title: Carbonic anhydrase 4 and crystallin α-B immunoreactivity may distinguish benign from malignant thyroid nodules in patients with indeterminate thyroid cytology.
abstract: BACKGROUND
  Thyroid nodules are present in 19%-67% of the population and carry a 5%-10% risk of malignancy. Unfortunately, fine-needle aspiration biopsies are indeterminate in 20%-30% of patients, often necessitating thyroid surgery for diagnosis. Numerous DNA microarray studies including a recently commercialized molecular classifier have helped to better distinguish benign from malignant thyroid nodules. Unfortunately, these assays often require probes for >100 genes, are expensive, and only available at a few laboratories. We sought to validate these DNA microarray assays at the protein level and determine whether simple and widely available immunohistochemical biomarkers alone could distinguish benign from malignant thyroid nodules.
  
  
  METHODS
  A tissue microarray (TMA) composed of 26 follicular thyroid carcinomas (FTCs) and 53 follicular adenomas (FAs) from patients with indeterminate thyroid nodules was stained with 17 immunohistochemical biomarkers selected based on prior DNA microarray studies. Antibodies used included galectin 3, growth and differentiation factor 15, protein convertase 2, cluster of differentiation 44 (CD44), glutamic oxaloacetic transaminase 1 (GOT1), trefoil factor 3 (TFF3), Friedreich Ataxia gene (X123), fibroblast growth factor 13 (FGF13), carbonic anhydrase 4 (CA4), crystallin alpha-B (CRYAB), peptidylprolyl isomerase F (PPIF), asparagine synthase (ASNS), sodium channel, non-voltage gated, 1 alpha subunit (SCNN1A), frizzled homolog 1 (FZD1), tyrosine related protein 1 (TYRP1), E cadherin, type 1 (ECAD), and thyroid hormone receptor associated protein 220 (TRAP220). Of note, two of these biomarkers (GOT1 and CD44) are now used in the Afirma classifier assay. We chose to compare specifically FTC versus FA rather than include all histologic categories to create a more uniform immunohistochemical comparison. In addition, we have found that most papillary thyroid carcinoma could often be reasonably distinguished from benign disease by morphological cytology findings alone.
  
  
  RESULTS
  Increased immunoreactivity of CRYAB was associated with thyroid malignancy (c-statistic, 0.644; negative predictive value [NPV], 0.90) and loss of immunoreactivity of CA4 was also associated with malignancy (c-statistic, 0.715; NPV, 0.90) in indeterminate thyroid specimens. The combination of CA4 and CRYAB for discriminating FTC from FA resulted in a better c-statistic of 0.75, sensitivity of 0.76, specificity of 0.59, positive predictive value (PPV) of 0.32, and NPV of 0.91. When comparing widely angioinvasive FTC from FA, the resultant c-statistic improved to 0.84, sensitivity of 0.75, specificity of 0.76, PPV of 0.11, and NPV of 0.99.
  
  
  CONCLUSIONS
  Loss of CA4 and increase in CRYAB immunoreactivity distinguish FTC from FA in indeterminate thyroid nodules on a thyroid TMA with an NPV of 91%. Further studies in preoperative patient fine needle aspiration (FNAs) are needed to validate these results.
authors.affiliation: Division of General Surgery, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ. Electronic address: davidoto@rutgers.edu.
authors.forename: Tomer
authors.initials: T
authors.lastname: Davidov
authors.affiliation: Mount Sinai Hospital, New York, NY.
authors.forename: Michael
authors.initials: M
authors.lastname: Nagar
authors.affiliation: St. Lukes Health Network in Bethlehem, PA.
authors.forename: Malca
authors.initials: M
authors.lastname: Kierson
authors.affiliation: Department of Pathology, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ.
authors.forename: Marina
authors.initials: M
authors.lastname: Chekmareva
authors.affiliation: Rutgers Cancer Institute of New Jersey, New Brunswick, NJ.
authors.forename: Chunxia
authors.initials: C
authors.lastname: Chen
authors.affiliation: Rutgers Cancer Institute of New Jersey, New Brunswick, NJ.
authors.forename: Shou-en
authors.initials: SE
authors.lastname: Lu
authors.affiliation: Rutgers Cancer Institute of New Jersey, New Brunswick, NJ.
authors.forename: Yong
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Division of General Surgery, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ.
authors.forename: Victoriya
authors.initials: V
authors.lastname: Chernyavsky
authors.affiliation: Division of General Surgery, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Potdevin
authors.affiliation: Division of General Surgery, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ.
authors.forename: Dena
authors.initials: D
authors.lastname: Arumugam
authors.affiliation: Department of Pathology, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ.
authors.forename: Nicola
authors.initials: N
authors.lastname: Barnard
authors.affiliation: Division of General Surgery, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ.
authors.forename: Stanley
authors.initials: S
authors.lastname: Trooskin
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D030741:Carbonic Anhydrase IV / Q000378:metabolism*; D002277:Carcinoma / Q000175:diagnosis* / Q000201:enzymology / Q000473:pathology; D002291:Carcinoma, Papillary; D003937:Diagnosis, Differential; D005260:Female; D006801:Humans; D008297:Male; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000201:enzymology / Q000473:pathology; D016606:Thyroid Nodule / Q000175:diagnosis* / Q000201:enzymology / Q000473:pathology; D046888:Tissue Array Analysis; D038203:alpha-Crystallin B Chain / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; C059215:CRYAB protein, human; D038203:alpha-Crystallin B Chain; D030741:Carbonic Anhydrase IV
keywords: CA4; CRYAB; Follicular neoplasm; Follicular thyroid carcinoma; Indeterminate cytology; Molecular markers; Thyroid