KARYOTYPE ANALYSIS OF ADVANCED THERAPY SPECIMENS IN A ROUTINE CYTOGENETIC LABORATORY

Abstract

Objectives: Cytogenetic study is a prerequisite for the therapeutic use of cellular/advanced therapy products required by regulatory agencies (ANVISA). This aims to guarantee the safety and quality of the cells for clinical use, preventing the use of extensive cell manipulations with chromosomal alterations. In this study we evaluated the frequency of cytogenetic abnormalities in samples from advanced therapy services received at the laboratory between March/2015 and January/2023, also analyzing the cellular origin of the samples and the purpose of the test (treatment, research). Material and methods: Samples of cells to be used as an advanced therapy product were evaluated by conventional karyotyping, performed according to the standard technique for cultures of adhered and suspension cells, GTW banding and reported according to ISCN standards. Results: 167 samples were evaluated, some of them coming from the same individual or cell lineage due to the use of cells in different culture passages. 101 (60.5%) samples were received from our own institution and 66 (39.5%) samples were sent by external services. The evaluated cell type [and tissue origin] were: 95 (56.9%) mesenchymal [30 bone marrow (18.0%), 17 muscle (10.2%), 14 palate periosteum (8.4%), 14 adipocytes (8.4%), 13 umbilical cord (7.8%), 7 tooth pulp (4.2%)], 17 (10.2%) NK cells [umbilical cord blood], 12 (7.2%) chondrocytes [cartilage], 11 (6.6%) cell lineage [7 HEK293-3F6 (4.2%), 3 K562 (1.8%) and 1 OCI-AML2 (0.6%)], 10 (6.0%) iPSC [urine epithelial cells], 8 (4.8%) lymphocytes [peripheral blood], 7 (4.2%) renal cells [kidney], 6 (3.6% ) CAR-T [peripheral blood] and 1 (0.6%) fibroblast. The application of cells was primarily designed for treatment [120 (71.9%)]: 39 (23.4%) chondral lesions and osteoarthritis, 37 (22.2%) urinary incontinence, 21 (12.6%) neoplasms hematological, 14 (8.4%) COVID lung injury and 9 (5.4%) cytomegalovirus ‒ post HSCT; and for research [47 (28.1%)]: 19 (11.4%) culture validation and cell expansion, 11 (6.6%) cell authenticity, 10 (6.0%) neurological processes associated with Down syndrome and 7 (4.2%) chronic kidney disease. Among the samples designated for treatment, 87 (72.5%) samples presented a normal karyotype result, 5 (4.2%) normal karyotype with non-clonal alterations, 9 (7.5%) absence of metaphases and 19 (15.8%) karyotype with clonal alterations. Among the clonal alterations detected, 10 (52.6%) cases had trisomies, 7 (36.8%) structural alterations, 1 (5.3%) tetraploidy and 1 (5.3%) loss of the Y chromosome. Among the samples designated for research, all samples that showed complex karyotype 10 (66.7%) were cells originating from a cell lineage for the purpose of evaluating cell authenticity; the others had trisomies 3 (20.0%) and structural alterations 2 (13.3%). Discussion: Trisomies are the most frequently seen alteration in cultures from advanced therapy. It is assumed that chromosome gain can lead to selective advantage and clonal expansion. Cell lineages can undergo different genetic alterations throughout their passages, an inherent characteristic of immortalized cells. Conclusion: Extensive manipulation of cells and advanced therapy products can contribute to instability, which may increase the risk of cytogenetic alterations and tumorigenic potential, suggesting that products with clonal cytogenetic alterations should be discarded.