Marie Olszewski

High WT1 gene expression before haematopoietic stem cell transplant in children with acute myeloid leukaemia predicts poor event-free survival.

WT1 gene expression has been proposed as a useful marker of minimal residual disease in leukaemia. Its utility in paediatric haematopoietic stem cell transplantation (HSCT) has not been studied. We studied the prognostic value of WT1 expression in peripheral blood prior to HSCT in 36 children with acute myeloid leukaemia (AML). Samples were obtained 2 weeks pre‐transplant to determine the level of WT1 expression. WT1 expression was normalized using K562 cells as a control and a relative value of 0·5 was chosen as the cut‐off point between high and low WT1 expression. The median level of pre‐transplant WT1 expression in the 36 patients was 0·09 (range 0·0001–11·0), with 11patients having WT1 ≥ 0·5 and 25, WT1 < 0·5. After HSCT, 76% of patients with high pre‐transplant WT1 expression relapsed, in contrast to 0% of the patients with low WT1 expression. Those with high WT1 expression had significantly lower 5‐year event‐free survival (EFS) (18%, 95% CI 0–40%) as compared to those with low WT1 expression (68%, 95% CI 50–86%, P = 0·007). Multivariate analysis showed that pre‐transplant WT1 level is the only significant prognostic factor for the difference in EFS. Our finding suggests that elevated WT1 gene expression before HSCT in paediatric AML predicts relapse and poor long‐term EFS. A larger prospective study is warranted to compare the value of high WT1 expression and other markers of minimal residue disease in predicting clinical outcomes after HSCT.

Hematopoietic stem-cell transplantation using unrelated cord-blood versus matched sibling marrow in pediatric bone marrow failure syndrome : One center's experience

Abstract: Hematopoietic stem‐cell transplantation (HSCT) is an effective mode of therapy in pediatrics for the treatment of both malignant and non‐malignant disorders. We compared the course of children transplanted with unrelated umbilical cord blood (UCB) to those transplanted with allogeneic sibling bone marrow (BM) for bone marrow failure syndromes. Thirteen patients with a median age of 6.3 years were transplanted for the following diseases between April 1992 and November 1997: myelodysplastic syndromes, aplastic anemia, Diamond–Blackfan anemia, myelofibrosis, paroxysmal nocturnal hemoglobinuria, osteopetrosis and dyskeratosis congenita. The stem cell source was BM in ten patients and UCB in three. We retrospectively examined the conditioning regimens, stem cell source and dose, days to engraftment, survival and complication rate to see whether there was a significant advantage in using one source over the other. The median time to an absolute neutrophil count > 500 per µL was 25 days for UCB patients and 16 days for BM patients. The median time to a platelet count > 20 000 per µL was 55 days for UCB patients and 22 days for BM patients. The 100‐day mortality was 66% in UCB patients and 20% in BM patients. The overall mortality rates were 66% and 40%, respectively. Three patients died prior to engraftment. Seven patients (54%) were still alive as of May 1999 with a median follow‐up of 1574 days post‐transplant. The patients transplanted with BM had faster engraftment and lower rates of graft‐versus‐host disease, 100‐day mortality and overall mortality. HLA‐matched sibling BM is preferred as a source but transplantation using unrelated UCB is still an option in treating pediatric bone marrow failure syndromes.

Expansion of megakaryocyte precursors and stem cells from umbilical cord blood CD34+ cells in collagen and liquid culture media

Umbilical cord blood (UCB) is now commonly used as a source of stem cells for hematopoietic reconstitution following myeloablative therapy in patients with a variety of diseases. Although UCB is a rich source of stem cells, platelet engraftment occurs at a median of 71 days which is significantly prolonged compared to allogeneic bone marrow. The number of megakaryocyte (MK) precursors in stem cell harvests appears to correlate inversely with the time to platelet engraftment. In an effort to increase the number of platelet precursors, we cultured CD34-selected cord blood mononuclear cells (MNC) in serum-free collagen medium with numerous cytokine combinations. The cells were cultured with four cytokines: interleukin-3 (IL-3), thrombopoietin (TPO), stem cell factor (SCF), and Flt-3); five cytokines, IL-3, TPO, SCF, Flt-3 plus granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo); or all six cytokines in combination. After 16 days, significant expansion of MK precursors (CD41(+)) and stem cells (CD34(+) and AC133(+) cells) were seen in cells cultured in IL-3, TPO, SCF, and Flt-3 with or without GM-CSF compared to the combinations that contained Epo (p < 0.05). Similar studies were performed using liquid culture medium, and after 14 days the number of MNCs, CD34(+), AC133(+), CD41(+), and CD61(+) cells were higher in the UCB cells cultured in IL-3, TPO, SCF, and Flt-3 compared to those cultured with those four cytokines plus GM-CSF. These results demonstrate that UCB stem cells can be effectively expanded ex vivo and enriched with platelet precursors using TPO, SCF, Flt-3, and IL-3, whereas the addition of Epo and GM-CSF is unnecessary.

