Total body irradiation is a crucial risk factor for developing secondary carcinomas after allogeneic hematopoietic stem cell transplantation in childhood.

Allogeneic hematopoietic stem cell transplantation (HSCT) has become a standard part of therapy for a variety of malignant and non-malignant disorders. With improved outcomes after HSCT, increasing attention has been drawn to late complications in long-term survivors. The development of secondary malignancies is recognized as one of the most serious complications. We have evaluated data from 426 patients (272 males, 154 females) who underwent allogeneic transplantation at a median age of 7.9 years from 1989 till 2017 and were alive more than one year after transplantation for the occurrence of secondary solid tumors. We have documented the occurrence of secondary solid tumors in 20 patients (4.7%). The median duration of the development of secondary solid cancer from HSCT was 11.7 (range, 5.4-21.5 years). 18 out of 20 patients (90%) had total body irradiation (TBI) 12-14.4 Gy as a part of a conditioning regimen. All but two had transplantation for malignant disease. All patients underwent surgery and/or chemo-radiotherapy. Eighteen are alive, and two died due to the progression of their secondary malignancy. The most frequent solid cancer was thyroid carcinoma (n=9). Cumulative incidence of secondary solid cancer in all groups was 15.2±3.9%, in a group using TBI based regimen 34.7±8.9%, in non-TBI (only chemo) group was 1.5±1.1%. Overall, the cumulative incidence is statistically significantly different between the TBI based and non-TBI (chemo only) group. The incidence and number of complications following allogeneic HSCT in childhood are increasing in time. The early diagnosis of secondary malignancies is one of the key tasks of long-life multidisciplinary post-transplant care.

Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.

Minimal residual disease (MRD) monitoring via quantitative PCR (qPCR) detection of Ag receptor gene rearrangements has been the most sensitive method for predicting prognosis and making post-transplant treatment decisions for patients with ALL. Despite the broad clinical usefulness and standardization of this method, we and others have repeatedly reported the possibility of false-positive MRD results caused by massive B-lymphocyte regeneration after stem cell transplantation (SCT). Next-generation sequencing (NGS) enables precise and sensitive detection of multiple Ag receptor rearrangements, thus providing a more specific readout compared to qPCR. We investigated two cohorts of children with ALL who underwent SCT (30 patients and 228 samples). The first cohort consisted of 17 patients who remained in long-term CR after SCT despite having low MRD positivity (<0.01%) at least once during post-SCT monitoring using qPCR. Only one of 27 qPCR-positive samples was confirmed to be positive by NGS. Conversely, 10 of 15 samples with low qPCR-detected MRD positivity from 13 patients who subsequently relapsed were also confirmed to be positive by NGS (P=0.002). These data show that NGS has a better specificity in post-SCT ALL management and indicate that treatment interventions aimed at reverting impending relapse should not be based on qPCR only.

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.

B-cell reconstitution after allogeneic SCT impairs minimal residual disease monitoring in children with ALL.

Minimal residual disease (MRD) detection using quantification of clone-specific Ig or TCR rearrangements before and after transplantation in children with high-risk ALL is an important predictor of outcome. The method and guidelines for its interpretation are very precise to avoid both false-negative and -positive results. In a group of 21 patients following transplantation, we observed detectable MRD positivities in Ig/TCR-based real-time quantitative PCR (RQ-PCR) leading to no further progression of the disease (11 of 100 (11%) total samples). We hypothesized that these positivities were mostly the result of nonspecific amplification despite the application of strict internationally agreed-upon measures. We applied two non-self-specific Ig heavy chain assays and received a similar number of positivities (20 and 15%). Nonspecific products amplified in these RQ-PCR systems differed from specific products in length and sequence. Statistical analysis proved that there was an excellent correlation of this phenomenon with B-cell regeneration in BM as measured by flow cytometry and Ig light chain-kappa excision circle quantification. We conclude that although Ig/TCR quantification is a reliable method for post transplant MRD detection, isolated positivities in Ig-based RQ-PCR systems at the time of intense B-cell regeneration must be viewed with caution to avoid the wrong indication of treatment.

