U zur Stadt

Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

New insights to the MLL recombinome of acute leukemias

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

The MLL recombinome of acute leukemias

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

Genotype–phenotype study of familial haemophagocytic lymphohistiocytosis due to perforin mutations

Background: PRF1 gene mutations are associated with familial haemophagocytic lymphohistiocytosis type 2 (FHL2). Genotype–phenotype analysis, previously hampered by limited numbers of patients, was for the first time performed by data pooling from five large centres worldwide. Patients and methods: Members of the Histiocyte Society were asked to report cases of FHL2 on specific forms. Data were pooled in a common database and analysed. Results: The 124 patients had 63 different mutations (including 15 novel mutations): 11 nonsense, 10 frameshift, 38 missense and 4 in-frame deletions. Some mutations were found more commonly: 1122 G→A (W374X), associated with Turkish origin, in 32 patients; 50delT (L17fsX22) associated with African/African American origin, in 21 patients; and 1090-91delCT (L364fsX), in 7 Japanese patients. Flow cytometry showed that perforin expression was absent in 40, reduced in 6 and normal in 4 patients. Patients presented at a median age of 3 months (quartiles: 2, 3 and 13 months), always with fever, splenomegaly and thrombocytopenia. NK activity was absent in 36 (51%), ⩽2% in 18 (26%), 3–⩽5% in 10 (14%), >5% in 4 (6%), “reduced” in 2 (3%) (not reported, n = 54). Nonsense mutations were significantly associated with younger age at onset (p<0.001) and absent natural killer activity (p = 0.008). Conclusion: PRF1 mutations are spread over the functional domains. Specific mutations are strongly associated with Turkish, African American and Japanese ethnic groups. Later onset and residual cytotoxic function are observed in patients with at least one missense mutation.

X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis.

X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.

Mutations in AP3D1 associated with immunodeficiency and seizures define a new type of Hermansky-Pudlak syndrome

Genetic disorders affecting biogenesis and transport of lysosome-related organelles are heterogeneous diseases frequently associated with albinism. We studied a patient with albinism, neutropenia, immunodeficiency, neurodevelopmental delay, generalized seizures, and impaired hearing but with no mutation in genes so far associated with albinism and immunodeficiency. Whole exome sequencing identified a homozygous mutation in AP3D1 that leads to destabilization of the adaptor protein 3 (AP3) complex. AP3 complex formation and the degranulation defect in patient T cells were restored by retroviral reconstitution. A previously described hypopigmented mouse mutant with an Ap3d1 null mutation (mocha strain) shares the neurologic phenotype with our patient and shows a platelet storage pool deficiency characteristic of Hermansky-Pudlak syndrome (HPS) that was not studied in our patient because of a lack of bleeding. HPS2 caused by mutations in AP3B1A leads to a highly overlapping phenotype without the neurologic symptoms. The AP3 complex exists in a ubiquitous and a neuronal form. AP3D1 codes for the AP3δ subunit of the complex, which is essential for both forms. In contrast, the AP3β3A subunit, affected in HPS2 patients, is substituted by AP3β3B in the neuron-specific heterotetramer. AP3δ deficiency thus causes a severe neurologic disorder with immunodeficiency and albinism that we propose to classify as HPS10.

A new functional assay for the diagnosis of X-linked inhibitor of apoptosis (XIAP) deficiency

X‐linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for nucleotide‐binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (TNF) production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18‐MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, six female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18‐MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while fewer than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.

KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia

High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18–30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.

MRD at the end of induction therapy in childhood acute lymphoblastic leukemia : outcome prediction strongly depends on the therapeutic regimen

MRD at the end of induction therapy in childhood acute lymphoblastic leukemia: outcome prediction strongly depends on the therapeutic regimen

Molecular analysis and clinical aspects of four patients with Chédiak‐Higashi syndrome (CHS)

To the Editor : Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by mutations in the CHS1 gene that consists of 51 coding exons and encodes for a 429-kD protein involved in intracellular trafficking (1–4). The disease was first mentioned by Beguez-Cesar (5), albeit the syndrome is named after Chédiak and Higashi, who first described its characteristic diagnostic findings (6, 7). More than two thirds of patients with CHS have the childhood form of the disease and experience at least one life-threatening ‘accelerated phase’, which corresponds to the clinical picture of hemophagocytic lymphohistiocytosis (HLH) (8). Without early stem cell transplantation most patients with CHS die in their first decade of life due to accelerated phases or severe infections (9). There are milder forms of CHS, the ‘adolescent’ and ‘adult’ form. These children survive until adulthood without experiencing an ‘accelerated phase’ and present with mostly neurological dysfunctions. We here describe the clinical and molecular characteristics of four patients with CHS. For polymerase chain reaction (PCR) amplification, genomic DNA was isolated from peripheral blood using standard methods. After PCR at an annealing temperature between 56 and 60◦C the products were extracted by agarose gel electrophoresis, purified, directly sequenced using BigDye Terminator v1.1 (Applied Biosystems, Weiterstadt, Germany), and run on ABI PRISM 310 or 3730 DNA analyzers. The exons containing a mutation in patients 1 and 3 were amplified in their parents as well. Primer sequences are available on request. The DNA mutation numbering was based on the numbering of (10). Patient 1 showed a homozygous 1 bp insertion in exon 7 (c.575insT). His parents showed equivalent heterozygous mutations. From birth on he suffered from recurrent skin infections and bruised easily. His hair was light blond, the eyes blue. After a cardiogenic shock due to ventricular tachycardia after diphtheria, pertussis, tetanus (DPT) immunization at the age of 4 months, the boy developed a severe pneumonia with atelectasis and a lung abscess at the age of 4 years. After surgical and antibiotic therapy he recovered. At this time the analysis of a blood smear led to the diagnosis of CHS. After a second pneumonia at the age of 6 years he received a long-term medication with antibiotics and antimycotics. Under this therapy severe infections no longer occurred. At present the patient is 17 years old, has a global development delay, but is in good physical condition. No accelerated phase has been observed until now. Patient 2 showed a homozygous 1 bp deletion in exon 17 (c.5004delA). His dark-grey silvery shining hair and a very pale skin colour were already noticed at birth. At the age of 1 year he presented with high fever, lymphadenopathy, pancytopenia and massive hepatosplenomegaly. CHS was diagnosed by bone marrow cytology. The accelerated phase was treated by immunosuppressive therapy with complete remission. Several mild infections followed. At the age of 2 years the boy presented with a second accelerated phase. He received a successful bone marrow transplantation (BMT) at the age of 3 years. His psychomotoric development within the following 2 years was normal. He is now 11 years old. Patient 3 showed a homozygous 1 bp deletion in exon 20 (c.5519 delC). His parents showed equivalent heterozygous mutations. The patient’s hair and skin were slightly grey and he had a partial ocular albinism. He first presented at the age of 3 years with an otitis media and mastoiditis without response to antibiotic therapy and slow recovery. After several milder infections, the boy presented again at the age of 8 years with an acute severe Epstein-Barr virus (EBV) infection. At this point a bone marrow cytology confirmed