Somatic mosaicism in patients with Fanconi anaemia: Proposal of alternative tissue for inconclusive diagnoses.

INTRODUCTION: Fanconi anaemia (FA) is a rare genetic disorder marked by progressive bone marrow failure, chromosomal fragility, and increased cancer susceptibility. Laboratory diagnosis includes chromosomal instability test and mutation investigation. A total of 15%-25% of all patients may have somatic mosaicism, characterized by two distinct haematopoietic cell populations, one resistant and one sensitive to agents that induce chromosomal breakage, which complicates the diagnosis by a high incidence of reverted cells leading to inconclusive or false-negative results. The study aimed to evaluate the use of bone marrow stromal mesenchymal cells (BM-MSCs) as an alternative, non-haematopoietic tissue for diagnosis. METHODS: Bone marrow mesenchymal stromal cells from 12 patients with positive diepoxybutane (DEB) tests were cultivated and analysed by cytogenetics and mutation investigation. RESULTS: The DEB test was performed at 0.1 and 0.01 µg/ml concentrations, with an index ranging from 0.24 to 1.00. At higher concentration, the metaphases number was lower, probably due to toxicity. Regarding the molecular investigation, all the mutations previously found in peripheral blood were identified on BM-MSC. CONCLUSION: This study demonstrated the possibility of using BM-MSCs as an alternative tissue for cytogenetic and molecular investigation. Future tests using an intermediate DEB concentration may lead to an optimal protocol that could be non-toxic to cells but provides conclusive results.

The Prognostic Role of CD8+ T Lymphocytes in Childhood Adrenocortical Carcinomas Compared to Ki-67, PD-1, PD-L1, and the Weiss Score.

Adrenocortical carcinoma (ACC) is a rare disease among children. Our goal was to identify prognostic biomarkers in 48 primary ACCs from children (2.83 ± 2.3 y; mean age ± SD) by evaluating the tumor stage and outcome for an age of diagnosis before or after 3 years, and association with ACC cluster of differentiation 8 positive (CD8+) cytotoxic T lymphocytes (CD8+-CTL) and Ki-67 immunohistochemical expression (IHC). Programmed death 1(PD-1)/Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) in ACC was analyzed in a second, partially overlapping cohort (N = 19) with a similar mean age. All patients and control children were carriers of the germline TP53 R337H mutation. Survival without recurrence for less than 3 years and death unrelated to disease were excluded. Higher counts of CD8+-CTL were associated with patients diagnosed with ACC at a younger age and stage I, whereas a higher percentage of the Ki-67 labeling index (LI) and Weiss scores did not differentiate disease free survival (DFS) in children younger than 3 years old. No PD-1 staining was observed, whereas weakly PD-L1-positive immune cells were found in 4/19 (21%) of the ACC samples studied. A high CD8+-CTL count in ACC of surviving children is compelling evidence of an immune response against the disease. A better understanding of the options for enhancement of targets for CD8+ T cell recognition may provide insights for future pre-clinical studies.