Erik W.A. Marijt

Generation and administration of HA-1-specific T-cell lines for the treatment of patients with relapsed leukemia after allogeneic stem cell transplantation: a pilot study

Since HA-1-specific T cells have been shown to make a significant contribution to the clinical responses in patients with relapsed leukemia, we investigated the feasibility of adoptive transfer of in vitro induced HA-1-specific CD8 positive T cells to patients with relapsed leukemia after allogeneic stem cell transplantation. The in vitro generation of clinical grade HA-1-specific T-cell lines from HA-1 negative donors was seen to be feasible and 3 patients were treated with HA-1-specific T-cell lines. No toxicity after infusion was observed. Although in one patient, during a period of stable disease, HA-1-specific T cells could be detected in the peripheral blood and bone marrow, these patients had no clear clinical response.

Effective Treatment of Refractory CMV Reactivation After Allogeneic Stem Cell Transplantation With In Vitro-generated CMV pp65-specific CD8+ T-cell Lines

To treat patients with refractory cytomegalovirus (CMV) reactivation after allogeneic stem cell transplantation, a phase I/II clinical study on adoptive transfer of in vitro-generated donor-derived or patient-derived CMV pp65-specific CD8+ T-cell lines was performed. Peripheral blood mononuclear cells from CMV seropositive donors or patients were stimulated with HLA-A*0201-restricted and/or HLA-B*0702-restricted CMV pp65 peptides (NLV/TPR) and 1 day after stimulation interferon-&ggr;)-producing cells were enriched using the CliniMACS Cytokine Capture System (interferon-&ggr;), and cultured with autologous feeders and low-dose interluekin-2. After 7–14 days of culture, quality controls were performed and the CMV-specific T-cell lines were administered or cryopreserved. The T-cell lines generated contained 0.6–17×106 cells, comprising 54%–96% CMV pp65-specific CD8+ T cells, and showed CMV-specific lysis of target cells. Fifteen CMV-specific T-cell lines were generated of which 8 were administered to patients with refractory CMV reactivation. After administration, no acute adverse events and no graft versus host disease were observed and CMV load disappeared. In several patients, a direct relation between administration of the T-cell line and the in vivo appearance of CMV pp65-specific T cells could be documented. In conclusion, administration of CMV pp65-specific CD8+ T-cell lines was found to be feasible and safe, and enduring efficacy of administered CMV pp65-specific CD8+ T-cell lines could be demonstrated.

Rapid identification of IDH1 and IDH2 mutations in acute myeloid leukaemia using high resolution melting curve analysis

Screening for recurrent mutations in leukaemia is becoming increasingly important because many of them have an impact on disease outcome. Nowadays, mutational detection is mostly based on DNA sequencing of PCR products or QPCR using allele specific probes. With the increasing identification of recurrent mutations in cancer, the development of fast and efficient approaches for mutational screening is needed. An alternative technique for mutation detection is high resolution melting (HRM) analysis (Wittwer, 2009). HRM analysis is a fast ‘single-well-technique’ combining PCR using a fluorescent saturating dye that is intercalated in the DNA, and melt curve analysis afterwards. As mutations cause different melting properties, they are identified when compared to non-mutated samples. Positive samples in HRM analysis can be sequenced subsequently to determine exact nucleotide changes. Recently, mutations in the NADP-dependent isocitrate dehydrogenase genes IDH1 and IDH2 were shown in acute myeloid leukaemia (AML), myeloid dysplastic syndromes and myeloproliferative diseases (Mardis et al, 2009; Abbas et al, 2010; Chou et al, 2010; Green & Beer, 2010; Kosmider et al, 2010; Marcucci et al, 2010; Wagner et al, 2010). We study the application of HRM analysis and direct sequencing to screen for these mutations in a cohort of 168 AML patients, using the real-time PCR platform of Applied Biosystems (Carlsbad, CA, USA). Sequence analysis detected IDH1 or IDH2 mutations in 32 patients (19%) (Tables SI and SII). Using the methods as described in the Data S1, mutations could also be detected by HRM analysis (used primer combinations are indicated in Table SIII). Different primers with and without M13sequences were tested, showing differential results for IDH1 or IDH2 mutations (Fig 1). Using HRM analysis, no mutations were missed. For IDH1 R132, 12 mutated samples (7Æ1%) were found both by HRM and sequence analysis while an additional three positive samples (1Æ8%) were found only by HRM analysis. To elucidate whether the extra mutations found by HRM analysis were false positives, we compared the sensitivity of both techniques. For this, a dilution series of genomic DNA from a mutated sample was measured by HRM analysis followed by direct sequencing. Positivity in HRM analysis was lost at an allele frequency of approximately 9%, whereas the mutation could still be detected at a percentage of 4% by sequencing (Fig S1). As sequencing was more sensitive, this suggests that the extra positive samples found by HRM analysis, that did not contain any SNPs, represent false positive results. Nineteen samples were found positive for the IDH2 R140 mutation (11Æ3%) by sequence analysis. Initial HRM analysis for this mutation identified many false positive results. However, exclusion of samples with low PCR yields (Ct>26Æ8, eight samples) significantly improved the discrimination between mutated and wild type samples. After exclusion, all positive samples found by sequencing were confirmed by HRM analysis while two cases remained false positive. R172 IDH2 mutations were found in two patients (1Æ2%) both by HRM and sequence analysis and two false positive samples were found by HRM (1Æ2%) (Tables SI and SII). One patient showed both an IDH1 R132C and IDH2 R140Q mutation. Allele frequencies of the mutations were measured by pyrosequencing (primer combinations are specified in Table SIV). The allele frequency of the IDH1 R132C mutation was 15Æ5%, while the IDH2 R140Q frequency was even lower (data not shown). Because IDH1 and IDH2 mutations are typically heterozygous and the total allele frequencies of both mutations was below 50%, we cannot conclude whether the mutations represent one and the same or independent clones. One patient exhibited a homozygous IDH2 R140W mutation, identified by both sequence and HRM analysis (Fig S2). SNP array analysis showed that the homozygous mutation was caused by uniparental disomy (UPD) on a large part of chromosome 15, including the IDH2 locus (Fig S3). Of the 32 patients with IDH mutations, we found cooccurrences with other chromosomal aberrations confirming data of other studies (Chou et al, 2010; Kosmider et al, 2010; Marcucci et al, 2010; Paschka et al, 2010; Wagner et al, 2010). Furthermore, none of the IDH1 or IDH2 mutated patients showed overexpression of MECOM (also known as EVI1) (Table SI). Low frequencies of mutated alleles may be missed using sequencing or HRM analysis. To test whether IDH1 mutations occur at frequencies under the detection limit of HRM analysis, we designed an allele specific QPCR for the most frequently occurring IDH1 R132H mutation (for primer