Prognosis of pediatric patients transplanted for Ph+ chronic myeloid leukemia in the period from 1989 to 2006 in the Czech Republic.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by clonal proliferation of primitive hematopoietic stem cell. The median age at diagnosis is 55 to 60 years with less than 10% of patients younger 20 years. Incidence of CML in children in the Czech Republic is 0.106 cases/100 thousands per year. Here we report outcome of 38 pediatric patients (median age 12.5 years; range 1.8 - 17.3) with Ph-positive CML diagnosed between years 1989 to 2006. Primarily chronic phase of the disease was diagnosed in 32 (84%) patients. 32 (84.2%) patients underwent hematopoietic stem cell transplantation (HSCT) with the median age at transplantation of 14.9 years (range 6.9 - 20.5 years). Out of transplanted patients 16 (50%) obtained graft from unrelated donor, 13 (41%) from matched sibling donor, 2 from haploidentical family donor and autologous transplantation has been performed in one case. 6 patients were not transplanted, 4 of them died (median 1.2 years from diagnosis), 2 are alive 0.6 and 17.8 years from the diagnosis. Overall survival (OS) in 25 patients after HSCT at our department during the whole period is 66.7% with 15/16 being in stable continuous molecular-genetic remission (94%). During the period of time results of transplantations have been significantly improved (p=0.0071). OS after HSCT until year 1997 is 25% while from year 1998 until now is 87.5%. All centers OS of patients after HSCT is 71%. Results of HSCT in children with CML obtained from the year 1998 at our center are fully comparable with results achieved in large and experienced centers. HSCT remains the only proven and effective method for the treatment of CML. Clinical studies assessing the role of tyrosine kinase inhibitors in children instead of early HSCT should be planned carefully in order to avoid sub-optimal outcomes.

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.

[EBV quantification in children after allogeneic hematopoietic stem cell transplantation]. / Kvantifikace EBV u detí po alogenní transplantaci hematopoetických kmenových bunek.

BACKGROUND: Patients undergoing allogeneic hematopoietic stem cell transplantation (AHSCT) are endangered by developing Epstein-Barr virus-related post-transplant lymfoprolipherative disease (EBV-LPD). The aims of the study were to retrospectively characterise the viral loads in four patients who died of this complication, and to test possible risk factors for EBV reactivation in a prospectively observed cohort of children after AHSCT. METHODS AND RESULTS: Serial DNA samples extracted from whole blood from four patients who died of post-transplant EBV-LPD in year 2000 were retrospectively analysed for EBV load using quantitative real-time PCR. First detection of EBV activation preceded death by 24-91 days. All four patients exceeded a viral load of one million EBV copies per 100,000 human genome equivalents. A cohort of 72 children undergoing AHSCT between 2001-2004 was prospectively followed-on using the same quantification method from regularly obtained samples of whole blood, and clinical and laboratory data were recorded on a weekly basis, totalling at 3,896 person-weeks of observation. Approximately one half of the cohort experienced at least one episode of EBV reactivation during the first 100 days after AHSCT, four of the episodes being accompanied with viral loads higher than our provisional threshold of 10,000 copies per 100,000 human genome equivalents. Three of the four patients developed EBV-LPD and were successfully treated by intravenous administration of anti-CD20 antibody. Testing of possible clinical and laboratory predictors of EBV reactivation did not reveal any clinically useful association. CONCLUSIONS: The cornerstone of predicting EBV-LPD in AHSCT is a regular monitoring of EBV viral load using quantitative methods. Using this strategy with a threshold of 10,000 EBV copies per 100,000 human genome equivalents was proved to be effective, as shown by no death of EBV for the study period, compared to four cases in the year before the quantitative monitoring.

[Familial haemophagocytic lymphohistiocytosis caused by perforin deficit can be successfully treated by haematopoietic stem cell transplantation--the first diagnosed case in the Czech Republic]. / Familiární hemofagocytující lymfohistiocytóza na podklade deficitu perforinu uspesne lécená transplantací hematopoetických kmenových bunek--první diagnostikovaný prípad v Ceské republice.

