Claudin 6 is a positive marker for atypical teratoid/rhabdoid tumors

Abstract

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive pediatric brain tumors characterized by the presence of rhabdoid cells and negative immunostaining for INI1 (BAF47). Histogenesis is unknown and diagnosis can be challenging because of their extreme morphological and immunophenotypic heterogeneity. Currently no signature markers other than INI1 loss have been identified. To search for possible candidate proteins of interest in AT/RTs, Affymetrix GeneChip microarrays were utilized to investigate nine AT/RTs vs. 124 other tumor samples. The most distinctive gene identified was claudin 6 (CLDN6), a key component of tight junctions. CLDN6 showed moderate or higher mRNA expression in eight of nine AT/RTs, with little to no expression in 114 of 115 other tumors. Average expression was 38-fold higher in AT/RTs vs. other samples. Immunohistochemical (IHC) staining of 33 tumor specimens found positive membrane staining in seven of seven AT/RTs, and was negative in 26 of 27 other brain tumor samples. Notably, none of the 16 medulloblastomas/primitive neuroectodermal tumors showed IHC staining for CLDN6. IHC staining results closely matched the level of mRNA expression detected by microarray. CLDN6 may be a useful positive marker to help further identify AT/RTs for diagnostic and treatment purposes.

Figure 1

mRNA Expression of selected genes as assessed by microarray. Dot plots of normalized log 2 gene expression as measured by Affymetrix HG U133 Plus 2 Gene Chip microarray for selected genes, by tumor diagnostic group. For specific diagnoses and counts of tumors included in each diagnostic group, see Table 2. A. Ubiquitin specific peptidase 44 (USP44). B. Sal‐like 4 (SALL4). C. Claudin 6 (CLDN6). Abbreviations: AT/RT = atypical teratoid/rhabdoid tumor; CPC/CPP = choroid plexus carcinoma/choroid plexus papilloma; EPN = ependymoma; HGG = High‐grade glioma; LGA = low‐grade astrocytoma; MEN = meningioma; MPNST = malignant peripheral nerve sheath tumor; PNET/MED = primitive neuroectodermal and medulloblastoma tumors.

Figure 2

Expression of INI1 and claudin 6 (CLDN6) in tumor samples as assessed by Western blot. Equal amounts of tumor sample lysates were analyzed by Western blot for the proteins indicated. Beta‐actin was used as a loading control. Abbreviations: AT/RT = atypical teratoid/rhabdoid tumor; CPP = choroid plexus papilloma; EPN = ependymoma; LARGE =  large‐cell medulloblastoma; MED = classic medulloblastoma; pGBM =  pediatric glioblastoma.

Figure 3

CLDN6 immunostaining in pediatric brain tumors. Photomicrographs illustrating immunostaining results for claudin 6 (CLDN6), with light hematoxylin counterstain. A. Low‐power view illustrates that some atypical teratoid/rhabdoid tumors (AT/RTs) manifested diffuse immunostaining for CLDN6 throughout the tumor. AT/RT, case 404, original magnification 100×. B. Low‐power view illustrates that other AT/RTs had immunostaining for CLDN6 in large islands of cohesive cells. AT/RT, case 514, original magnification 100×. C. Low‐power view illustrates that one AT/RT case possessed only small groups of cells that were immunoreactive for CLDN6 in a field of otherwise negatively staining cells. AT/RT, case 605, original magnification 100×. D. Medium‐power view illustrates that positive immunostaining occurred more often in rhabdoid cells and in cells with larger cytoplasmic volume. AT/RT, case 517, original magnification 400×. E. Medium‐power view illustrates that CLDN6 immunostaining could be found in cells without obvious rhabdoid features. AT/RT, case 605, original magnification 600×. F. Medium‐power view shows absence of CLDN6 immunostaining in some rhabdoid cells (arrow). AT/RT, case 605, original magnification 600×. G. High‐power view highlights the strong membranous pattern of CLDN6 staining in AT/RTs. AT/RT, case 515, original magnification 1000×. H. High‐power view emphasizes that even in immunoreactive cells with smaller cytoplasmic volume the pattern of immunostaining for CLDN6 remained membranous. AT/RT, case 517, original magnification 1000×. I. Medium‐power view illustrates that almost all non‐AT/RT cases had negative immunoreactivity for CLDN6. Large cell medulloblastoma, original magnification 400×.

Figure 4

CLDN6 and INI1 protein expression in choroid plexus papilloma (CPP) case 451. Photomicrographs illustrating immunostaining results for claudin 6 (CLDN6) and INI1, with light hematoxylin counterstain. A. Medium‐power view illustrates that a few areas of positive immunoreactivity for CLDN6 could be found in one choroid plexus papilloma. Staining is limited to the membranes at the lumenal and lateral cell surfaces. CPP, case 451, original magnification 400×. B. Medium‐power view from the same tumor area as seen in Figure 4A, immunostained for INI1 (BAF47), showing complete retention of INI1 nuclear protein. CPP, case 451, original magnification 400×.