Short Tandem Repeat PCR Research Articles

Isolation of a Low Number of Sperm Cells from Female DNA in a Glass-PDMS-Glass Microchip via Bead-Assisted Acoustic Differential Extraction.

We report an improved separation method for the isolation of sperm cells from dilute, "large volume" samples containing female DNA using bead-assisted acoustic trapping. In an enclosed glass-PDMS-glass (GPG) resonator, we exploit a three-layer microfluidic architecture to generate "trapping nodes" in ultrasonic standing waves. We investigate the dependence of trapping efficiency on particle concentration for both sperm cells and polymeric beads. After determination of the critical concentration of polymeric beads required to seed the trapping event, sperm cells in dilute solution are trapped as a result of the enhanced secondary radiation force (SRF). Sperm-cell-containing samples with volumes up to 300 μL and cell concentrations as low as ∼10 cells/μL are amenable to effective trapping in the presence of an abundance of female DNA in solution. Complete processing of samples is accomplished with separation of the female and male fractions within 15 min. We demonstrate that the collected fractions are amenable to subsequent DNA extraction, short tandem repeat PCR, and the generation of STR profiles for the isolated sperm cells.

Short Tandem Repeat-Polymerase Chain Reaction (STR-PCR) with Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) Method Using for Chimerism Analysis.

Recently molecular chimerism analysis after allogeneic hematopoietic stem cell transplantation (AHSCT) has become more important. The use of quantitative chimerism methods aims to assess the kinetics of engraftment to determine graft rejection and failure or relapse of the underlying disease after AHSCT. An accurate and sensitive determination of chimerism status is mandatory after AHSCT. This study aimed to compare two chimerism methods: Multiplex Short Tandem Repeat-Polymerase Chain Reaction (STR-PCR) and quantitative Real Time-PCR (qRT-PCR). Thirty-nine blood samples at +28 day were used to extract DNA. Most patients had been diagnosed with acute leukemia (74.3 %) and other hematological diseases. For Multiplex STR-PCR method, PCR products were separated on an ABI 3130 Genetic Analyzer (Applied Biosystem, USA) and for qRT-PCR, an ABI 7500 (Applied Biosystem, USA) Plate System Real Time Analyzer was used to determine the quantification of chimerism per-centage. Of the 39 analyzed samples, 82% concordant chimerism results were detected for both STR-PCR and qRT-PCR methods. Ten mixed chimerisms (MC) were found by qRT-PCR whereas of only 3 MC cases were detected by STR-PCR. In the discordant group of 7 by qRT-PCR, we observed Acute Graft versus Host Disease (aGVHD). Three MC cases that were detected by both STR- and qRT-PCR methods died because of relapse. The quantitative chimerism method along with multiplex STR-PCR method is important for early detection of MC. qRT-PCR methods can be valuable options in the prevention of graft failure and assisting with fast and early treatment strategies for patients undergoing AHSCT.

Impact of Day 14 Peripheral Blood Chimerism after Allogeneic Hematopoietic Stem Cell Bone Transplantation on the Treatment Outcome of Non-Malignant Disease.

BackgroundThe impact of early peripheral blood chimerism on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unclear. We aimed to determine whether day 14 peripheral blood chimerism after allo-HSCT predicts outcomes in patients with non-malignant diseases.MethodsData from 56 patients who received allo-HSCT between April 2007 and March 2016 were retrospectively analyzed. Chimerism was evaluated using short-tandem repeat polymerase chain reaction, with mixed chimerism (MC) defined as greater than 1% recipient cells which was further categorized into low-level MC (> 1% and < 15% of recipient-derived cells) and high-level MC (≥ 15% of the recipient-derived cells).ResultsThirty-six patients showed complete donor chimerism (CC), 14 low-level MC, and 6 high-level MC at day 14 post-transplant. The estimated 5-year event-free survival (EFS) was higher in the CC or low-level MC groups than in the high-level MC group (86.1% vs. 71.4% vs. 33.3%; P = 0.001). In BM or peripheral blood stem cell (BM/PBSC) transplants, the 5-year EFS was higher in the CC or low-level MC group than in the high-level MC group (93.1% vs. 66.7% vs. 0%; P < 0.001). However, in cord blood transplants, the 5-year OS and EFS according to the day 14 peripheral blood chimerism did not reach statistical significance.ConclusionAlthough CC is not always necessary after allo-HSCT for non-malignant diseases, our data suggest that day 14 peripheral blood chimerism may predict outcomes in patients with non-malignant diseases who underwent BM/PBSC transplants.

Identification of the Skeletal Remains of a Murder Victim in Forensic Casework by DNA Typing: A Case Report

We report the identification of skeletal remains using DNA technology. DNA was isolated from severely decomposed human remains using pre-extraction decalcification followed by optimized phenol/chloroform DNA extraction procedure. Additionally DNA from the reference samples of the relatives was extracted using the Automate Express DNA extraction system. Quantifiler™ Human DNA quantification kit was used for DNA quantification. AmpFl STR Identifiler plus PCR amplification kit was used to simultaneously amplify 15 autosomal Short Tandem Repeat (STR) loci alongwith the gender specific locus amelogenin.