Identification of Varicella-Zoster Virus-Specific CD8 T Cells in Patients after T-Cell-Depleted Allogeneic Stem Cell Transplantation

ABSTRACT To study the role of CD8 T cells in the control of varicella-zoster virus (VZV) reactivation, we developed multimeric major histocompatibility complexes to identify VZV-specific CD8 T cells. Potential HLA-A2 binding peptides from the putative immediate-early 62 protein (IE62) of VZV were tested for binding, and peptides with sufficient binding capacity were used to generate pentamers. Patients with VZV reactivation following stem cell transplantation were screened with these pentamers, leading to the identification of the first validated class I-restricted epitope of VZV. In 42% of HLA-A2 patients following VZV reactivation, these IE62-ALW-A2 T cells could be detected ex vivo.

Exploratory analysis of 1936 Snps in Adme genes for association with busulfan clearance in adult hematopoietic stem cell recipients

Background Busulfan is used in preparative regimens before stem cell transplantation. There is significant interpatient variability in busulfan pharmacokinetics (PK) and exposure is related to outcome. Polymorphisms in genes encoding glutathione-S-transferases have been associated with busulfan PK but only explain a limited portion of the observed variability. Aim The aim of this study is to identify additional genetic variants associated with busulfan PK by interrogating 1936 variants in 225 genes involved in drug absorption, distribution, metabolism, and excretion (ADME). Materials and methods In an exploratory cohort (n=65), patients who received busulfan were genotyped with the DMET array. Top SNPs and haplotypes associated with busulfan clearance were validated in an independent validation cohort (n=78). Results In the exploratory cohort, seven variants were identified to be associated with busulfan clearance (P<0.001). In the validation cohort, only GSTA5 (rs4715354 and rs7746993) remained significantly associated with busulfan clearance (P=0.025). Conclusion This is the first study using an exploratory pharmacogenetic approach to explain the interindividual variability in busulfan PK. The role of glutathione-S-transferases was confirmed, but no additional genetic markers involved in drug ADME appear to be associated with busulfan PK.

Genome wide molecular analysis of minimally differentiated acute myeloid leukemia

This study used single nucleotide polymorphism (SNP)-array technology to study copy number changes and to determine regions of loss of heterozygosity in minimally differentiated acute myeloid leukemia. Several chromosomal regions were found to be deleted or duplicated, and mutations in 163gene were the most frequent mutations detected. Background Minimally differentiated acute myeloid leukemia is heterogeneous in karyotype and is defined by immature morphological and molecular characteristics. This originally French-American-British classification is still used in the new World Health Organization classification when other criteria are not met. Apart from RUNX1 mutation, no characteristic molecular aberrations are recognized. Design and Methods We performed whole genome single nucleotide polymorphism analysis and extensive molecular analysis in a cohort of 52 patients with minimally differentiated acute myeloid leukemia. Results Many recurring and potentially relevant regions of loss of heterozygosity were revealed. These point towards a variety of candidate genes that could contribute to the pathogenesis of minimally differentiated acute myeloid leukemia, including the tumor suppressor genes TP53 and NF1, and reinforced the importance of RUNX1 in this leukemia. Furthermore, for the first time in this minimally differentiated form of leukemia we detected mutations in the transactivation domain of RUNX1. Mutations in other acute myeloid leukemia associated transcriptions factors were infrequent. In contrast, FLT3, RAS, PTPN11 and JAK2 were often mutated. Irrespective of the RUNX1 mutation status, our results show that RAS signaling is the most important pathway for proliferation in minimally differentiated acute myeloid leukemia. Importantly, we found that high terminal deoxynucleotidyl transferase expression is closely associated with RUNX1 mutation, which could allow an easier diagnosis of RUNX1 mutation in this hematologic malignancy. Conclusions Our results suggest that in patients without RUNX1 mutation, several other molecular aberrations, separately or in combination, contribute to a common minimally differentiated phenotype.

Immune surveillance by autoreactive CD4-positive helper T cells is a common phenomenon in patients with acute myeloid leukemia

The importance of autologous T‐cell responses in immune surveillance against acute myeloid leukemia (AML) remains unclear. Therefore, we investigated the presence and functional reactivity of autoreactive T‐cell responses against autologous AML blasts.