Familial haemophagocytic lymphohistiocytosis (FHL) is an inherited disorder characterized by an impaired cytotoxicity of T lymphocytes and NK cells typically manifesting within first few months after birth. If not treated adequately, it is inevitably fatal within several months. The incidence in Caucasians has been estimated to 1: 50 000 births. Haematopoietic stem cell transplantation represents the only curative treatment for FHL. Recently, several genetic defects underlying molecular defects in FHL have been identified. In approximately 30% of patients FHL is caused by mutations in PRF1 gene coding for perforin. Further 30% of patients were found to have mutations in UNC13D coding for hMunc13-4 protein. Very recent report has identified another cause of FHL, mutations in STX11 gene on chromosome 6, coding for syntaxin 11. Absence of any of those proteins severely impairs the process of exocytosis of cytotoxic granules. We describe patient with clinical symptoms of FHL. Immunological and molecular biology methods led to the identification of perforin mutation as a cause of the disease. Patient received an allogeneic SCT from HLA-matched unrelated donor. SCT was followed by rapid normalization of clinical symptoms and laboratory findings. In patient described in this study, FHL manifested with typical clinical and laboratory symptoms. Adequate immunosuppressive treatment and subsequent SCT led to the sustained remission of FHL and correction of molecular defect. This is the first case of FHL in Czech Republic where perforin mutation was identified as a molecular cause both at cellular and molecular level.

Long-term follow-up of chimerical state of the patients transplanted for different haematologic diseases.

Long-term follow-up of peripheral cellular chimerism in patients treated with BMT or PBSCT revealed the usefulness of their continuous monitoring at molecular level. Our results are based on monitoring of 120 patients, who were followed for at least 24 months. Comparison of the patients treated for chronic myelogenous leukemia (CML), acute myelocytic leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS) and aplastic anaemia (AA) revealed that mixed chimerism was practically absent in MDS and relatively long-lasting in ALL and AA (regardless to substantially different post-transplantation treatment). The first disease relapses signalized by molecular checking of mixed peripheral chimerism were observed also after a period of remission lasting for several years. Molecular watching enables us to detect relapses at their very beginning that would remain hidden to less sensitive methods. We believe that all of the transplanted patients ought to be monitored for residual disease i.e. cellular chimerism using molecular methods without time limits. On the other hand low level of mixed cellular chimerism is not necessarily a sign of disease progression and can remain unchanged as "status quo" for a very long period.

[Transplantation of highly purified CD34+ hematologic peripheral stem cells from haploidentical related donors in the treatment of children with non-malignant diseases]. / Transplantace vysoce cistených CD34+ kmenových bunek periferní krve od haploidentických rodinných dárcu v lécbe detí s nemaligními onemocneními.

BACKGROUND: Children suffering from rare inherited disorders can be cured using stem cell transplantation (SCT). For patients where HLA-identical donor could not be found, there is an excellent chance of identifying a family member who shares an identical haplotype with the patient but whose second haplotype is different. This situation is called an HLA-full haplotype mismatch. Risks of haploidentical transplantation are graft rejection and severe graft-versus-host disease (GVHD). Histocompatibility barriers can be overcome by infusing high doses of T-cell depleted peripheral CD34+ stem cells. METHODS AND RESULTS: Between December 1995 and March 2000, 5 children with rare inherited disorders were transplanted using highly purified CD34+ stem cells from haploidentical parents at the 2nd Department of Pediatrics, University Hospital Motol, Prague. Two children suffered from severe combined immunodeficiency (SCID), one child from malignant osteopetrosis, Wiskott-Aldrich syndrome and hemophagocytic lymphohistiocytosis, respectively. Positive selection of peripheral CD34+ stem cells from G-CSF stimulated donors was performed using the method of immunoabsorbtion (CellPro) (n = 2) or immunomagnetic separation (CliniMACS) (n = 3). Donor type engraftment was achieved in 4 children. In one of them early graft failure has developed successfully managed by second SCT from the same donor. One child with SCID had primary graft failure. Mild acute GVHD with good response to steroid therapy developed in 2 children. Three children are alive and 2 of them are cured. Two children died due to post-transplant complications--CMV pneumonia and encephalitis. CONCLUSIONS: Transplantation of highly purified CD34+ stem cells from haploidentical parents is a reasonable therapeutic option for children with some specific nonmalignant disorders lacking HLA identical donor. Risks of this type of SCT are the graft failure and severe infectious complications due to slow immunological reconstitution in comparison with SCT from HLA identical donors.