Evaluation of TIM-3+ LSCs Is a Highly Sensitive MRD Monitoring Strategy to Predict AML Relapse after Allogenic Stem Cell Transplantation

Relapse of acute myeloid leukemia (AML) is a critical problem in clinics. Particularly, the prognosis of patients who relapsed after allogeneic stem cell transplantation (allo-SCT) is still poor. Self-renewing leukemic stem cells (LSCs), which are mainly enriched within CD34+CD38- fraction, cause re-growth of leukemia and eventually lead to recurrence. Therefore, evaluation of residual LSCs is crucial to estimate the efficacy of therapies including allo-SCT. As we previously described, T-cell immunoglobulin mucin-3 (TIM-3) is a useful marker for discrimination of normal and malignant HSCs. We, therefore, prospectively evaluated the frequencies of LSCs at multiple time points and assessed the validity of TIM-3 as a minimal residual disease (MRD) marker in the setting of allo-SCT.50 AML patients who had been detected CD34+CD38-TIM-3+ LSCs in BM sample at least once before SCT were analyzed. All 50 patients underwent allo-SCT at Fukuoka Blood and Marrow Transplantation Group (FBMTG)-related hospitals from July 2015 to May 2018. 43 out of 50 patients achieved complete donor chimerism confirmed by short tandem repeat PCR (STR-PCR) at the time of engraftment (day 15-36), and these 43 patients was prospectively evaluated the frequencies of residual CD34+CD38-TIM-3+ LSCs using multi-color flow cytometry. Median age of the evaluated 43 AML patients at SCT was 44.8 years (28-67). 26 patients underwent SCT on disease and 17 patients at hematological CR. Relapse-free survival (RFS) was calculated from the time of SCT until last follow-up or documentation of relapse.In each timing (at diagnosis, relapse, engraftment, etc.), we tracked the frequencies of TIM-3+ cells within CD34+CD38- fraction in BM. Of note, the proportion of CD34+CD38- fraction was 0.016 [0.0056 - 0.0267] % of BMMNC at engraftment. Then, according to the frequency of TIM-3+ cells within CD34+CD38- fraction, we classified patients into 3 groups; ‘high’ (> 90 % of CD34+CD38- cells were positive for TIM-3, n=5), ‘intermediate’ (61-90 % of CD34+CD38- cells were positive for TIM-3, n=11), and ‘low’ (< 60 % of CD34+CD38- cells were positive for TIM-3, n=27). This classification revealed that the recurrence rate within the observation period (median 334.4 days) was 100 % (5 of 5) in ‘high’ group, 45.4 % (5 of 11) in ‘intermediate’ group and 14.8 % (4 of 27) in ‘low’ group. Relapse-free survival was 83 (72-92) days in ‘high’ group, 368 (56-869) days in ‘intermediate’ group and 482.6 (45-1029) days in ‘low’ group, respectively (p<0.01) (Figure 1). Of note, all of 5 patients in ‘high’ group exhibited hematological relapse within 100 days. Thus, our new classification using TIM-3 as LSC-specific marker could isolate the patients at high risk for early relapse in allo-SCT settings even if they were considered as hematological CR and maintained complete donor chimerism assessed by clinically available methods (three-color FACS, STR-PCR, FLT3-ITD status and fusion gene specific qPCR) at the same timepoint. It suggests that monitoring of LSCs using TIM-3 should be a more sensitive and versatile strategy to predict early relapse of AML than aberrant surface markers monitoring by multi-color FACS because conventional FACS did not detect MRD population at engraftment while our strategy detected (we could detect LSCs (CD34+CD38-TIM-3+ fraction) within BMMNC in 0.0053 [0 - 0.012] %), and, the cases whose aberrant markers could be traced were only 28 cases of 43 in this study.We also confirmed that the identical driver mutations were detected at both the initial diagnosis and relapse using whole exome sequencing (WES) of purified LSCs. As a typical example, WES detected CEBPA mutation (c.11dupG, 72.7 % and 50.0 %) and WT1 mutation (c.1091_1092insTTGTACGGTC, 41.9 % and 39.5 %) in a single patient. Additionally, we also validated that purified TIM-3+ LSCs at engraftment harbored the identical mutations by amplicon sequencing. It indicates that CD34+CD38-TIM-3+ cells should represent LSCs throughout the clinical course, from diagnosis to relapse.In summary, TIM-3 expression represents the clones of functional LSCs are involved in relapse. Evaluation of TIM-3+ LSCs by multi-color FACS should be a highly sensitive strategy to predict relapse in clinical allo-SCT settings, and might enable us to appropriately intervene to overcome the poor clinical outcomes of AML. [Display omitted] DisclosuresAkashi:Taiho Pharmaceutical: Research Funding; Novartis pharma: Research Funding; Pfizer: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eisai: Research Funding; Celgene: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; sanofi: Research Funding; MSD: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Chugai Pharma: Research Funding; Eli Lilly Japan: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi-kasei: Research Funding.

