Gill Wilson

c-kit proto-oncogene exon 8 in-frame deletion plus insertion mutations in acute myeloid leukaemia.

Genomic DNA from 60 cases of acute myeloid leukaemia (AML) was screened for mutations in the c‐kit gene. DNA from all 21 exons was subjected to polymerase chain reaction (PCR) amplification and analysis by conformation sensitive gel electrophoresis (CSGE); exons showing altered CSGE patterns were then sequenced. Mutations were identified only in those patients with inv(16) (3/7 cases) or t(8;21) (1/2 cases) and comprised three in‐frame deletion plus insertion mutations (exon 8) and one point mutation (exon 10, GTA → ATA, Val530Ile). Exons 8 and 10 were then analysed in 31 further cases of inv(16) (n = 14) and t(8;21) (n = 17), revealing four additional exon 8 in‐frame deletion plus insertion mutations, all of which were in cases of inv(16). All exon 8 in‐frame deletion plus insertion mutations (n = 7) involved the loss or repacement of the codon for Asp419 which is highly conserved cross species and is located in the receptors extracellular domain. The high frequency of the c‐kit proto‐oncogene exon 8 deletion plus insertion mutations in AML suggests an essential role for this region of the receptors extracellular domain. The association with inv(16) invites speculation as to the link between these two changes in the pathogenesis of AML.

Identification of novel regions of amplification and deletion within mantle cell lymphoma DNA by comparative genomic hybridization

Summary. We have carried out comparative genomic hybridization (CGH) analysis on archival biopsy material from a series of 30 UK mantle cell lymphomas. The most frequent aberrations were gains of 3q (21 cases), 6p (19 cases), 7q (8 cases), 12p (8 cases), 12q (9 cases) and 17q11q21 (8 cases), and losses of 1p13p32 (10 cases), 5p13p15.3 (9 cases), 6q14q27 (11 cases), 8p (7 cases), 11q13q23 (8 cases) and 13q (18 cases). Nineteen cases (63%) had a common region of amplification at 3q28q29, which was highly amplified in three cases, suggesting the presence of a mantle cell lymphoma (MCL)‐related oncogene in this region. There was a minimal common region of deletion at 6q25q26 in nine cases (30%). No MCL‐specific locus has previously been identified on chromosome 6 and this region may contain a tumour suppressor gene specifically implicated in the development of this subtype of lymphoma. An increased number of chromosome aberrations, gain of Xq and loss of 17p were all significantly associated with a worse prognosis. A greater understanding of the genetics of mantle cell lymphoma may allow the identification of prognostic factors which will aid the identification of appropriate treatment regimens.

Cytogenetically cryptic AML1–ETO and CBFβ–MYH11 gene rearrangements: incidence in 412 cases of acute myeloid leukaemia

The rearrangements t(8;21)(q22;22) and inv(16)(p13q22) are two of the most frequently seen in acute myeloid leukaemia (AML), accounting for 8% and 4% of cases respectively. Detection of these abnormalities is important for disease management as both are associated with good responses to conventional chemotherapy and prolonged disease‐free survival. Recent reports using reverse transcriptase polymerase chain reaction (RT‐PCR) suggest that significant proportions of AML cases without a visible t(8;21) or inv(16) show expression of an abnormal fusion gene transcript and, consequently, they could not be detected using conventional cytogenetic analysis alone. We present here a four centre study involving 412 cases of AML screened using both standard cytogenetics and RT‐PCR for AML1–ETO and CBFβ–MYH11. We detected a cytogenetic t(8;21) in 31 out of 412 (7·5%) cases and an inv(16) or t(16;16) variant in 27 out of 412 (6·6%) cases. RT‐PCR detected only two cases (0·5%) of cryptic t(8;21) and no instances of cryptic inv(16). Both cryptic t(8;21) cases had the classic M2 FAB morphology for this type of disease. Our data concur with the established FAB type distribution of the rearrangements and indicate that cryptic t(8;21) and inv(16) may be much less frequent than reported elsewhere.

Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia

Molecular testing for the BCR‐ABL1 fusion gene by real time quantitative polymerase chain reaction (RT‐qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT‐qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

Mutational analysis of class III receptor tyrosine kinases (C-KIT, C-FMS, FLT3) in idiopathic myelofibrosis.