Validation of chimerism in pediatric recipients of allogeneic hematopoietic stem cell transplantation (HSCT) a comparison between two methods: Real-time PCR (qPCR) vs. variable number tandem repeats PCR (VNTR PCR)

Post-hematopoietic stem cell transplantation (HSCT) chimerism monitoring is important to assess relapse and therapeutic intervention. The purpose of our study is to compare two methods variable number tandem repeats (VNTR) vs. quantitative real- time polymerase chain reaction (qPCR) in terms of determining chimerism. 127 (peripheral blood n = 112, bone marrow n = 15) samples were simultaneously tested by VNTR using APO-B, D1S80, D1S111, D17S30, gene loci SRY and ZP3 and qPCR using 34 assays (CA001-CA034) that are designed to a bi-allelic insertion/deletion (indel) polymorphism in the human genome. Samples were separated in three subsets: total WBC, T-cell and Myeloid cells. Extraction of DNA was performed then quantified. We analyzed column statistics, paired t-test and regression analysis for both methods. There was complete correlation between the two methods. The simplicity and rapidity of the test results from the qPCR method is more efficient and accurate to assess chimerism.

Red cell salvage and reinfusion in pediatric bone marrow donors.

We evaluated the use of a semi-automated processing technique to salvage red blood cells from pediatric bone marrow donors to minimize the risk of severe anemia following bone marrow harvest and ABO incompatibility in the recipient. Sixty healthy, HLA-matched, pediatric donors of bone marrow hematopoietic cells with a median age 8.0 years (2–19) were studied. Thirteen of the donor–recipient pairs were ABO incompatible. There were 60 recipients with a median age of 8.6 years (2 months to 20.8 years). Bone marrow was harvested under general anesthesia, filtered in the operating room and then transferred to the stem cell laboratory for processing. Samples were obtained for cell count, CD34+ quantification, colony assay, viability, and bacteriologic cultures before and after processing. The cells were processed in a semi-automated closed system (Stericel, Terumo) by density gradient separation with Ficoll–Hypaque and then washed. Two aliquots were obtained: one containing the mononuclear cell layer to be infused to the recipient and the other the washed red cells to be infused to the donor. The median volume harvested was 608 ± 40.42 ml (278–1409), while the final volume infused was 174 ± 10.75 ml (30.2–380) P < 0.0001, representing a decrease of 72% of the volume infused. the nucleated cell count harvested was 1.6 × 1010 ± 0.1 (0.56–3.2), while the count infused was 6.9 × 109 ± 0.1 (0.12–5.4) P < 0.0001. the median mononuclear cell count (mnc) per kg harvested was 0.67 × 108 ± 0.05 (0.18–2.0) vs an infused cell number of 1.3 × 108 MNC/kg ± 0.1 (0.6–33.6) P < 0.0001. the cd34+ cells harvested were 2.8 × 106/kg ± 0.1 (0.25–10.2) vs an infused number of 6.0 × 106/kg ± 0.5 (0.84–31.0) P < 0.0001. the viability before and after processing was 99%. red cell salvage performed in a semi-automated closed system is safe and reduces the risk of post-bone marrow harvest anemia in pediatric donors, decreases the volume infused into the donor and enriches the mononuclear and cd34+ cell population, without affecting hematopoietic reconstitution.

Induction of a transient graft vs. leukemia effect following unrelated cord blood transplantation

Abstract: Umbilical cord blood (UCB) has become a frequent source of allogeneic hematopoietic stem cells for transplantation. Of theoretical concern is a potential decrease in the graft vs. leukemia (GvL) effect, given the lesser degree of graft vs. host disease (GvHD) with this donor source. We report a case of recurrent acute non‐lymphoblastic leukemia (ANLL) following stem cell transplantation with unrelated mismatched UCB, which responded to the induction of GvHD. The response was documented both morphologically and by evaluation of chimeric engraftment by molecular DNA techniques. In addition, WT‐1, a purported marker of minimal residual disease in acute leukemia, correlated with remission status in this patient. In summary, the GvL effect is seen with allogeneic UCB transplantation and has the potential to be induced along with GvHD.