OR29. Higher percent recipient chimerism in bone marrow than peripheral blood is risk for relapse

Aim In patients after allogeneic stem cell transplant, chimerism is monitored on peripheral blood (PB). Occasionally, bone marrow (BM) samples are tested for chimerism and results are often similar. We investigated the significance of results that differ between PB and BM. Methods Retrospective review of post-allogeneic stem cell transplant patients, who were tested for chimerism on both PB and BM (total cells, unfractionated) within 7 days interval. The % recipient difference between PB and BM was compared with relapse/remission status (BM biopsy results, cytogenetics, molecular testing, CBC, and clinical follow-up documents. For patients with multiple time points of concurrent BM and PB results, only the first point was included in this evaluation. Results Concurrent BM and PB chimerism results were compared in 74 patients. Median age at transplant was 54 years (range: 3–76 years). Date of PB and BM sample post-transplant was median 207 days (range: 27–3102). Test methods were XY FISH (N = 9), short tandem repeat (N = 55) and quantitative PCR (N = 10). Diagnoses were myeloproliferative/dysplastic disease (N = 61), aplastic anemia (N = 2), lymphoma/lymphocytic leukemia (N = 11). % Recipient was higher in BM than PB (difference: Δ ⩾ 2 % ) in 11 patients, and similar ( Δ 2 % ) in 63 patients. Of the 11 patients with Δ ⩾ 2 % , disease relapse was detected in 5 patients at BM collection and two more patients within the following 2 months (total N = 7/11). Of the 63 patients with Δ 2 % , disease relapse was detected in 2 patients at BM collection and five more patients in the following 2 months (total N = 7/63). In the patients with Δ ⩾ 2 % compared to the patients with Δ 2 % , relapse was higher at BM collection (p = 0.0005 by Fisher exact) and cumulatively in the following 2 months (p = 0.000004, chi-square). Mixed chimerism status (recipient ⩾5%) in PB was observed in 4/11 patients with Δ ⩾ 2 % and 9/63 patients with Δ 2 % , not statistically different (p = 0.76). Mixed chimerism was associated with relapse in the patients with Δ 2 % (p = 0.022, chi-square), but not in patients with Δ ⩾ 2 % (p = 0.55, Fisher exact). Conclusions Higher % recipient in BM than in PB indicates risk of concurrent/imminent relapse, regardless of PB mixed/complete chimerism status. This status may reflect increasing mixed chimerism.

Anti-CD36 Mediated Platelet Transfusion Refractoriness and Related Cases After Stem Cell Transplantation

To analyse the cases of platelet transfusion refractoriness after received HLA-matched unrelated donor hematopoietic stem cell transplantation, to analyze and identify the phenotype and genotype of CD36 in both the patient and stem cell donor, as well as the characteristic of antibody induced platelet transfusion refractoriness, and to analyse the efficacy of matched CD36-deficiency platelets transfusions. The CD36 expression on platelet and monocyte was analyzed by flow cytometry (FCM) in both patient and donor. Polymerase chain reaction sequence-based typing (PCR-SBT) was used to analyze the exons sequence of CD36 and HPA. Fast monoclonal antibody-specific immobilization of platelet antigen (F-MAIPA) and FCM were used to identify platelet antibodies in the patient. Short tandem repeat polymerase chain reaction (STR-PCR) was applied to monitor engraftment evidence. The platelet level was monitored. CD36- deficiency donor's platelets were selected from CD36- deficiency donor blood bank. The donor was CD36 positive and the patient was typed I CD36 deficiency. The anti-CD36 antibody was identified in patient's serum (after transplantation), while the HLA and HPA-related antibodies were excluded. Sequence analysis of CD36 exon in the patient showed Exon 6 -1G>C(Change in splicing site) homozygote, which was a novel CD36 mutation. STR, HPA and CD36 of the patient (complete chimerism) were conversed to that of donor gene types on day 18 after allo-HSCT. The positive CD36 expression on platelet and monocyte in the patient was observed on day 96 after allo-HSCT. The patient showed the platelet transfusion refractoriness which was significantly improved after platelets transfusions from CD36 deficiency donors. Stem cell transplants resulted in anti-CD36 and caused platelet transfusion refractoriness, that was first reported in China. To ensure the efficacy of platelet transfusion, the CD36-deficiency patient should receive CD36 deficiency platelets for transfusion.

Droplet Digital PCR-Based Chimerism Analysis for Primary Immunodeficiency Diseases.

In the current study, we aimed to accurately evaluate donor/recipient or male/female chimerism in samples from patients who underwent hematopoietic stem cell transplantation (HSCT). We designed the droplet digital polymerase chain reaction (ddPCR) for SRY and RPP30 to detect the male/female chimerism. We also developed mutation-specific ddPCR for four primary immunodeficiency diseases. The accuracy of the male/female chimerism analysis using ddPCR was confirmed by comparing the results with those of conventional methods (fluorescence in situ hybridization and short tandem repeat-PCR) and evaluating dilution assays. In particular, we found that this method was useful for analyzing small samples. Thus, this method could be used with patient samples, especially to sorted leukocyte subpopulations, during the early post-transplant period. Four mutation-specific ddPCR accurately detected post-transplant chimerism. ddPCR-based male/female chimerism analysis and mutation-specific ddPCR were useful for all HSCT, and these simple methods contribute to following the post-transplant chimerism, especially in disease-specific small leukocyte fractions.