Summary. Genomic DNA from patients with idiopathic myelofibrosis (IMF) was screened by polymerase chain reaction (PCR) and conformation sensitive gel electrophoresis (CSGE) for mutations in the C‐KIT gene (60 patients), as well as the C‐FMS and FLT3 genes (40 patients). Intronic primers were used to amplify the entire coding region of both the C‐KIT and C‐FMS genes, and selected regions of the FLT3 gene. CSGE and direct DNA sequencing detected all previously reported as well as several novel polymorphisms in each of the genes. A novel c‐fms exon 9 mutation (Gly413Ser) was detected in two patients. Its functional significance remains to be determined. The c‐kit mutation Asp52Asn, previously described in two of six IMF patients in Japan, was not detected in this study. In addition, the reported c‐fms mutations involving codons 301 and 969 were not identified. Therefore, in contrast to acute myeloid leukaemia, mutations in RTKs class III do not appear to play a significant pathogenetic role in idiopathic myelofibrosis.

A certified plasmid reference material for the standardisation of BCR–ABL1 mRNA quantification by real-time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR–ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR–ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR–ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Are aberrant BCR-ABL transcripts more common than previously thought?

We report the use of multiplex polymerase chain reaction (PCR), using 4% polyacrylamide gel electrophoresis (PAGE) for the detection of BCR‐ABL transcripts in Philadelphia‐positive disease. Three out of 50 cases [two out of 37 chronic myeloid leukaemia (CML), one out of 13 acute lymphoblastic leukaemia (ALL)] possessed rare breakpoints; an e19a2 and e13a3 in CML and an e1a3 in the ALL. We suggest that multiplex PCR using 4% PAGE and optimized for smaller transcript detection may lead to a higher detection rate of rare BCR‐ABL breakpoints. Multiplex PCR, however, failed to distinguish e13a2 from e1a3 transcripts. Finally, the presence of e13a3 in CML supports the view that abl exon 2 sequences are unnecessary for the pathogenesis of ‘classic’ CML.

Mutations in PTPN11 are uncommon in adult myelodysplastic syndromes and acute myeloid leukaemia