Two-Day Collection and Pooling of Peripheral Blood Stem Cells with Semiautomated Density Gradient Cell Separation

Autologous and allogeneic PBSC collection and cryopreservation have been shown to be feasible in the pediatric population. This technique may be associated with complications, including volume overload and DMSO toxicity. To decrease these risks, we developed a technique by which PBSC are collected over a 2-day period and pooled prior to cryopreservation. PBSC are harvested on day 1 and stored with tissue culture medium on a rocker at ambient temperature. On day 2, a second PBSC harvest is performed, and the two harvests are pooled, separated on a Ficoll gradient in a semiautomatic closed system, and frozen with 10% DMSO after a soft spin for volume reduction. Cells from each days harvest are tested for cell count and viability. A total of 36 collections in 26 patients were performed. This technique resulted in a 73%+/-0.0% reduction in volume (mean +/- SEM) and an 88%+/-0.9% depletion of RBC. Mononuclear cell (MNC) count recovery was 88%+/-2.6%, and the MNC dose delivered to the patient was 3.1+/-0.6x10(8) cells/kg. Cell viability was >98% before and after processing. Seventeen patients have been transplanted thus far, and all these patients engrafted with minimal toxicity. These data indicate that storing PBSC for up to 24 h after harvest does not decrease PBSC viability or delay engraftment.

The acute phase inflammatory response to maximal exercise testing in children and young adults with sickle cell anaemia

Although individuals with sickle cell anaemia (SCA) have elevated baseline inflammation and endothelial activation, the acute phase response to maximal exercise has not been evaluated among children with SCA. We measured the acute phase response to maximal exercise testing for soluble vascular cell adhesion molecule (sVCAM) as well as interleukin 6 (IL6), total white blood cell (WBC) count, C‐reactive protein (CRP) and D‐dimer in a cohort of children with SCA and matched controls at baseline, immediately after, and 30, 60 and 120 min following exercise. Despite higher baseline levels of all biomarkers except CRP, the acute phase response from baseline to immediately after exercise was significantly greater in subjects versus controls for CRP (2·1 vs. 0·2 mg/l, P = 0·02) and D‐dimer (160 vs. 10 μg/l, P < 0·01) only. Similar between‐group trends were observed over time for all biomarkers, including sVCAM, IL6, total WBC, CRP and D‐dimer. Lower fitness, defined by peak oxygen consumption (VO2), was independently associated with greater acute phase responses to exercise for sVCAM. Our results suggest maximal exercise may not be associated with any greater escalation of endothelial activation or inflammation in SCA and provide preliminary biomarker evidence for the safety of brief, high‐intensity physical exertion in children with SCA.

A flow cytometric technique using thiazole orange to detect platelet engraftment following pediatric stem-cell transplants

BACKGROUND Thiazole orange (TO) is a nucleic-acid-specific dye that enters cells without pretreatment. When it binds to either RNA or DNA, there is an increase in fluorescence emission. This property has been utilized to measure the amount of newly released platelets using flow cytometry. These newly released platelets differ from more mature platelets because they still contain residual amounts of RNA, and have become known as reticulated platelets. METHODS Peripheral blood samples were collected at least 48 h following platelet infusion. For validation, manual reticulocyte counts obtained in the laboratory were compared with results obtained using TO and flow cytometry. Following validation, experiments using platelet-rich plasma were performed to evaluate the presence of reticulated platelets in the sample. RESULTS Validation experiments comparing the manual and flow cytometric reticulocyte counts gave a strong relationship between the two values (r(2) = 0.92). Reticulated platelet studies performed on platelet-rich plasma samples yielded the following results. Patients who did not engraft within 4 days were significantly different from patients who did engraft within 4 days, idiopathic thrombocytoperic purpura (ITP) patients, and donor platelet segments (all P < 0.0008). Patients who engrafted within 4 days, ITP patients, and donor platelet segments were all statistically similar (all P > 0.08). DISCUSSION The statistical difference between patients who did engraft within 4 days and those who did not suggests that this method could have an important clinical impact in determining those patients who are still in need of platelet support. However, great care must be taken when performing and analyzing the results.

Platelet chimerism by polymerase chain reaction (PCR) utilizing variable number of tandem repeats (VNTR) in allogeneic stem cell transplant in children: a new novel approach to full chimerism analysis.

Summary:Evaluation of chimerism following allogeneic transplantation has been performed traditionally focusing on two cellular compartments, namely lymphoid and myeloid. However, none has been described so far to evaluate platelet chimerism. In order to achieve full chimerism in all three cellular compartments, we prospectively obtained 138 samples of peripheral blood in 55 patients at different post transplant periods following allogeneic hematopoietic transplantation. Evaluation of chimerism was performed utilizing tests of variable number of tandem repeat (VNTR) and sex determination by quantitative polymerase chain reaction (PCR). Tests for platelet chimerism using platelet-rich plasma were simultaneously analyzed with samples for T-cell lymphoid and myeloid compartments. Complete donor chimerism was noted in 49 of 55 patients (89%), while the remaining six have split chimerism ranging from 34 to 98%. There is significant difference (P=0.0004) between the percentages of donor DNA in all three cellular compartments comparing the means±s.e.m. (myeloid 95.60±0.9, T-cell lymphocytes 87.6±1.9, and the platelets 90.8±1.5); however, comparison between the medians is not statistically significant. This study represents an additional step towards achieving full chimerism and the observation may help reduce the number of unnecessary platelet transfusions once chimerism is noted in that cellular compartment.