GBM is a heterogeneous disease and stratification of patients based upon predicted response to specific agents may be critical for the success of targeted therapeutics. Because current diagnostic neuropathology relies on the analysis of FFPE material, broad applicability of a new assay currently requires it to be paraffin-based. Using FFPE material, we used FISH to identify tumors with deletions of RB1 and IHC to identify tumors with loss of RB1 protein expression. In combination, the 2 methods provided information regarding potential mechanisms of RB1 alteration and RB1 status. Our data demonstrate loss of RB1 protein in roughly 10% of GBMs, consistent with previous estimates (11
Loss of heterozygosity of the RB1 gene on chromosome 13q is a relatively common event in astrocytoma (11
). In a subset of tumors, alteration of the remaining RB1 allele and subsequent inactivation of RB1 disrupts the p16-CDK4/6-RB1 pathway (8
). The most common alteration of this pathway, homozygous CDKN2A deletion, results in suppression of RB1 protein function via elevated CDK4/6 activity. CDK4/6 is an attractive therapeutic target in tumors lacking CDKN2A/p16 function, but not in tumors lacking RB1 protein. Using IHC on clinical tumor samples we stratified patients into an RB1-intact group that was expected to benefit from CDK4/6 inhibitors and a group with loss of RB1 expression that would not be expected to benefit. IHC was a reliable method to detect RB1 status as predicted by our analysis of RB1 copy number by FISH and large-scale genomic data from TCGA, including expression array, array CGH, and sequencing (11
). These data support the use of IHC to identify RB1 status for potential stratification of patients for clinical trials. Furthermore, they confirm the robust nature of the TCGA data and demonstrate its usefulness in the development and validation of clinical tests for patient stratification. In fact, expression array data available on the TCGA Data Portal (downloaded October 24, 2011) predict that 47 of 424 (11.1%) tumors analyzed have loss of RB1 expression. This estimate is consistent with our data showing loss of RB1 protein expression in 9 of 85 (10.6%) GBMs.
Two of 33 tumors were identified with hemizygous gene deletion by FISH but absent protein expression. Typical of tumor suppressor genes, multiple mechanisms may explain complete loss of RB1 protein in the setting of a hemizygous deletion, including point mutation, deletion, or alterations in transcriptional or translational regulation (27
). Consistent with this line of reasoning, sequencing of one of these cases demonstrated a novel nonsense point mutation in the remaining intact copy of RB1 predicted to result in a premature stop codon. Sequencing data was not available from the second case. We did not identify any tumors with loss of RB1 protein expression in the absence of gene deletion, but such occurrences have been described in other cancers (29
). Our data indicate that determination of RB1 protein status in tumor tissue reliably identifies the majority of cases with homozygous RB1 gene inactivation. It is possible, however, that in some cases epitope expression may persist in the context of a truncated, dysfunctional protein, yielding a false negative result. Such results are anticipated to be very infrequent.
We did not identify an association between RB1 status and survival in either our subset of tumors analyzed by IHC or in the 170 TCGA tumors analyzed by expression array. Although some reports have suggested such an association in GBM (31
), RB1 may have a more direct correlation with prognosis in lower-grade astrocytomas including World Health Organization grade II and III tumors (14
). Our finding that alterations in RB1 are more common in the proneural subtype of GBM may reflect similarities between genetic alterations in a subset of GBMs and lower-grade astrocytomas. Future studies will address this possibility and determine the utility of RB1 FISH and IHC for patient stratification in both low- and high-grade astrocytomas.