The PTPN11 gene encodes the ubiquitously expressed nonreceptor-type protein tyrosine phosphatase SHP-2 (src homology region 2-domain phosphatase-2) (Neel, 1993). SHP-2 is a key molecule in the cellular response to growth factors, hormones, cytokines, and cell adhesion molecules and is required for the activation of the RAS/MEK/ERK kinase cascade (Cunnick et al, 2002). Recently, PTPN11 has been identified as the Noonan syndrome disease gene, through use of a positional candidacy approach (Tartaglia et al, 2001). As a result of the rare association of juvenile myelomonocytic leukaemia (JMML) and Noonan syndrome, Tartaglia et al (2003) screened non-syndromic JMML patients for PTPN11 mutations and documented somatic changes in 34% of cases. Furthermore, the same group reported mutations in 10% and 4% of children with myelodysplasic syndromes (MDS) and de novo acute myeloid leukaemia (AML) respectively. These acquired mutations were predicted to cause gain-of-function of SHP-2 through preferential occupation of the activated state of the phosphatase. To assess the pathogenetic relevance of PTPN11 mutations in adult myeloid disorders, we have undertaken mutational analysis of exons 2, 3, 4, 7, 8 and 13 of PTPN11 in MDS and AML. Genomic DNA was obtained from peripheral blood or bone marrow from 107 cases of MDS, refractory anaemia (RA; n 1⁄4 20), RA with ringed sideroblasts (RARS; n 1⁄4 20), RA with excess blasts (RAEB; n 1⁄4 30), RAEB in transformation (RAEBt; n 1⁄4 2) and chronic myelomonocytic leukaemia (n 1⁄4 35). Genomic DNA was also obtained at presentation of 64 cases of AML entered into the Medical Research council (MRC) AML X and XII Trials. The cases were classified according to the French–American–British (FAB) criteria, as: M0 (n 1⁄4 4), M1 (n 1⁄4 8), M2 (n 1⁄4 13), M3 (n 1⁄4 10), M4 (n 1⁄4 13), M5 (n 1⁄4 10), M6 (n 1⁄4 6). Genomic DNA was also prepared from five cases of JMML [four de novo, one associated with neurofibromatosis type 1 (NF1)], and from the peripheral blood of 35 normal individuals using the Nucleon Biosciences BACC II kit. Genomic DNA was amplified using polymerase chain reaction and the coding sequences and intron/exon boundaries corresponding to exons 2, 3, 4, 7, 8 and 13 were screened as described by Tartaglia et al (2001). Conformation sensitive gel electrophoresis (CSGE) was used to screen for mutations and samples displaying abnormal CSGE profiles were purified (Qiagen) and sequenced (MWG Biotech). Screening 64 cases of AML revealed a single missense mutation (1Æ5%). The patient, classified as M2 with normal cytogenetics, exhibited an exon 3 C fi G transition at nucleotide 218 that is predicted to result in a threonine to isoleucine substitution at codon 73. Interestingly, this individual did not possess a RAS (N-, or Ki), c-FMS, FLT3-ITD, FLT3835 or c-KIT816 mutation. A silent change was identified in exon 2 in another AML patient (nucleotide 48A fi G; Ala16Ala). None of the 107 MDS cases exhibited a PTPN11 mutation. However, one of the four de novo JMML cases possessed an exon 3 G fi A transversion at position 226, which is predicted to result in a glutamic acid to lysine substitution at codon 76. A further de novo JMML case possessed an N-RAS12 mutation but was negative for a PTPN11 mutation, while the NF1-associated JMML was negative for both RAS and PTPN11 mutations. All reported mutations affect residues located at the NSH2 and PTP interacting surfaces and result in gain-offunction of SHP-2 through preferential occupation of the phosphatase’s activated state (Tartaglia et al, 2001, 2003). The two mutations identified in this study, which are located in the N-SH2 domain, have previously been associated with de novo and Noonan syndrome-associated JMML, as well as with paediatric MDS (Tartaglia et al, 2003). Our results suggest that PTPN11 mutations are not common in adult AML, occurring in approximately 1Æ5% of cases. Interestingly, the Thr73Ile mutation, which occurred in a case of AML M2, was not associated with a RAS, cFMS, FLT3 or c-KIT mutation, supporting the concept of mutual exclusivity of RTK/RAS pathway mutations. In contrast to the 10% reported frequency of PTPN11 mutations in paediatric MDS (Tartaglia et al, 2003), none of the 107 adult MDS cases possessed a mutation, suggesting a difference in the pathogenesis of adult and paediatric MDS. Finally, the results from our small study of JMML patients supports the findings of Tartaglia et al (2003), that defects in the regulatory components of the mitogen-activated protein kinase cascade, namely RAS, neurofibromin and SHP-2, appear to be mutually exclusive.

Long-term dual donor derived haematopoietic reconstitution following double unrelated cord blood transplantation – single unit dominance is not always the case

