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Immunohistochemical Detection of p63 and XIAP in Thymic Hyperplasia and Thymomas

Maoxin Wu MD, PhD, Katherine Sun MD, PhD, Joan Gil MD, Li Gan MD, David E. Burstein MD
DOI: http://dx.doi.org/10.1309/AJCPVTZ06DHKCOGP 689-693 First published online: 1 May 2009


We subjected 23 formalin-fixed, paraffin-embedded tissue blocks (11 cases of thymic hyperplasia and 12 thymomas [3 encapsulated, 8 with capsular invasion, and 1 atypical]) to incubation with monoclonal anti–X-linked inhibitor of apoptosis protein (XIAP) (BD Biosciences, San Jose, CA) and monoclonal anti-p63 (4A4, Santa Cruz, Santa Cruz, CA). Granular or heterogeneous cytoplasmic XIAP staining and nuclear p63 staining were considered positive. We compared thymic hyperplasia with thymoma and capsulated thymoma with thymoma with capsular invasion or atypia.

p63 was positive in virtually all thymic epithelial cells in hyperplasia and thymoma. XIAP was negative in all hyperplasia cases except one. Of 12 thymomas, 9 were XIAP+ with focal/weak to diffuse/strong positivity: 2 of 3 encapsulated and 7 of 8 thymomas with capsular invasion were XIAP+. One atypical thymoma was XIAP–. XIAP expression differed significantly between hyperplasia and thymoma (P = .0007) but not between capsulated and invasive thymomas (P = .3797).

p63 is consistently positive in nonneoplastic and neoplastic thymic epithelium. XIAP expression in thymoma suggests a possible role in the pathogenesis of thymoma and may be helpful in differentiating thymic hyperplasia from thymoma, especially in small biopsy specimens. However, the level of expression does not correlate with capsular invasion or atypia.

Key Words:
  • XIAP
  • p63
  • Thymic hyperplasia
  • Thymoma

Thymoma is defined as a neoplasm comprising cells that show differentiation toward keratin-containing, p63+ thymic epithelium, which manifest no more than moderate cytologic atypia.19 Although the World Health Organization (WHO) histological classification is helpful,10 distinguishing thymomas from nonneoplastic thymic tissue or thymic hyperplasia, particularly in biopsy specimens, can be difficult based on morphologic features alone. The significance of capsular invasion in establishing aggressive behavior has been recently challenged based on a meta-analysis of a large number of published cases.11 A recent study reported expression of X-linked inhibitor of apoptosis protein (XIAP) in normal human tissues, including thymus.12 Immunoexpression of XIAP in a number of tumors has been reported1319; however, there has not been any study comparing XIAP expression in thymic hyperplasia and thymomas. We report an immunohistochemical survey of XIAP and p63 in human thymic hyperplasia and thymomas, including cases with and without capsular invasion and atypia.

Materials and Methods

We selected 23 cases from the Mount Sinai Medical Center (New York, NY) pathology archives from 2004 to 2006, comprising 11 cases of thymic hyperplasia (3 cases of true thymic hyperplasia and 8 cases of thymus with lymphoid hyperplasia) and 12 thymomas, of which 3 (1 B2 and 2 AB) were encapsulated and 9 were invasive, including 8 (6 B1 and 2 AB) with capsular invasion and 1 with atypia (B3). The thymomas, initially classified based on the 2004 WHO histologic classification,10 were regrouped as encapsulated and invasive in an attempt to correlate tumor behavior with detection of XIAP and p63. All routinely stained slides were reviewed to confirm the diagnosis and classification.

A representative formalin-fixed, paraffin-embedded tissue block was selected from each case. For the study, 4-μm-thick sections were cut and subjected to citrate-based antigen retrieval and then incubated with monoclonal anti-XIAP (No. 610763, BD Biosciences, San Jose, CA), dilution 1:250 at 4°C for 72 hours; for p63, monoclonal anti-p63 (4A4, Santa Cruz, Santa Cruz, CA) diluted 1:4,000 in phosphate-buffered saline with 0.1% bovine serum albumin and 5% nonfat dry milk was incubated with the slides at room temperature for 16 hours. The slides were developed using EnVision+ reagents (DAKO, Carpinteria, CA) with diaminobenzidine as the chromogen. Squamous cell carcinoma and poorly differentiated colonic carcinoma were used as positive control samples for p63 and XIAP, respectively.

Granular or heterogeneous cytoplasmic staining for XIAP and nuclear staining for p63 were considered positive. Weak homogeneous staining for XIAP was considered negative. Slides were scored as 1+ (focal/weak), 2+ (moderate intensity and extent), or 3+ (strong and extensive).


Expression of p63 was uniformly strong (3+) in all cases of hyperplasia and thymoma Image 1A, Image 2A, Image 3A, and Image 4A. p63 nuclear staining was seen in nearly all epithelial cells. Most of the positive cells were scattered. Of the 11 cases of hyperplasia, 2 showed a focal nesting pattern (Image 1A). In Hassall corpuscles, p63 was negative in the center and weakly positive peripherally (Image 1A, inset). p63 positivity was noted sporadically in some germinal center lymphoid cells (5 cases). As in hyperplasia, p63 expression in all thymomas was diffusely strong and expressed in virtually all epithelial cells. The only difference in p63 expression between hyperplasia and thymoma was that p63 staining boldly highlighted the sheets (Image 2A) and the invasive (Image 3A) and pseudoglandular (Image 4A) patterns in thymomas.

