Discrimination Power Assessment of STR Genotyping in Parentage Investigation

OBJECTIVE Nowadays, the application of DNA-typing in laboratory medicine is increasing rapidly for paternity/maternity disputes. The goal of this study was to evaluate the use of polymorphic microsatellite marker DNA analysis and to establish this analysis as the method of choice for parentage investigations.SUBJECTS AND METHODS Among 708 civil parentage tests addressed to our Laboratory previously examined for HLA class I (-A*,-B*,-Cw*), and class II (-DRB1*,-DQB1*,-DPB1*) alleles using PCR-SSOP and/or PCR-SSP methodologies, a cohort of 50 cases (137 individuals) of disputed parentage was selected. In these cases DNA-typing was generated from co-amplification of 15 autosomal STR DNA markers (D3S1358, HUMTH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, HUMCSF1PO, Penta D, HUMvWA, D8S1179, HUMTPOX, HUMFGA and the sex determining Amelogenin marker HUMAMEL), using fragment analysis methodology.RESULTS The evaluation of the results showed that 15 out of 50 cases, were sufficient for exclusion of fatherhood by both approaches (HLA and STRs). In all remaining 35 non-excluded cases, the PI value using HLA genotyping ranged from 76 to 6,452,794, whereas using aSTR genotyping ranged from 15,173 to 9.2 x 1010. In one non-excluded motherless case the alleged father showed one genetic discrepancy with the child at D21S11 locus, due to a mutation event.CONCLUSION The use of DNA-typing with 15 aSTR loci for parentage testing provides an accurate and high-sensitivity method which is simpler to perform and more rapid than an accepted standard technology, such as HLA genotyping. The analysis of aSTR loci offers a highly discriminating test suitable for trio paternity testing, increasing the W rate in comparison to HLA genotyping. Nevertheless, when a mutation event occurs in motherless cases, combination of HLA and STR polymorphisms offers high level of information, and also diminishes the possibility of false exclusion due to aSTRs mutations

Repetitively Administered Low-Dose Donor Lymphocyte Infusion for Prevention of Relapse after Allogeneic Stem Cell Transplantation in Patients with High-Risk Acute Leukemia

Background: Patients with high-risk acute leukemia have a high risk of relapse after allogeneic stem cell transplantation (allo-SCT). In an effort to reduce the relapse rate, various therapeutic methods have been implemented into clinical practice. Among them, prophylactic donor lymphocyte infusion (pro-DLI) has shown significant efficacy. However, the widespread application of pro-DLI has been restricted mostly due to concerns regarding the development of graft versus host disease (GVHD). In the present study, we tested the safety and efficacy of a novel method of prophylactic-DLI based by repetitive administration of low lymphocyte doses. Methods: DLI was administered to patients with high-risk acute leukemia at a dose of 2 × 106/kg CD3-positive cells. DLI at the same dose was repeated every two months for at least 36 months post-allo-SCT, or until relapse or any clinical or laboratory feature suggested GVHD, whichever occurred first. Forty-four patients with a median age of 53 years (range 20–67) who underwent allo-SCT between 2011 and 2020 were included in our study. Thirty-three patients with high-risk acute myeloid leukemia (AML) and 11 with high-risk acute lymphoblastic leukemia (ALL) after allo-SCT from a matched sibling (MSD, no = 38 pts) or a matched-unrelated donor (MUD, no = 6 pts) received pro-DLI. Twenty-three patients were in CR1, all with unfavorable genetic features; 12 patients were in CR2 or beyond; and 9 patients had refractory disease at the time of transplant. Ten out of 23 patients in CR1 had detectable minimal residual disease (MRD) at the time of allo-SCT. Disease risk index (DRI) was high and intermediate in 21 and 23 patients, respectively. Conditioning was myeloablative (MAC) in 36 and reduced intensity (RIC) in 8 patients, while GVHD prophylaxis consisted of cyclosporine-A in combination with low-dose alemtuzumab in 39 patients or with low-dose MTX in 5 patients, respectively. Results: Thirty-five patients completed the scheduled treatment and received a median of 8 DLI doses (range 1–35). Fifteen out of 35 patients received all planned doses, while DLI was discontinued in 20 patients. Reasons for discontinuation included GVHD development in nine, donor unavailability in seven, disease relapse in three, and secondary malignancy in one patient, respectively. Nine patients were still on treatment with DLI, and they received a median of four (range 2–12) doses. Fourteen percent of patients developed transient grade-II acute GVHD while 12% developed chronic GVHD post-DLI administration. Acute GVHD was managed successfully with short course steroids, and four out of five patients with cGVHD were disease-free and off immunosuppression. With a median follow-up of 44 months (range 8–120), relapse-free (RFS) and overall survival (OS) were 74%, (95% CI, 54–87%) and 78%, (95% CI, 58–89%) respectively, while the cumulative incidence of non-relapse mortality (NRM) was 13% (95% CI, 4–28%). The cumulative incidence of relapse in patients with intermediate and high DRI is 7% and 15%, respectively. Conclusion: Prolonged—up to three years—low-dose pro-DLI administered every two months is safe and effective in reducing relapse rate in patients with high-risk acute leukemia. The low-dose repetitive administration DLI strategy reduced the risk of DLI-mediated GVHD, while the prolonged repeated administration helped in preventing relapse, possibly by inducing a sustained and prolonged immunological pressure on residual leukemic cells. This novel strategy deserves testing in larger cohort of patients with high-risk acute leukemia