, and p27 Triple Immunostaining Pattern for Diagnosis of Indeterminate Thyroid Nodules in Cytology With Correlation to Histology.

Zhang L, Krausz T, DeMay RM - Appl. Immunohistochem. Mol. Morphol. (2015)

Illustration of triple-stained cases. Case #16 showed very subtle nuclear clearing and slightly overcrowded nuclei on cytology (Diff Quik). This patient has been aspirated 3 times with 2 AUS and 1 FLUS cytology diagnoses. Triple staining on cytology revealed coexpression of Galectin-3 and HBME-1 in the same cell with no p27 labeling. The histology diagnosis is PTC. Case #4 showed mixed macrofollicles and microfollicles with very scant colloid on cytology (Diff Quik). The cytology diagnosis is FLUS. Triple staining on cytology demonstrated colabeling of p27 and HBME-1 in the same cell. Histology diagnosis is multiple nodular goiter. Control staining from liquid-based preparation and 1 PTC formalin-fixed paraffin-embedded section is shown in the first column. Case #9 showed microfollicles with very rare trabecular architecture on cytology (Pap stain). The cytology diagnosis is FLUS and histology diagnosis is follicular adenoma. Triple staining highlights the nuclei of benign follicular cells in both cytology and histology. The vascular stromal cells are positive for Galectin-3 on histology. AUS indicates atypia of undetermined significance; FLUS, follicular lesion of undetermined significance; PTC, papillary thyroid carcinoma.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4530731&req=5

Figure 1: Illustration of triple-stained cases. Case #16 showed very subtle nuclear clearing and slightly overcrowded nuclei on cytology (Diff Quik). This patient has been aspirated 3 times with 2 AUS and 1 FLUS cytology diagnoses. Triple staining on cytology revealed coexpression of Galectin-3 and HBME-1 in the same cell with no p27 labeling. The histology diagnosis is PTC. Case #4 showed mixed macrofollicles and microfollicles with very scant colloid on cytology (Diff Quik). The cytology diagnosis is FLUS. Triple staining on cytology demonstrated colabeling of p27 and HBME-1 in the same cell. Histology diagnosis is multiple nodular goiter. Control staining from liquid-based preparation and 1 PTC formalin-fixed paraffin-embedded section is shown in the first column. Case #9 showed microfollicles with very rare trabecular architecture on cytology (Pap stain). The cytology diagnosis is FLUS and histology diagnosis is follicular adenoma. Triple staining highlights the nuclei of benign follicular cells in both cytology and histology. The vascular stromal cells are positive for Galectin-3 on histology. AUS indicates atypia of undetermined significance; FLUS, follicular lesion of undetermined significance; PTC, papillary thyroid carcinoma.
Mentions: Representative triple Galectin-3/HBME-1/p27 immunocytochemical stains and immunohistochemical stains are illustrated in Figure 1 (see online supplementary Fig. 1, Supplemental Digital Content 1, http://links.lww.com/AIMM/A59, for more pictures and quantification of staining). Positive HBME-1 and Galectin-3 stains are shown in case #16 (AUS in cytology and diagnosis of PTC in histology), and the cells that are simultaneously positive for both markers are better visualized in cytology staining. Case #4 (FLUS in cytology and colloid nodule in histology) shows positive HBME-1 and p27 staining in the same cells, again better demonstrated in cytology preparation. Case #9 (SFN in cytology and FA in histology) is positive for p27 only. HBME-1 and Galectin-3 positive individually or in combination (case #16) indicates malignancy. p27 positive only (case #9) supports diagnosis of benign lesion. An algorithm is required for Galectin-3/HBME-1 and p27 dual-positive or triple-positive cases.

Bottom Line: Sensitivity is increased to 95.5% (0.751, 0.998), but specificity is decreased to 69.0% (0.490, 0.840), if Galectin3 and HBME1 are both used in combination as markers for malignancy.However, the level of specificity is increased to 86.2% (0.674, 0.955) and sensitivity remains high 100% (0.808, 1) if in addition, using the Galectin3/HBME1:p27 ratio (ratio ≥2 indicating malignancy) for 2 or 3 markers positive cases.Further studies and validation in different larger populations are warranted.

View Article: PubMed Central - PubMed

Affiliation: Departments of *Pathology, Cytopathology Division †Pathology, Surgical Pathology Division, The University of Chicago, Chicago, IL.

ABSTRACT
Indeterminate thyroid nodules form a heterogenous group of lesions that constitute 5% to 30% of thyroid cytology diagnoses. We introduce a triple immunostaining protocol for subtyping. Galectin-3, HBME-1, and p27 triple immunostaining, performed on destained cytology slides and formalin-fixed paraffin-embedded tissue, was developed and applied to 51 patients retrospectively with preoperative cytologic diagnoses of follicular lesion of undetermined significance (n=40), atypia of undetermined significance (n=6), and suspicious for follicular neoplasm (n=5). The malignant rate in this series was 43.1% (22/51). A hierarchal evaluation algorithm was generated based on digital image quantitation of triple-stained histologic sections, and applied to both cytology and histology specimens. Fifty of 51 cytology cases have triple staining validated by internal controls. In cytology specimens, the individual sensitivities and specificities of p27, Galectin3, and HBME1 for cancer with 95% confidence interval are: 86.2% (0.674, 0.955)/66.7% (0.431, 0.845); 77.3% (0.542, 0.913)/72.4% (0.525, 0.866); and 72.7% (0.496, 0.884)/93.1% (0.758, 0.988), respectively. Sensitivity is increased to 95.5% (0.751, 0.998), but specificity is decreased to 69.0% (0.490, 0.840), if Galectin3 and HBME1 are both used in combination as markers for malignancy. However, the level of specificity is increased to 86.2% (0.674, 0.955) and sensitivity remains high 100% (0.808, 1) if in addition, using the Galectin3/HBME1:p27 ratio (ratio ≥2 indicating malignancy) for 2 or 3 markers positive cases. Thus, the triple staining method on cytology slides and histology sections shows a similar sensitivity/specificity/positive predictive value/negative predictive value of 100.0%/86.2%/84.0%/100.0% and 95.5%/86.2%/84.0%/96.2%, respectively (P=0.92). Overall, p27 is the most frequent single positive marker (19/50, 38% in cytology), consistent with benign nature of most indeterminate thyroid nodules. Galectin-3 and HBME-1 colocalization (positive in the same cell) was demonstrated in thyroid cancer in 45.5% (10/22) of histology sections, but in none of the normal thyroid tissues and benign thyroid lesions. This supports the notion that synchronous activation of Galectin-3 and HBME-1 occurs in thyroid malignancy and is highly specific for malignancy. We have demonstrated the performance and pattern of triple immunostaining for subtyping indeterminate thyroid nodules. Further studies and validation in different larger populations are warranted.

No MeSH data available.


Related in: MedlinePlus