Petra Keslova

HHV-6 DNA throughout the tissues of two stem cell transplant patients with chromosomally integrated HHV-6 and fatal CMV pneumonitis

Two patients with the characteristic high human herpesvirus 6 (HHV‐6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV‐6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV‐6 DNA was detected at high levels throughout the organs with the exception of donor‐derived haematopoietic tissue. In individuals with chromosomal integration, HHV‐6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.

Signature profiles of CMV-specific T-cells in patients with CMV reactivation after hematopoietic SCT

Depletion of cellular immunity as a consequence of conditioning before allogeneic hematopoietic SCT (HSCT) frequently results in CMV reactivation, which may in turn lead to life-threatening infections and require timely antiviral treatment. We have investigated the functional signatures of CMV-specific CD4+ and CD8+ T-cells in 191 samples from 118 individuals. We compared healthy donors with both patients with high and undetectable viral loads, and those who controlled and did not control their CMV reactivations. Polychromatic flow-cytometric measurements of CD154 (CD40L), intracellular cytokines (IFNγ, IL2) and a degranulation marker (CD107a) allowed us to assess the functional status of various T-cells simultaneously. We found that dual IFNγ/IL2-producing CD8+ T-cells were present in patients controlling their CMV reactivations but absent from non-controllers. CD8+ T-cells that produce only IFNγ were the most abundant subtype, but they most likely represent non-protective memory cells. Distinct functional signatures were examined by hierarchical clustering, and this revealed that, unlike polyfunctional CD8+ T-cells, CD8+ T-cells that produce IFNγ alone were not functioning in concert with other subsets. In conclusion, our study revealed functional signatures that may be useful for immune monitoring, and it could change the interpretation of previous studies that assessed only IFNγ.

Unrelated partially matched lymphocyte infusions in a patient with complete DiGeorge/CHARGE syndrome.

Abstract:  We present an infant with cDGS overlapping with CHARGE syndrome, who suffered from T‐cell deficiency treated with screened healthy DLI from an unrelated donor (8/10 match). The first dose of DLI (1.1 × 106 CD3+/kg) was administered at the age of six months, the second one (0.9 × 106 CD3+/kg) 36 days later. No conditioning was employed, GvHD prophylaxis consisting of CsA was used only during the second infusion. Since day+10 after the first DLI, split chimerism showing T‐cell engraftment has been documented. Proliferative response to PHA was detected on day+145. The treatment was complicated by severe acute GvHD (grade II‐III) after the first DLI and prolonged chronic liver cholestatic GvHD developing after the second DLI. Vigorous EBV proliferation four wk after the second DLI was accompanied by peripheral expansion of CD8+ donor cells. The patient, 26‐months old, is clinically well and has slowly started to gain his developmental milestones. We believe that infusions of small doses of DLI from an unrelated donor represent a potentially helpful therapeutic option in patients with cDGS/CHARGE phenotype.

Cytomegalovirus encephalitis/retinitis in allogeneic haematopoietic stem cell transplant recipient treated successfully with combination of cidofovir and foscarnet.

Abstract:  We report an 18‐yr‐old female patient with repeated CMV reactivations after HSCT treated by several pre‐emptive courses of virostatic therapy. Seven months after HSCT, she developed CMV encephalitis/retinitis. Initial therapy with GCV and hyperimmune globulin failed, and later on GCV‐resistant strain was detected. Continual increase of CMV DNA in peripheral blood led us to combined therapy with CDV and FCV, which was successful and free of severe renal toxicity. To our best knowlegde, this is the first reported case of successful CMV treatment with a combination of CDV and FCV.

Low mortality of children undergoing hematopoietic stem cell transplantation from 7 to 8/10 human leukocyte antigen allele-matched unrelated donors with the use of antithymocyte globulin.

Human leukocyte antigen (HLA)-matched sibling donor hematopoietic stem cell transplantation (HSCT) is available for only approximately 30% patients needing HSCT. Use of alternative donors is associated with a high incidence and severity of graft-versus-host disease (GVHD). Here we report our experience with GVHD prophylaxis using pre-transplant rabbit antithymocyte globulin (rATG), in addition to post transplant cyclosporin A and methotrexate. Seventy-five children received unmanipulated grafts from 7 to 10/10 HLA allele-matched unrelated donors. Median follow-up was 25 months (range, 6–65 months). Only 2/75 patients (2.5%) developed acute GVHD grades III–IV, and 17/75 (25%) developed extensive chronic GVHD. Overall survival was 79%. It was similar in patients receiving grafts from 7 or 8/10 to 9 or 10/10 allele-matched donors, and similar in patients receiving peripheral blood stem cells and marrow. Six (11%) patients died owing to relapse, and 10 (13%) due to transplant-related complications. The addition of rATG appears to result in a low incidence of severe GVHD and overall mortality.