In the recent Cord Blood Special Issue of the British Journal of Haematology, several reviews discussed double unrelated cord blood transplantation in detail (Delaney et al, 2009; Fernandez, 2009; Gluckman, 2009). The reviews highlighted the ultimate haematopoietic dominance of one cord blood unit over the other. Single unit cord unit dominance occurs relatively early post-transplant in the majority of patients and appears to be universal within 1 year post-transplant, irrespective of the intensity of conditioning regimen (Barker et al, 2005; Ballen et al, 2007; Brunstein et al, 2007). Potential mechanistic aspects were also discussed, although the biological factors which determine the ‘winning’ cord unit remain unclear (Delaney et al, 2009; Gluckman, 2009). At odds with these observations, we describe a patient treated with double Umbilical Cord Blood (UCB) transplantation in which there was full donor hematopoietic reconstitution with substantial and sustained contributions from both UCB sources well beyond 1 year post-transplant. In March 2005, a 21-year-old Caucasian female was diagnosed with high risk acute promyelocytic leukaemia (white cell count 170 · 10/l). Cytogenetic and molecular analysis confirmed t(15:17) translocation with PML–RARA fusion gene. Despite intensive treatments [PETHEMA (Programa para el Estudio y Tratamiento de las Hemopatias Maligna) regimen, gemtuzumab ozogamicin, arsenic trioxide and idiarubicin], she remained persistently minimal residual disease (MRD) positive both in blood and bone marrow by real-time quantitative polymerase chain reaction (RQ-PCR). In November 2006, magnetic resonance imaging confirmed leptomeningeal enhancement consistent with central nervous system (CNS) disease, whilst bone marrow examination confirmed medullary relapse. Two cycles of salvage chemotherapy (high dose methotrexate with high dose cytarabine) achieved both complete haematological and molecular remission, and clearance of CNS disease. With no siblings available, repeated worldwide unrelated donor registry search failed to identify a suitable donor and two suitable mismatched UCB units were identified for the patient (HLA-A*0301, 2402; B*1518, 1801; Cw*0501, 0704; DRB1*0407, 1301; DQB1*0301/09, 0603). Unit A was an 3/8 allele match (A*0301, 2403; B*3501/42, -;Cw*0401/09N, -; DRB1*0407, 1301; DQB1* NT) with a nucleated cell dose 4Æ89 · 10/kg. Unit B was a 5/10 allele match (A*0301, 2402; B*3801, 3548; Cw*0702, 1203; DRB1*0407, 1301; DQB1*0302, 0603) with a nucleated cell dose 3Æ2 · 10/kg. The units were selected to be allele matched at the HLA-DRB1 locus and no more than two antigen mismatched at the HLA-A and B loci combined between both patient and units as well as between units. In February 2007, the patient underwent double UCB transplant after conditioning with cyclophosphamide 120 mg/ kg and total body irradiation (12 Gy) and alemtuzumab (total dose 100 mg). Additional radiotherapy (5Æ4 Gy in three fractions) was given to the cranium before the transplant. Neutrophil engraftment (>0Æ5 · 10/l on two consecutive days) occurred at day +44 and platelet engraftment (platelet count>20 · 10/l without platelet support) on day +67. There was no acute graft-versus-host disease or severe infective episodes. At 32 months post-transplant, the patient remained well. Bone marrow and peripheral blood RQ-PCR remained molecularly negative for the PML-RARA transcript. Donor chimerism was analysed using the Promega Powerplex 16 System (Promega, Madison, WI, USA), which co-amplifies 15 short tandem repeat loci and amelogenin, fluorescently detected on an abi prism 3730 system with GeneMapper software (Applied Biosystems, Foster City, CA,

Is routine molecular screening for common α-thalassaemia deletions necessary as part of an antenatal screening programme?

Antenatal sickle and thalassaemia screening programmes are now established in most high prevalence areas in England. Although screening reliably detects β-thalassaemia trait, in many cases, results state that α-thalassaemia trait cannot be excluded. The detection of couples at risk of a child with hydrops fetalis is one of the aims of the national programme. We, therefore, performed polymerase chain reaction (PCR) for the common α-thalassaemia gene deletions to assess the usefulness of this technique in routine screening practice. Between August 2001 and August 2002, of the 5092 women booked at the antenatal clinic, 425 were found to have a mean corpuscular haemoglobin (MCH) <27 pg in the absence of β-thalassaemia trait; 189 (44.5%) had an MCH <25 pg. All 425 patients underwent PCR analysis for the common deletions: –SEA (South-East Asian), –MED (Mediterranean), −α 20.5, –FIL (Filipino), −α 3.7 and −α 4.2 genotypes. In total, 130 (31%) women were positive for α-thalassaemia deletion; 86 (24.7%) were heterozygous for −α 3.7, 19 (4.4%) were homozygous for −α 3.7, 12 (2.8%) were heterozygous for −α 4.2, 1 (0.2%) was homozygous for −α 4.2, 11 (2.6%) were heterozygous for –SEA and one (0.2%) was heterozygous for the –MED genotype. Although the detection rate for α +-thalassaemia was high, a strategy of selective screening using MCH <25 pg and ethnic group (SEA, Middle East or Eastern MED) would have identified all individuals heterozygous for α 0-thalassaemia. Routine molecular screening for all forms of α-thalassaemia trait is unjustified in antenatal screening.