Fuchs' Endothelial Corneal Dystrophy and RNA Foci in Patients With Myotonic Dystrophy.

PurposeThe most common cause of Fuchs' endothelial corneal dystrophy (FECD) is an intronic CTG repeat expansion in TCF4. Expanded CUG repeat RNA colocalize with splicing factor, muscleblind-like 1 (MBNL1), in nuclear foci in endothelium as a molecular hallmark. Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG repeat expansion in the 3′-untranslated region (UTR) of DMPK. In this study, we examine for RNA-MBNL1 foci in endothelial cells of FECD subjects with DM1, test the hypothesis that DM1 patients are at risk for FECD, and determine prevalence of TCF4 and DMPK expansions in a FECD cohort.MethodsUsing FISH, we examined for nuclear RNA-MBNL1 foci in endothelial cells from FECD subjects with DM1. We examined 13 consecutive unrelated DM1 patients for FECD using slit-lamp and specular microscopy. We genotyped TCF4 and DMPK repeat polymorphisms in a FECD cohort of 317 probands using short-tandem repeat and triplet repeat-primed PCR assays.ResultsWe detected abundant nuclear RNA foci colocalizing with MBNL1 in endothelial cells of FECD subjects with DM1. Six of thirteen DM1 patients (46%) had slit-lamp and specular microscopic findings of FECD, compared to 4% disease prevalence (P = 5.5 × 10-6 ). As expected, 222 out of 317 (70%) FECD probands harbored TCF4 expansion, while one subject harbored DMPK expansion without prior diagnosis of DM1. ConclusionsOur work suggests that DM1 patients are at risk for FECD. DMPK mutations contribute to the genetic burden of FECD but are uncommon. We establish a connection between two repeat expansion disorders converging upon RNA-MBNL1 foci and FECD.

P070 A post-transplant chimerism study protocol that incoporated both STR and QPCR methods

Aim To monitor post-stem cell transplant chimerism status, most clinical laboratories use commercial kits for short tandem repeat (STR) or quantitative real-time PCR (qPCR). We present a protocol that combines both systems to maximize monitoring performance. Methods PowerPlex Fusion STR kit (Promega) was used to genotype 22 autosomal STR markers. Donor (%) was assigned as an average from five informative loci (Types 1 and 2). Fragment analysis used 3130xl Genetic Analyzer (Applied Biosystems) and GeneMapper software (Thermo Fisher Scientific). KMRType kit (GenDx) was used for genotyping 29 insertion/deletion markers (4-color multiplex) on QuantStudio 12K Flex Real-Time PCR instrument (Thermo Fisher). Two informative loci (insertion in recipient -deletion in donor) selected were used to determine minor allele (%) (one-color) by KMRTrack kit (GenDx) on the Roche LightCycler480 Real-Time PCR instrument (KMRTrack). The average from the two loci was assigned as minor allele (%). Results The average number of STR informative loci for a patient-donor pair was 18.3 (range: 16–21) for unrelated (N = 30) and 9.0 (range: 6–16) for related (N = 30). The number of qPCR informative loci was 6.0 (range: 4–9) for unrelated (N = 28) and 3.5 (range: 3–4) for related (N = 2). In qPCR, sensitivity increases when more DNA is used per reaction and surpasses STR sensitivity of 3–5% when >2 ng DNA is used per reaction. We validated the qPCR method using 183 different donor-recipient-marker combinations and 14 ng whole blood DNA/reaction, which achieved sensitivity of 0.5%. Correlation coefficient (r) of intended % donor (mixed DNA) vs. assay-determined was 0.999 by STR (N = 12 for range: 5–95% donor) and 0.91 by qPCR (range: 0.5–10% minor allele). Average %CV was similar at low minor allele range; STR (29.6% for 5% donor detection) vs. qPCR (average 27.8% for detecting 0.5%–10% minor allele). At high minor allele range, %CV decreased for STR (CV Conclusions The combination of STR and qPCR can improve a post-transplant monitoring protocol. STR (higher precision) is used immediately after transplantation when high recipient (minor) alleles are present. Testing will be switched to qPCR (higher sensitivity) once complete chimerism is achieved.