The importance of therapeutic drug monitoring (TDM) for parenteral busulfan dosing in conditioning regimen for hematopoietic stem cell transplantation (HSCT) in children.

BACKGROUND Series of observations indicate PK/PD variability challenging the accuracy of the body-weight based busulfan (Bu) dosing schedule for (HSCT) conditioning therapy. The purpose of this communication is to describe the frequency of dose changes in initially body-weight-based fixed IV Bu dose and to emphasize the importance of TDM. MATERIAL AND METHODS Sixty-two children (ages 2 months-18 years) were treated with IV busulfan doses based on body weight for myeloablation. TDM utilizing a limited sample strategy (trough concentration immediately before the 5th dose, followed by samples immediately after the end of the 2-h infusion peak, 4 h, and 6 h from initiation of the infusion) was performed in 46 of 62 subjects. Busulfan concentrations were determined by high-performance liquid chromatography (HPLC). AUC was calculated according to the trapezoidal rule. RESULTS We observed trough levels of 25-1244 µg/L, peak levels of 849-4586 µg/L, and AUC of 2225-12818 µg/L·h following body weight-based high-dose busulfan. The doses were changed in 54% of cases. AUC in 5 of 9 patients with VOD were within target, in 3 patients AUS was higher, and in 1 patient AUC was lower. One of the 2 patients with neurotoxicity had higher AUC. Engraftment was 100%, but relapse occurred in 25% of cases. CONCLUSIONS Our results demonstrate that even with IV busulfan, intra-individual PK/PD variability is challenging. Although AUC does not necessarily correspond with outcomes (due to the role of other factors the fact that doses were changed in 54% of cases underlines the importance of TDM.

The TREC/KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome.

DiGeorge syndrome (DGS) presents with a wide spectrum of thymic pathologies. Nationwide neonatal screening programs of lymphocyte production using T-cell recombination excision circles (TREC) have repeatedly identified patients with DGS. We tested what proportion of DGS patients could be identified at birth by combined TREC and kappa-deleting element recombination circle (KREC) screening. Furthermore, we followed TREC/KREC levels in peripheral blood (PB) to monitor postnatal changes in lymphocyte production. Methods TREC/KREC copies were assessed by quantitative PCR (qPCR) and were related to the albumin control gene in dry blood spots (DBSs) from control (n = 56), severe immunodeficiency syndrome (SCID, n = 10) and DGS (n = 13) newborns. PB was evaluated in DGS children (n = 32), in diagnostic samples from SCID babies (n = 5) and in 91 controls. Results All but one DGS patient had TREC levels in the normal range at birth, albeit quantitative TREC values were significantly lower in the DGS cohort. One patient had slightly reduced KREC at birth. Postnatal DGS samples revealed reduced TREC numbers in 5 of 32 (16%) patients, whereas KREC copy numbers were similar to controls. Both TREC and KREC levels showed a more pronounced decrease with age in DGS patients than in controls (p<0.0001 for both in a linear model). DGS patients had higher percentages of NK cells at the expense of T cells (p<0.0001). The patients with reduced TREC levels had repeated infections in infancy and developed allergy and/or autoimmunity, but they were not strikingly different from other patients. In 12 DGS patients with paired DBS and blood samples, the TREC/KREC levels were mostly stable or increased and showed similar kinetics in respective patients. Conclusions The combined TREC/KREC approach with correction via control gene identified 1 of 13 (8%) of DiGeorge syndrome patients at birth in our cohort. The majority of patients had TREC/KREC levels in the normal range.

Allogeneic stem cell transplantation in children with leukemia using human leukocyte antigen-mismatched unrelated donors

Abstract:  Allogeneic HSCT is a curative treatment, when chemotherapy fails, for certain malignant diseases. In Europe, only 15% of the indicated children have an HLA‐matched sibling available; in 65–70% of others, HLA allele‐matched (9–10/10) UDs can be identified. For the rest, it is necessary to identify other alternative donors (HLA‐mismatched family or unrelated cord blood). We present our data of HSCT using HLA partially allele‐mismatched (7–8/10) UDs in 24 children with leukemia. Uniform GvHD prophylaxis was used (rATG, CsA and MTX). Acute GvHD grade II was diagnosed in 70.8% of the patients and grade III–IV in 12.5%. Overall incidence of chronic GvHD was 38.7% (extensive in 30%). The probability of EFS was 60.3% (95% CI 35.5–78.1) and OS was 74.9 (95% CI 49.1–88.9). No difference in survival between PBSC and BM recipients was observed. TRM at day + 100 was 4%, and overall was 12.5%. We conclude that used combination of drugs for GvHD prophylaxis is efficient even for patients transplanted with grafts from a HLA‐mismatched UDs. It enables stable engraftment, good control of GvHD, full reconstitution of immunity, and is not connected with unacceptable transplant‐related mortality.