Image 1

Expression of p63 and X-linked inhibitor of apoptosis protein (XIAP) in thymic hyperplasia. A, p63 positivity in thymic hyperplasia and in Hassall corpuscles (x200; inset, x400). B, Absence of XIAP staining in thymic hyperplasia (x200).

Image 2

Expression of p63 and X-linked inhibitor of apoptosis protein (XIAP) in thymoma. A, p63 positivity in encapsulated thymoma (x200). B, Focal XIAP positivity in encapsulated thymoma (arrows) (x200).

The detection of XIAP in the thymic lesions is described in Table 1, and representative photomicrographs are shown in Image 1B, Image 2B, Image 3B, and Image 4B. XIAP expression was virtually negative (Image 1B) in all cases of thymic hyperplasia in cortical and spindle-type epithelial cells except in 1 case, in which rare spindle-shaped cells were weakly stained for XIAP but were considered negative. In 3 of 6 thymic hyperplasia cases, the germinal center lymphoid cells were also positive for XIAP. No XIAP expression was noted in Hassall corpuscles. In contrast, 9 (75%) of 12 thymomas were XIAP+, ranging from focal/weak (Image 2B), to diffuse weak with focal strong (Image 3B), to regional strong (Image 4B). An unpaired t test comparing the XIAP score between thymic hyperplasia (n = 11) and thymoma (n = 12) revealed a highly significant difference (2-tailed P = .0007).

Image 3

Expression of p63 and X-linked inhibitor of apoptosis protein (XIAP) in thymoma. A, p63 positivity in invasive thymoma (* capsular invasion) (x200). B, Weak to focally strong XIAP positivity (arrows) in invasive thymoma (* capsular invasion) (x200).

Image 4

Expression of p63 and X-linked inhibitor of apoptosis protein (XIAP) in thymoma. A, p63 positivity in invasive thymoma (pseudoglandular pattern) (x200). B, XIAP positivity in invasive thymoma (pseudoglandular pattern) (x200).

View this table:
Table 1

More specifically, within the encapsulated cases, XIAP was expressed in the epithelial component of 2 WHO type AB thymomas but negative in the type B2 case. Among 8 thymomas with capsular invasion, the epithelial component of 5 of 6 type B1 cases was positive for XIAP with relatively high scores (range 1+ to 3+), and 2 type AB cases were positive but had the same score (1+) as the encapsulated type AB. The invasive type appeared to have more diffuse and regionally stronger expression of XIAP compared with the encapsulated type (Image 3B; Image 4B, pseudoglandular pattern). However, unpaired t test comparing the XIAP score between encapsulated thymomas (n = 3) and thymomas with capsular invasion or atypia (n = 9) revealed a nonsignificant 2-tailed P value of .3797.

Of interest, the positive expression of both markers in Image 4 was concentrated around the vasculature, creating a palisading or pseudoglandular staining pattern.


The present study demonstrated p63 positivity in virtually all thymic epithelial cells, including hyperplastic and neoplastic types. This result is in agreement with all previously published results.15 p63 was also weakly positive in the peripherally located nuclei of Hassall corpuscles but negative in the nuclei in the more differentiated cells in the center of Hassall corpuscles. This pattern is similar to that seen in keratinizing squamous pearls seen in squamous cell carcinoma and is consistent with the role of p63 in maintaining the basal cell phenotype. Overall, p63 staining seems to suggest that thymus epithelium is likely to share the origin or property of squamous and basal reserve cells and p63 positivity could be helpful in differentiating thymoma and thymic carcinoma from other thoracic tumors such as mesothelioma, which is negative for p6320,21 (and our unpublished data, 2004), and many adenocarcinomas.

XIAP, the most potent inhibitor of the apoptosis protein family, has been shown to be expressed in many different types of tumors and has been linked to more aggressive histopathologic and clinical behavior.1319 The statistically highly significant differential expression of XIAP seen in thymoma compared with thymic hyperplasia suggests a role in the pathogenesis of thymoma; although XIAP expression appeared to be stronger and more frequent in invasive thymoma compared with encapsulated thymoma, the difference was not statistically significant. This finding is compatible with the recent report of a meta-analysis of 2,451 published thymoma cases with a follow-up period of more than 5 years in which transcapsular invasion was found not to be a significant prognostic feature.11

Practically, XIAP immunoexpression could be a helpful tool in distinguishing thymomas from thymic hyperplasia and normal thymus and, perhaps, B1 thymoma from well-differentiated lymphocytic lymphoma. XIAP expression was also reported in interdigitating cells and macrophages (3+), medullary epithelium (2+), and Hassall corpuscles (3+) in normal thymus.12 These findings seem to contrast with the present study in which XIAP was found to be positive only in rare spindle cells but not in other elements in thymic hyperplasia. Differences in sources and concentrations of anti-XIAP antibodies may explain the discrepancy. Regardless of the difference in technique, both studies demonstrated XIAP negativity in the cortical epithelial cells of normal and hyper-plastic thymus.


Upon completion of this activity you will be able to:

  • list the types of cells in the thymus likely to be reactive with antibodies to p63.

  • define possible use of X-linked inhibitor of apoptosis protein (XIAP) as an immunohistochemical marker in thymic specimens.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 745. Exam is located at www.ascp.org/ajcpcme.


  • Supported by a generous bequest from the Estate of Hilda Leveen (Dr Burstein).


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