The allele combinations of three loci based on, liver, stomach cancers, hematencephalon, COPD and normal population: A preliminary study

The purpose of this study was to examine the specific allele combinations of three loci connected with the liver cancers, stomach cancers, hematencephalon and patients with chronic obstructive pulmonary disease (COPD) and to explore the feasibility of the research methods. We explored different mathematical methods for statistical analyses to assess the association between the genotype and phenotype. At the same time we still analyses the statistical results of allele combinations of three loci by difference value method and ratio method. All the DNA blood samples were collected from patients with 50 liver cancers, 75 stomach cancers, 50 hematencephalon, 72 COPD and 200 normal populations. All the samples were from Chinese. Alleles from short tandem repeat (STR) loci were determined using the STR Profiler plus PCR amplification kit (15 STR loci). Previous research was based on combinations of single-locus alleles, and combinations of cross-loci (two loci) alleles. Allele combinations of three loci were obtained by computer counting and stronger genetic signal was obtained. The methods of allele combinations of three loci can help to identify the statistically significant differences of allele combinations between liver cancers, stomach cancers, patients with hematencephalon, COPD and the normal population. The probability of illness followed different rules and had apparent specificity. This method can be extended to other diseases and provide reference for early clinical diagnosis.

Polymorphisms of Estrogen Metabolism-Related Genes and Prostate Cancer Risk in Two Populations of African Ancestry.

BackgroundEstrogens are thought to play a critical role in prostate carcinogenesis. It has been suggested that polymorphisms of genes encoding enzymes involved in estrogen metabolism are risk factors for prostate cancer. However, few studies have been performed on populations of African ancestry, which are known to have a high risk of prostate cancer.ObjectiveWe investigated whether functional polymorphisms of CYP17, CYP19, CYP1B1, COMT and UGT1A1 affected the risk of prostate cancer in two different populations of African ancestry.MethodsIn Guadeloupe (French West Indies), we compared 498 prostate cancer patients and 565 control subjects. In Kinshasa (Democratic Republic of Congo), 162 prostate cancer patients were compared with 144 controls. Gene polymorphisms were determined by the SNaPshot technique or short tandem repeat PCR analysis. Logistic regression was used to estimate adjusted odds ratios (OR) and 95% confidence intervals (CI).ResultsThe AA genotype and the A allele of rs4680 (COMT) appeared to be inversely associated with the risk of prostate cancer in adjusted models for both Afro-Caribbean and native African men. For the A allele, a significant inverse association was observed among cases with low-grade Gleason scores and localized clinical stage, in both populations.ConclusionsThese preliminary results support the hypothesis that polymorphisms of genes encoding enzymes involved in estrogen metabolism may modulate the risk of prostate cancer in populations of African ancestry.

Does Day 14 Peripheral Blood Chimerism after Allogeneic Hematopoietic Stem Cell Transplantation Predict Treatment Failure in Children with Non-Malignant Disease?

The impact of early time peripheral blood chimerism on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unclear. This study was aimed to identify if day 14 peripheral blood chimerism is associated with transplant outcomes in children with non-malignant disease. Data from 52 patients who received allo-HSCT between October 2006 and April 2015 at Samsung Medical Center were retrospectively analyzed. Chimerism was evaluated using short-tandem repeat polymerase chain reaction, with mixed chimerism defined as greater than 1% of recipient cells. Event was defined as death, primary engraftment failure, late rejection, and progression of primary disease. A total of 52 patients underwent 56 transplantations and their median follow-up duration was 28 months (range, 1-91). Of 56 transplants, 24 (42.9%) showed mixed chimerism (MC) and 32 (57.1%) showed complete donor chimerism (CC) at day 14 post-transplant. The estimated event-free survival (EFS) at 2-year was higher in day 14 CC group as compared with MC group (80.5% vs. 53.3%, P = 0.047) while their 2-year overall survivals were similar (80.5% vs. 72.8%, P = 0.711). Among 34 transplants achieving bone marrow CC at 1 mo post-transplant (28 CC and 6 MC at day 14), those with day 14 peripheral blood CC showed better EFS than those with MC (81.4% vs. 20.8%, P= 0.026). Although CC is not always necessary after allo-HSCT for non-malignant disease, our data suggest that early peripheral blood chimerism may serve as a predictor of transplant outcomes in children with non-malignant disease.

Cytomegalovirus and Effect on Early Chimerism in Patients with Myeloid Disorders Undergoing Stem Cell Transplantation Using Reduced Toxicity Ablative Conditioning Regimen

