Background: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia and congenital anomalies, notably skeletal defects. DBA is known in the majority of cases to be caused by haploinsufficiency of one of the small or large ribosomal protein (rp) subunits. In addition, there is a cancer predisposition in DBA. Indeed, somatic and germline rp mutations have been associated with a number of malignancies. In DBA patients one of the common malignancies is osteogenic sarcoma (OS). It is proposed that selective pressure caused by faulty translation may lead to clonal expansion of cells with acquired interdicting mutations leading to cancer.

Purpose: To quantitate the incidence of cancer in patients with DBA and to examine osteoblast formation in vitro using a mouse embryonic stem (mES) cell model haploinsufficient for ribosomal protein Rps19 to identify putative bone defects that may promote tumorigenesis.

Methods: The DBA Registry of North America (DBAR), the largest established patient cohort with prospective follow-up since 1991, was interrogated to ascertain the incidence of cancer in DBA. For in vitro studies, mES cells were differentiated towards osteoblasts using 1,25-dihydroxy-vitamin D3, dexamethasone, ascorbic acid and β-glycerophosphate for 10 days. qRT-PCR and histologic stains (alizarin red, alcian blue and alkaline phosphatase) were used to assess osteoblast production in wild type and Rps19+/- cultures.

Results: The DBAR reported the first quantitative assessment of cancer incidence in DBA. The significantly elevated observed to expected ratios were 287, 36, 33 and 28 respectively, for myelodysplastic syndrome (MDS), colon carcinoma, OS and acute myeloid leukemia (AML). As of Nov 2015 (N = 751), with a median patient age of 20 years, 11 patients with gastrointestinal carcinoma, 5 with OS, 2 with AML and 8 with MDS are reported to the DBAR. The incidence of gastrointestinal carcinoma and OS is highly significant in this young population. We elected to study OS due to the skeletal defects and poor growth encountered in DBA patients. Using mES cells induced to differentiation towards osteoblasts, we observed reduced mineralization and therefore, bone formation, by alizarin red staining in Rps19+/- cells. Furthermore, rp haplo-insufficient cultures displayed a significant increase in alcian blue staining, a marker for chondrogenesis; these results suggest that regulation of osteochondrogenic potential was altered. In support of this phenotype, qRT-PCR analyses revealed that Rps19 haploinsufficiency induced abnormal expression of the master transcription factors and p53 involved in osteoblast and chondrocyte development.

Conclusions: The most common cancers in DBA are AML, OS and adenocarcinoma of the colon. With regards to OS, we are now creating a conditional Rps19 CRISPR murine model, in order to clarify the contribution of both rp and p53 in the etiology of OS.

Citation Format: Lionel Blanc, Brian Dulmovits, Julien Papoin, Jessica Kang, Eva Atsidaftos, Jeffrey M. Lipton, Adrianna Vlachos. Ribosomal protein haploinsufficiency in Diamond-Blackfan anemia potentially leads to osteogenic sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2457.