<b><i>Background:</i></b> Sensitive and accurate methods to detect hematopoietic chimerism after hematopoietic stem cell transplantation (HSCT) are essential to evaluate engraftment and to monitor response to therapeutic procedures such as donor lymphocyte infusion. Continuous long-term follow up, however, requires large amounts of pre-HSCT samples limiting the application of many widely used techniques for sensitive chimerism monitoring. <b><i>Methods: </i></b>DNAs from 42 normal healthy donors and 16 HSCT donor/recipient pairs were employed to validate the use of allele-specific insertion/deletion (indel) quantitative real-time polymerase chain reaction (qPCR) to quantify chimerism in samples with low amounts of DNA. Consequently, indel-qPCR analyses of samples from 16 HSCT patients were compared to short-tandem repeat (STR) specific PCR analyses. <b><i>Results</i></b>: Typing with reduced amounts of input DNA (15 vs. 60 ng) allowed for the reliable distinction of positive (mean threshold cycle (ct) 28.05) and negative (ct >36) signals. The high informativity of primer/probe sets, with 12 out of 19 markers exceeding 20% informativity, was confirmed in our cohort (n = 74). Importantly, a fourfold reduction of input DNA compared to published protocols did not alter PCR efficiencies and allowed for a more sensitive detection of chimerism in 7 of 16 HSCT patients compared to results obtained by STR-PCR. <b><i>Conclusions</i></b>: Our data suggest that indel-qPCR is a more sensitive technique for the detection of hematopoietic chimerism compared to STR-PCR and works efficiently for samples with low amounts of DNA.
POEMS syndrome is a rare plasma cell dyscrasia characterised by polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes. The pathogenesis of the disease is unclear. However, certain features are similar to multicentric Castleman's disease and HHV-8 infection. We report the case of a patient with POEMS syndrome progressing to multiple myeloma that was unresponsive to treatment with rituximab and standard chemotherapy but improved considerably after ASCT. The 28 year old patient presented with severe polyneuropathy. A low level IgG lamda monoclonal gradient and a slight plasma cell infiltration of the marrow was found. Hepatosplenomegaly, thrombocytosis, polycythemia, plethora and low TSH was consistent with POEMS syndrome. The patient had anti-HHV-8 antibodies and HHV-8-DNA was found in her initial marrow examination. While reported to be effective in Castleman's disease, the patient was treated with 4 doses of rituximab, but did not respond. During the following 3 months the patient progressed to multiple myeloma with a bone marrow involvement of > 20% plasma cells. Atypical plasma cells and a hypercellular megakaryopoesis were found. However, no osteolytic or osteosclerotic bone lesions were detectable. HHV-8 DNA was no longer detectable in marrow or blood. The patient received 4 cycles of idarubicine/dexamethasone. The response to this treatment was minimal and an ASCT was planned. After mobilisation chemotherapy with iphosphamide, epirubicine and etoposide, 29.65 x 106 CD34-positive progenitor cells/kg could be harvested with one leukapheresis. ASCT was performed after high dose therapy with melphalan 200 mg/m2. Reinfusion of 14.83 × 106 CD34-positive progenitor cells/kg and medication with G-CSF resulted in engraftment on day +9. Restaging 5 weeks later showed an excellent response. Bone marrow plasma cell infiltration was no longer detectable and hypercellularity of megakaryopoesis was markedly reduced. The platelet counts had returned to normal and the pleural effusions had vanished. IgG and serum electrophoresis were normal while the immunofixation remained positive for IgG lamda. The polyneuropathy of the patient is continuously improving. In conclusion, this case may represent an evolution from possibly HHV-8 related POEMS syndrome to multiple myeloma. Despite lack of response to standard chemo- and immunotherapy ASCT seems to be a useful therapeutic option in patients with signs of POEMS syndrome.
Introduction Allogeneic stem cell transplantation is used to cure hematologic malignancies or deficiencies of the hematopoietic system. It is associated with severe immunodeficiency of the host early after transplant and therefore early reactivation of latent herpesviruses such as CMV and EBV within the first 100 days are frequent. Small studies and case series indicated that application of herpes virus specific T cells can control and prevent disease in this patient population. Methods We report the results of a randomized controlled multi centre phase I/IIa study (MULTIVIR-01) using a newly developed T cell product with specificity for CMV and EBV derived from the allogeneic stem cell grafts used for transplantation. The study aimed at prevention and preemptive treatment of both viruses in patients after allogeneic stem cell transplantation targeting first infusion on day +30. Primary endpoints were acute transfusion reaction and acute-graft versus-host-disease after infusion of activated T cells. Results Thirty-three patients were screened and 9 patients were treated with a total of 25 doses of the T cell product. We show that central manufacturing can be achieved successfully under study conditions and the product can be applied without major side effects. Overall survival, transplant related mortality, cumulative incidence of graft versus host disease and number of severe adverse events were not different between treatment and control groups. Expansion of CMV/EBV specific T cells was observed in a fraction of patients, but overall there was no difference in virus reactivation. Discussion Our study results indicate peptide stimulated epitope specific T cells derived from stem cell grafts can be administered safely for prevention and preemptive treatment of reactivation without evidence for induction of acute graft versus host disease. Clinical trial registration https://clinicaltrials.gov , identifier NCT02227641.
Abstract After allogeneic hematopoietic stem cell transplantation (HSCT), patients are repetitively vaccinated to reduce the risk of infection caused by the immune deficiency following allogeneic HSCT. By the vaccination of transplanted patients, the humoral memory function can be restored in the majority of cases. It is unknown, however, to what extent memory B cells derived from the donor contribute to the mobilization of antibody-secreting cells and long-term humoral memory in patients after allogeneic HSCT. We therefore analyzed patients after allogeneic HSCT for memory B cell responses 7 days after single vaccination against tetanus toxoid (TT), diphtheria toxoid (DT), pertussis toxoid (PT), Haemophilus influenzae type b (Hib), and poliovirus. Patients showed an insufficient mobilization of plasmablasts (PB) after vaccination, whereas healthy subjects (HD, n = 13) exhibited a significant increase of PB in the peripheral blood. Regarding vaccine-specific antibody-secreting PB, all HD responded against all vaccine antigens, as expected. However, only 65% of the patients responded with a measurable increase in IgG-secreting PB against TT, 65% against DT, 33% against PT, and 53% against poliovirus. Correspondingly, the antibody titers on day 7 after vaccination did not increase in patients. A significant increase of serum titers for the vaccine antigens was detectable in the majority of patients only after repetitive vaccinations. In contrast to the low mobilization of vaccine-specific PB after vaccination, a high number of PB before vaccination was detectable in patients following allogeneic HSCT. High frequencies of circulating PB correlated with the incidence of moderate/severe chronic GVHD. In summary, patients showed a weak mobilization of antigen-specific PB and an inadequate increase in antibody titers 7 days after the first vaccination. Patients with moderate or severe chronic GVHD in their history had a significantly higher percentage of IgG-secreting PB prior to vaccination. The antigen specificity of these IgG-secreting PB is currently unknown.