Cytomegalovirus (CMV) remains a significant cause of morbidity after allogeneic hematopoietic stem cell transplantation (SCT). The influence of CMV on the chimerism in reduced toxicity ablative conditioning SCT in myeloid disorders is ill defined. A recent report published in Blood by Sellar et al showed that in patients who received alemtuzumab-based regimen, the group of patients who were recipient positive (R+)/ Donor negative (D-) had CMV-specific T cells that are exclusively of recipient origin and significantly influenced the chimerism status toward recipients. To explore the impact of seropositivity and seronegativity of CMV in recipients and donors on early chimerism, we undertook a retrospective analysis of patients with myeloid disorders who received four days of fludarabine and busulfan with or without anti-thymocyte globulin (ATG) at our center in the last 10 years.Methods: Chimerism assay was performed using a quantitative fluorescence-based short tandem repeat-polymerase chain reaction (STR-PCR) with capillary electrophoresis for PCR product resolution.Results: 42 patients were identified and included in the study. All patients received fludarabine (40 mg/m2/day x 4 doses), busulfan (3.2mg/kg/dose IV x 4 doses). Of these 42 patients, 25 had anti-thymocyte globulin. There were 28 male and 14 female patients with a median age of 62 years (range 48-74yrs). Median time to follow up was 8 months (0.8-54 months). Disease risk was considered advanced in 21 patients, intermediate in 4 and early in 17. Median blast number at time of SCT was 5%. Stem cell source included peripheral blood in all patients. There were no primary graft failures. Total recipient cell chimerism showed increase or persistence of recipient chimerism in 5/11 (45%) of R+D- vs 2/6 (33.3%) of R-D- in the group of patients who received ATG, p=1.0, with a mean of recipient chimerism at day 100 of 20.4% in the R+D- group compared to a mean of 17% in the R-D- group. In the group who did not get ATG, recipient chimerism persistence or increase was not that different between the R+D- patients 3/4 (75%) compared to 4/4 (100%) in patients who were seronegative for CMV (R-), p=1.0. The mean of recipient chimerism at day 100 in the R+D- no ATG group was 23.25% with a median of 12% while the mean and median at day 100 in the R- no ATG group were 35.25% and 19.5% respectively (p=0.573).When looking at the persistence or increase in recipient chimerism in the group of patients who were R+D-, in those who got ATG it was 45% increase vs 75% increase in those who did not get ATG (p=0.569) with a median of 12% vs 0% respectively (p=0.49). Also increase or persistence of recipient chimerism was 33.3 % in patients who were R- and got ATG vs 100% in R- no ATG patients (p=0.076)with median at day 100 of 0 vs 19.5% (p=0.098).Conclusion: In this small cohort from a single center, we found that in patients with myeloid disorders who received reduced toxicity ablative conditioning regimen, the group of patients who received ATG, there was no statistically significant increase in recipient chimersim in the R+D- group compared to R-D- group. This is different from what Sellar et al found in a small group of patients who received alemtuzumab. These results may indicate a difference between ATG and alemtuzumab in the effect of CMV seropositivity and negativity on the recipient chimerism, which need to be studied further in a larger retrospective or prospective study. This is especially important in myeloid disorders since persistent or increase in recipient chimerism may identify high-risk patient cohorts who may benefit from additional therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Relationship between gene polymorphisms and prostate cancer risk.

To investigate the relationship between genetic factor and prostate cancer (Pca) risk and the possible cause in it. The polymorphisms of cytochrome P450 family 17 (CYPl7) rs743572, p27 V109G and androgen receptor (AR) gene CAG repeat length in peripheral blood from 70 cases and 70 controls were detected through the polymerase chain reaction-restriction fragment length polymorphism technique or short tandem repeat-polymerase chain reaction technique. Then, according to the results of case-control study, the recombinant plasmids containing the wild/mutant p27 gene were constructed and transfected Pca LNcap cells. After 24 and 72h of transfection, the cell proliferative activity was determined by MTT method, cell cycle distribution and apoptosis was detected by flow cytometry, and the expression level of bcl-2, caspase-3 and p27 protein was determined by Western-blot. In three target polymorphisms, only p27 V109G polymorphism was related to Pca risk (P=0.030, OR=0.202, 95% CI=0.042-0.973). Pca risk of p27-109G allele was lower than-109V allele (P=0.006, OR=0.285, 95% CI=0.110-0.737). Cells transfected with wild/mutant p27 gene both showed the higher cells apoptosis rate and the lower cell proliferative activity than mock cells (P<0.05 or 0.01), the regulatory effect of mutant p27 on cell proliferation and apoptosis was stronger than the wild p27 (P<0.05). p27-109G allele that could cause higher p27 protein expression than-109V allele in LNcap cells, maybe is the protective factor of Pca.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR

BackgroundChimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. MethodsSNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). ResultsDroplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. ConclusionThe droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation

Analysis of hematopoietic chimerism after allogeneic stem cell transplantation represents a crucial method to evaluate donor-cell engraftment. Whereas sensitivity of classical approaches for chimerism monitoring is limited to ≥1%, quantitative polymerase chain reaction (qPCR)-based techniques readily detect one patient cell in >1,000 donor cells, thus facilitating application of chimerism assessment as a surrogate for minimal residual disease. However, due to methodologic specificities, qPCR combines its high sensitivity with limited resolution power in the state of mixed chimerism (e.g., >10% patient cells). Our aim was to overcome this limitation by employing a further development of qPCR, namely digital PCR (dPCR), for chimerism analysis. For proof-of-principle, we established more than 10 dPCR assays detecting Indel polymorphisms or Y-chromosome sequences and tested them on artificial cell mixtures and patient samples. Employing artificial cell mixtures, we found that dPCR allows exact quantification of chimerism over several orders of magnitude. Digital PCR results proved to be highly reproducible (deviation<5%), particularly in the "difficult" range of mixed chimerism. Excellent performance of the new assays was confirmed by analysis of multiple retrospective blood samples from patients after allogeneic stem cell transplantation, in comparison with established qPCR (14 patients) and short-tandem repeat PCR (4 patients) techniques. Finally, dPCR is easy to perform, needs only small amounts of DNA for chimerism assessment (65ng corresponds to a sensitivity of approximately 0.03%), and does not require the use of standard curves and replicate analysis. In conclusion, dPCR represents a very promising method for routine chimerism monitoring.