Hematopoietic stem cell transplantation in children with hematological malignancies across HLA barriers--reasonable alternative?

The aim of this study was to evaluate the efficiency and risks of T-cell depletion in prevention of graft versus host disease (GVHD) using HLA haploidentical family donors as an alternative source of hematopoietic stem cells (HSC) in children with hematological malignancies without suitable matched donor. Ten children, median age 12 years (range, 3-17), were transplanted from haploidentical family donors for acute lymphoblastic leukemia (n = 4), acute myelogenous leukemia (n=2), chronic myelogenous leukemia (n = 2), non-Hodgkin lymphoma (n = 1) and myelodysplastic syndrome (n = 1). Parents were donors for nine, sibling for one patient. T-cell depletion of HSC was performed using CellPro followed by antiCD2/CD3 depletion in 7, and CliniMacs magnetic sorting in 3 grafts. Primary engraftment was achieved in nine patients. Patient with graft failure was successfully re-grafted. Primary acute GVHD was diagnosed in one patient who got higher amount of T-cells in the graft. Secondary GVHD was induced by add-backs of lymphocytes in four patients. Three patients developed chronic GVHD. Four patients died due to transplant related mortality (40%), one from veno-occlusive disease, two due to CMV pneumonia and one of aspergillosis with extensive chronic GVHD. Four patients relapsed with leukemia within 35-98 days post transplant, three without previous signs of GVHD, and all died. Two patients are alive and well 26 and 42 months after transplant. Haploidentical family donors appear to be a reasonable alternative option for patients with urgent indications for allogeneic transplant and/or without a matched donor.

Detailed monitoring of hematopoietic chimerism in a child treated by adoptive immunotherapy for high risk of relapse after BMT for acute myeloid leukemia.

We report a case of a 13-year-old boy who was transplanted for relapse of acute myeloid leukemia (AML). A detailed study of hematopoietic chimerism was performed using polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) at very short time intervals. We used discontinuation of post-transplant immunosuppression and donor lymphocyte infusions (DLI) in order to prevent leukemia relapse that was indicated by a progressive increase in autologous hematopoiesis. Despite the fact that the boy relapsed 10 months after BMT, we could see a significant influence of adoptive immunotherapy on the mixed chimerism status during the post-transplant period.

Prediction and reversion of post-transplant relapse in patients with chronic myeloid leukemia using mixed chimerism and residual disease detection and adoptive immunotherapy.

In the prospective study, we examined hematopoietic mixed chimerism (using polymerase chain reaction (PCR) of variable number of tandem repeat-VNTR sequences) and minimal residual disease (MRD) status (using qualitative and in the case of positivity quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the BCR/ABL fusion mRNA) in serial peripheral blood samples taken from 25 patients after bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Increasing mixed chimerism in correlation with increasing signal of MRD was detected in 10 patients. In two patients mixed chimera status and BCR/ABL rearrangement led to hematologic relapse, in five patients molecular relapse was followed by reappearance of Ph chromosome and three patients developed molecular relapse only. Adoptive immunotherapy-donor lymphocyte infusion (DLI), interferon (INF) and discontinuation of post-transplant immunosupression-separately or in different combinations was used in nine patients with molecular, cytogenetic or hematologic relapse of CML. The results demonstrate that significant response at the molecular level can be achieved for a majority of CML patients and that using of all forms of adoptive immunotherapy controlled by MC and MRD is more efficient in patients treated in early molecular relapse-with minimal disease burdens.