Molecular RB1 gene detection of minimal residual disease in metastatic retinoblastoma

Proc Amer Assoc Cancer Res, Volume 47, 2006 4349 Purpose: Cure of metastatic retinoblastoma (RB) depends on chemotherapy with or without radiation to first reduce the systemic and central nervous system tumor load, followed by marrow-ablative chemotherapy and marrow rescue with stem cell transplant. We propose that the search for RB1 mutant alleles may be useful for assessing the completeness of the remission at the molecular level. This also allows monitoring for the absence of contaminating tumor cells in the stem cells harvested prior to transplant. Method: We developed a highly sensitive allele-specific PCR assay, to detect RB1 mutant alleles in the tumor cells, which are not present in the normal cells, in two children with metastatic but non-germline RB. Child A, age 2.6 years, had tumor infiltrating the orbit and extending intracranially with tumor cells in the cerebrospinal fluid (CSF), 1.6 years after enucleation of one eye for unilateral RB. Child B, age 2.5 years, had tumor infiltrating soft tissues and bone at the base of the skull with bone marrow involvement, 0.5 years after enucleation of one eye for unilateral RB. Results: RB1 mutations of both recurrent tumors were first identified by sequence analysis. The tumor of Child A was homozygous for R251X, and the tumor of Child B was homozygous for R358X. The allele-specific PCR assay was then used to detect the tumor RB1 mutant alleles. Both children had no tumor RB1 mutant alleles detectable in the normal cells in their peripheral blood. In Child A, the assay resolution was adequate to detect the R251X mutation at as low as 1:12,800 dilution, but serial CSF samples during and after chemotherapy showed no detectable R251X mutant. In Child B, the R358X mutation could be detectable at the 1:500 dilution level in the initial bone marrow sample, but serial bone marrow samples during and after chemotherapy showed no detectable R358X mutant. The peripheral blood stem cells harvested from both children also did not show their specific RB1 mutant alleles. Absence of RB1 mutant signals in post-chemotherapy CSF or bone marrow samples and the harvested stem cells supported the suitability of both children proceeding to consolidation with autologous stem cell transplant aimed at curing their metastatic RB. Conclusion: Molecular assaying for the tumor-specific RB1 mutations is complementary to conventional cytological and immunohistochemical evaluations of RB involvement of the CSF and bone marrow. This novel diagnostic tool is very sensitive for assessing minimal residual disease, detecting extraocular RB tumor cells in numbers much lower than possible by conventional methods. Molecular management holds particular promise for evaluating the efficacy of systemic treatment strategies for metastatic RB.

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