High Rates of Early Donor Chimerism in Intermediate and Poor Risk Patients Undergoing Stem Cell Transplantation Using Reduced Toxicity Ablative Conditioning Regimen Incorporating Busulfan Pharmacokinetics

Abstract Reduced toxicity ablative conditioning regimens are increasingly used for allogeneic stem cell transplantation (allo-SCT). The impact of early donor cell chimerism on outcomes of T cell–replete reduced-intensity conditioning SCT in myeloid disorders is ill defined. To explore the impact of measuring busulfan pharmacokinetics in conditioning regimens on early donor chimerism, we undertook a retrospective analysis of patients with myeloid disorders who received four days of fludarabine and busulfan with or without measuring busulfan pharmacokinetics at our center in the last 10 years. Methods: Chimerism assay was performed using a quantitative fluorescence-based short tandem repeat– polymerase chain reaction (STR-PCR) with capillary electrophoresis for PCR product resolution. Results: Thirty patients were identified and included in the analysis. All patients were conditioned with fludarabine (40 mg/m2/day x 4 doses), busulfan (3.2mg/kg/dose IV x 4 doses). Of these thirty patients, 7 had busulfan pharmacokinetics measured. There were 21 male and 9 female patients with a median age of 62 years (range 48–72yrs). Median time to follow up was 13.3 months. Diagnoses included AML (N=17), MDS (N=10), MPN (N=1), CMML (N=2). Disease risk was considered advanced in 17 patients, intermediate in 3 and early in 10. All patients in the busulfan pharmacokinetic group had advanced disease except one had early disease. Regarding cytogenetics, all patients in the busulfan pharmacokinetics group had high or intermediate risk cytogenetics. Median blast number at time of SCT was 5%. Stem cell source included peripheral blood in all patients. Eleven patients received matched related donor (MRD) SCT, 15 had matched unrelated donor (MUD) SCT and 4 had mismatched unrelated donor (MMUD) SCT. Prophylaxis of GVHD consisted of tacrolimus and methotrexate in MRD, and tacrolimus, methotrexate and anti-thymocyte globulin in MUD and MMUD. All patients engrafted neutrophils and platelets promptly (median 13 and 14 days, respectively). There were no primary graft failures. Total Donor cell Chimerism analysis in the Busulfan pharmacokinetics group showed 100% donor at both time points (days 30, 100) in all patients except in one who relapsed at day 30 (85.7%). While in the non-pharmacokinetics group only 7 out of 23 (30%) patients had complete chimerism at day 30 and day 100. Complete donor chimerism at day 100 in the non-pharmacokinetics group was 47.8% compared to 85.7% in the pharmacokinetic group (p=0.18). Ten out of 23 patients (43.5%) in the non-pharmacokinetics group had decreasing donor chimerism by day 100, while in the pharmacokinetic group only one patient (14%) who relapsed had decreasing donor chimerism by day 100 with an odds ratio of 0.241 (95% Confidence Interval =0.025-2.357; p-value=0.22). None developed sinusoidal obstructive syndrome. Conclusion: In this small cohort of consecutive patients from a single center, we found that patients with myeloid disorders who received fludarabine busulfan for 4 days incorporating busulfan pharmacokinetics had a trend for higher rates of early complete donor chimerism and less decreasing donor chimerism by day100, although not statistically significant, despite having intermediate or high risk disease at time of SCT. Longer follow up is needed for our patients to see if there is effect on relapse or survival but previous studies have showed that low or decreasing donor chimerism early after SCT is an independent risk factor for relapse and impaired survival. This is especially important in myeloid disorders. Busulfan pharmacokinetics may help target better level for inducing early total donor chimerism and donor chimerism may identify high-risk patient cohorts who may benefit from additional therapeutic interventions. These results should be interpreted with caution given the retrospective design of the study and the small number of patients. Disclosures No relevant conflicts of interest to declare.

High Rates of Early Donor Chimerism in Intermediate and Poor Risk Patients Undergoing Stem Cell Transplantation Using Reduced Toxicity Ablative Conditioning Regimen Incorporating Busulfan Pharmacokinetics