Allo-SCT in children with high-risk leukemia using unmanipulated grafts from alternative donors.

Allogeneic HSCT is a curative treatment for high-risk leukemia. In Europe, approximately 15% of children have an HLA-matched sibling, but in 65–70% HLA allele-matched (9–10/10) unrelated donors (UD) can be identified. Transplantation using an HLA partially mismatched donor, unrelated cord blood or haploidentical family donor with graft manipulation is then considered with preference on the basis of local experience and/or availability. Here we evaluate the outcomes of 87 consecutive patients with leukemia transplanted with unmanipulated graft from matched or partially mismatched UD or cord blood (CB) at our institution between January 2001 and December 2007. Within the median follow-up of 30 months, the acute GVHD grade II was diagnosed in 70.9% patients; grades III–IV only in 4.6%. The overall incidence of chronic GVHD was 43.3% (extensive in 34.9%). The probability of 3-year EFS was 59.5% and that of 3-year overall survival was 66.9%. TRM at day +100 was 4.5%, and overall it was 13.8%. Fourteen patients (16.1%) died as a consequence of post-transplant leukemia relapse. We conclude that the prognosis of patients transplanted for leukemia using unmanipulated grafts from HLA-matched or partially mismatched UD or CB is comparable and satisfactory. TRM and relapse rate are lower than in the earlier period.

Incidence of HHV7 in donors and recipients of allogeneic hematopoietic stem cell transplantation.

To the Editor: We read with the great interest the article of by Khanani et al. [1] and were surprised with the extremely low incidence of detected Human Herpesvirus 7 (HHV7) in paediatric hematopoietic stem cell transplant (HSCT) recipients, which is not in agreement with our experience. The incidence in the allogeneic setting was 5.5% (9/163) with the median of detection 21 days after HSCT. It would have been interesting to see the total number of tested samples in different categories (blood, plasma, other body fluids, tissue) and the separation of that cohort concerning patients on ganciclovir (GCV) prophylaxis as possible explanation for the low HHV7 incidence. In the article and in the reference about the PCR methods [2] we were not able to find the sensitivity of theHHV7 assay. The lower incidencemay be then also explained by the lower sensitivity of assay. In our institution,we have testedHHV7 in similar clinical setting with the exception of GCV prophylaxis. All patients received acyclovir prophylaxis, while GCV was used to treat CMV preemptively based on the results of weekly PCR monitoring. Viral detection is based on DNA extraction fromwhole blood. The results are normalized to 10,000 human genome equivalents quantified using the albumin gene. The sensitivity of both the HHV7 and albumin gene detection are down to 5 copies per reaction. Up to May 2007, we tested for HHV7 2,546 blood samples obtained from125 patients transplanted between February 2000 and May 2006 (median age at HSCT 9.13 years). Two hundred seventyeight samples from 56 patients (44.8%) contained more than 1 normalized viral copy (NVC), more than 10 NVCs contained samples from 26 patients (20.8%) and more than 100 NVCs were detected in only 5 patients (0.04%). In 72 patients from our cohort, we found no impact of viral reactivation on clinical features (e.g., presence of fever, pneumonia, thrombocytopenia). With regard to the predictors of the reactivation of HHV7 at level 10 NVCs, we have found that a higher dose of CD34þ cells in the graft and use of busulfan increased the probability for HHV7 detection during posttransplant surveillance. HSCT with related donor compared to unrelated resulted in a 4.5-fold increase in risk of HHV7 reactivation. Compared to Khanani et al. [1], we found a positive association of HHV7 and HHV6 detection while there was no relation between HHV7 and CMVand EBV. Interestingly, we were able to detect HHV7 DNA in 9 unrelated and 5 related donors out of 30 donors tested (21 unrelated and 9 related donors) and observed no clinical consequence for the recipient. The quantity of HHV7 DNA in these cases was below 100 NVCs (median 7.5, range 0.6–88). Because the graft for HSCT is obtained in donors without any clinical sign of infection, we suggest this level as a non-clinically important in term of latent HHV7, at least for immunocompetent host. Compared to Khanani et al., higher incidence observed in our cohort is similar to other publishedworks [3,4] andmay be caused by the higher sensitivity of our PCR assay.