Alterations of RB1 gene in embryonal and alveolar rhabdomyosarcoma: special reference to utility of pRB immunoreactivity in differential diagnosis of rhabdomyosarcoma subtype

Abstract

Purpose: Rhabdomyosarcoma (RMS), which is the most common pediatric soft tissue sarcoma, is classified into two major histologic subtypes, embryonal RMS (ERMS) and alveolar RMS (ARMS). RMS is occasionally reported to be the second neoplasm of hereditary retinoblastoma. Osteosarcoma is known as the most common second neoplasm of hereditary retinoblastoma, and tumorigenesis of osteosarcoma has been proven in previous studies to be related to the RB gene (RB1) alteration. Therefore, there might be a correlation between the tumorigenesis of RMS and RB1 alteration.

Methods: We examined the RB protein (pRB) expression and RB1 alteration such as allelic imbalance (gain or loss) and homozygous deletion, using immunohistochemistry, microsatellite makers, and quantitative real-time PCR in 57 sporadic RMS.

Results: Allelic imbalance was more frequently detected in ERMS (13/27), than in ARMS (3/20) (P = 0.04). Homozygous deletion on the protein-binding pocket domain of RB1 was found in 6 of 27 ERMS and in 2 of 20 ARMS (P = 0.24). Furthermore, immunohistochemical pRB labeling indexes (LI) in 31 ERMS (median value, 31%) were significantly reduced in comparison with those observed in 26 ARMS (median value, 85%) (P < 0.0001).

Conclusions: Our results support the assertion that tumorigenesis of RMS may be associated with RB1 alteration especially in ERMS, as previously reported for osteosarcoma. As for the RB pathway, each subtype of RMS may have a different tumorigenesis. In addition, immunohistochemical pRB LI may have the potential to be a useful ancillary tool in the differential diagnosis of RMS subtypes.

Fig. 1

Estimation of allelic imbalance (AI) from the output of the ABI 310 genetic analyzer: chromatograms showing the genotypes of two normal-tumor tissue pairs. AI is evident in case A (2 years old, embryonal rhabdomyosarcoma): AI index = (T2/T1)/(N2/N1) <0.7. The tumor in case B (24 years old, alveolar rhabdomyosarcoma) is unaltered: AI index >0.7

Fig. 2

Boxplot of retinoblastoma protein (pRB) expression. The median values of the pRB labeling index in embryonal and alveolar rhabdomyosarcoma are 31 and 85%, respectively (P < 0.0001)

Fig. 3

Immunohistochemical staining of retinoblastoma protein (pRB) expression. a The numbers of tumor cells with a positive nuclear reaction are reduced; the labeling index is 37% (1 year old, embryonal rhabdomyosarcoma). b Most tumor cells show the immunoreaction in the nuclei; the labeling index is 88% (5 years old, alveolar rhabdomyosarcoma)

Fig. 4

Solid variant of alveolar rhabdomyosarcoma (1 month old, orbit). a Although the characteristic alveolar structures are not present, cellular nests are still separated by fibrovascular septa. b PAX3-FKHR fusion gene transcript is detected in this case. c Most tumor cell nuclei show retinoblastoma protein (pRB) immunopositivity