▪Reduced toxicity ablative conditioning regimens are increasingly used for allogeneic stem cell transplantation (allo-SCT). The impact of early donor cell chimerism on outcomes of T cell–replete reduced-intensity conditioning SCT in myeloid disorders is ill defined. To explore the impact of measuring busulfan pharmacokinetics in conditioning regimens on early donor chimerism, we undertook a retrospective analysis of patients with myeloid disorders who received four days of fludarabine and busulfan with or without measuring busulfan pharmacokinetics at our center in the last 10 years.Methods: Chimerism assay was performed using a quantitative fluorescence-based short tandem repeat– polymerase chain reaction (STR-PCR) with capillary electrophoresis for PCR product resolution.Results: Thirty patients were identified and included in the analysis. All patients were conditioned with fludarabine (40 mg/m2/day x 4 doses), busulfan (3.2mg/kg/dose IV x 4 doses). Of these thirty patients, 7 had busulfan pharmacokinetics measured. There were 21 male and 9 female patients with a median age of 62 years (range 48–72yrs). Median time to follow up was 13.3 months. Diagnoses included AML (N=17), MDS (N=10), MPN (N=1), CMML (N=2). Disease risk was considered advanced in 17 patients, intermediate in 3 and early in 10. All patients in the busulfan pharmacokinetic group had advanced disease except one had early disease. Regarding cytogenetics, all patients in the busulfan pharmacokinetics group had high or intermediate risk cytogenetics. Median blast number at time of SCT was 5%. Stem cell source included peripheral blood in all patients. Eleven patients received matched related donor (MRD) SCT, 15 had matched unrelated donor (MUD) SCT and 4 had mismatched unrelated donor (MMUD) SCT. Prophylaxis of GVHD consisted of tacrolimus and methotrexate in MRD, and tacrolimus, methotrexate and anti-thymocyte globulin in MUD and MMUD.All patients engrafted neutrophils and platelets promptly (median 13 and 14 days, respectively). There were no primary graft failures. Total Donor cell Chimerism analysis in the Busulfan pharmacokinetics group showed 100% donor at both time points (days 30, 100) in all patients except in one who relapsed at day 30 (85.7%). While in the non-pharmacokinetics group only 7 out of 23 (30%) patients had complete chimerism at day 30 and day 100. Complete donor chimerism at day 100 in the non-pharmacokinetics group was 47.8% compared to 85.7% in the pharmacokinetic group (p=0.18). Ten out of 23 patients (43.5%) in the non-pharmacokinetics group had decreasing donor chimerism by day 100, while in the pharmacokinetic group only one patient (14%) who relapsed had decreasing donor chimerism by day 100 with an odds ratio of 0.241 (95% Confidence Interval =0.025-2.357; p-value=0.22). None developed sinusoidal obstructive syndrome.Conclusion: In this small cohort of consecutive patients from a single center, we found that patients with myeloid disorders who received fludarabine busulfan for 4 days incorporating busulfan pharmacokinetics had a trend for higher rates of early complete donor chimerism and less decreasing donor chimerism by day100, although not statistically significant, despite having intermediate or high risk disease at time of SCT. Longer follow up is needed for our patients to see if there is effect on relapse or survival but previous studies have showed that low or decreasing donor chimerism early after SCT is an independent risk factor for relapse and impaired survival. This is especially important in myeloid disorders. Busulfan pharmacokinetics may help target better level for inducing early total donor chimerism and donor chimerism may identify high-risk patient cohorts who may benefit from additional therapeutic interventions. These results should be interpreted with caution given the retrospective design of the study and the small number of patients. DisclosuresNo relevant conflicts of interest to declare.

Graft Versus HIV-1 Reservoir Effect after Allogeneic Stem Cell Transplantation

Background: The use of a stem cell source homozygous for the CCR5 delta32 mutation is of particular interest in HIV+ patients in need of an allogeneic hematopoietic stem cell transplantation (SCT) given the high natural resistance against HIV-1 acquisition. In the absence of such cell source, the impact of allogeneic SCT on human immunodeficiency virus type 1 (HIV-1) reservoirs in patients receiving combination antiretroviral therapy (cART) is unknown.Methods: Size of HIV-1 reservoirs was investigated in 3 patients with hematological malignancies receiving cART, who underwent allogeneic SCT (Table 1). Patient 1 received a myeloablative single cord blood (wild type (WT) CCR5), supported with third party HLA-mismatched CD34+ cells (haplo-cord SCT). Patients 2 and 3 underwent reduced intensity conditioning SCT of an HLA-matched sibling (heterozygous CCR5 delta32 donor and WT CCR5 donor, respectively). In-depth analysis of the HIV-1 reservoir was performed in the post-transplant period using droplet digital PCR and hemi-nested qPCR (proviral DNA copies/million PBMC). Chimerism analysis was performed by short tandem repeat PCR (STR-PCR).Results: Proviral DNA showed a decreasing dynamics after allogeneic SCT and became undetectable in all 3 patients at 9, 18 and 1 months, respectively (Table 1). In patient 1, negativity of proviral HIV-1 DNA coincided with achievement of complete chimerism while being free of immunosuppression. Patient 2 showed undetectable proviral DNA together with the development of skin chronic GvHD. Finally, undetectable proviral DNA and complete chimerism was achieved in patient 3 early after transplantation. All patients remained in complete hematological remission.Conclusion: All our patients showed a small HIV-1 reservoir after allogeneic SCT. The coincidence of achievement of negative proviral HIV-1 DNA and complete chimerism and/or development of GvHD suggests a role of Ògraft versus HIV-1 reservoir effectÓ in the control of HIV infection. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.