Michelle McBean

Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

BackgroundThe JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity.MethodsWe developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3′dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2.ResultsWe showed that the addition of the 3′dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10-4 per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay.ConclusionsQuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.

KIT D816V mutation detection: a comparative study using peripheral blood, bone marrow aspirate and bone marrow trephine samples for detection of KIT mutations in patients with systemic mastocytosis

KIT D816V mutation detection: a comparative study using peripheral blood, bone marrow aspirate and bone marrow trephine samples for detection of KIT mutations in patients with systemic mastocytosis Rishu Agarwal, Michelle McBean, Chelsee Hewitt & David A. Westerman a Division of Cancer Medicine, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Australia b University of Melbourne, Parkville, Australia Published online: 01 Jun 2015.

Comprehensive genomic characterization dissects the complex biology of a case of synchronous Burkitt lymphoma and myeloid malignancy with shared hematopoietic ancestry

Burkitt lymphoma (BL) is a germinal center B-cell-derived malignancy characterized by oncogenic MYC activation through translocation with one of the immunoglobulin loci. This highly aggressive lymphoproliferative disorder typically responds to chemoimmunotherapy with 70–80% of patients achieving long-term remission [1]. Those that relapse tend to do so early with very few relapses after 24 months [1]. We present a case of a patient diagnosed with two separate incidences of BL five years apart along with comprehensive genomic assessment, demonstrating that the second episode of BL was derived from an unrelated clone that also gave rise to a therapy-related myeloid neoplasm. A 54-year-old otherwise healthy man with no significant family history of malignancy presented to his general practitioner with right lower facial and jaw pain. Investigation with magnetic resonance imaging (MRI) demonstrated widespread malignant bony infiltration involving the skull vault and mandible. A computed tomography (CT) scan from neck to pelvis revealed additional extensive skeletal lytic lesions. Bone marrow biopsy showed replacement of normal hematopoiesis with a monomorphic population of medium-sized cells with an open chromatin pattern and the immunophenotype CD10þ, BCL2 , BCL6þ, CD19þ, CD20þ, TdT with lambda light chain expression and a Ki-67 of 100%. Fluorescent in situ hybridization (FISH) testing demonstrated the presence of an IGH-MYC fusion. A diagnosis of BL was made and the patient was treated with rituximab, cyclophosphamide, vincristine, doxorubicin, and methotrexate (R-CODOX-M) alternating with rituximab, ifosfamide, etoposide, and cytarabine (R-IVAC) for four cycles in total, attaining complete remission. He remained in remission for five years and then represented with night sweats and loss of weight. A right iliac fossa mass was demonstrated on CT scan and a Ffluorodeoxyglucose (FDG) PET scan showed diffuse FDG uptake throughout the peritoneal cavity. Biopsy of the right iliac fossa mass again showed a monomorphic population of medium-sized cells with an open chromatin pattern and with the immunophenotype CD10þ, BCL2 , BCL6þ, CD20þ, TdT with a Ki-67 of 100%. No IGH-MYC fusion was detected by FISH using a MYC break-apart probe. Despite the absence of an IGH-MYC fusion, a diagnosis of BL was made based on morphological and immunophenotypic appearances. Bone marrow biopsy was morphologically unremarkable with no involvement by BL. The patient again underwent treatment with R-CODOX-M/R-IVAC and achieved a complete remission. He was monitored in remission for six months when he was noted to have progressive pancytopenia. A bone marrow biopsy showed trilineage morphological dysplasia with 15% blasts by morphology and a hypodiploid karyotype (44,XY,add(1)(q?32),add(2)(q34),del(2)(p11.2), del(5)(q13q33),add(7)(q11.2),add(10)(q22),add(13)(q?34), 17, ?t(19;22)(q13.3;q13), 21,þmar1). There was no evidence of relapsed BL on bone marrow biopsy or repeat FDGPET scan. A diagnosis of a therapy-related myeloid neoplasm (t-MN) was made and he commenced treatment with subcutaneous azacytidine. Repeat bone marrow biopsy after four cycles of therapy demonstrated less than 5% blasts by morphology and he underwent a matched unrelated donor allogeneic hematopoietic progenitor cell transplant with reduced intensity conditioning. We first sought to determine whether the two episodes of BL in this patient were clonally related. PCRbased B-cell receptor clonality analysis of the immunoglobulin heavy chain (IGH) was performed on DNA extracted from bone marrow with morphological involvement of BL at diagnosis (BL1) and an appendiceal tissue biopsy from the second episode of BL (BL2). The clonal

Incidental detection of germline variants of potential clinical significance by massively parallel sequencing in haematological malignancies

Massively parallel sequencing (MPS) technology has become routinely available for diagnosis, prognostication and therapeutic decision-making in haematological malignancies. However, increased throughput and wider coverage of genes can have unintended consequences. Germline variants of potential clinical significance (GVPCSs) detected during cancer testing may have implications for patients and families beyond the biological evaluation of a specific tumour. 721 reports generated from MPS panels used in the routine testing of myeloid and lymphoid malignancies were reviewed and variants within genes of potential germline relevance (TP53, RUNX1, GATA2 and WT1 in all contexts and CBL, KRAS and NRAS in the setting of juvenile myelomonocytic leukaemia) were analysed. A variant allele fraction threshold of ≥33.09% for considering germline origin of variants within cancer samples was established. The detection rate of incidental, pathogenic germline variants was 0.42%. Patient education and confirmatory germline sample testing of GVPCSs in appropriate circumstances are recommended.

Detection of clinically relevant early genomic lesions in B‐cell malignancies from circulating tumour DNA using a single hybridisation‐based next generation sequencing assay

Green, T.M., Young, K.H., Visco, C., Xu-Monette, Z.Y., Orazi, A., Go, R.S., Nielsen, O., Gadeberg, O.V., Mourits-Andersen, T., Frederiksen, M., Pedersen, L.M. & Møller, M.B. (2012) Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Journal of Clinical Oncology, 30, 3460–3467. Swerdlow, S.H., Campo, E., Pileri, S.A., Harris, N.L., Stein, H., Siebert, R., Advani, R., Ghielmini, M., Salles, G.A., Zelenetz, A.D. & Jaffe, E.S. (2016) The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood, 127, 2375–2390. Zhou, Z., Sehn, L.H., Rademaker, A.W., Gordon, L.I., Lacasce, A.S., Crosby-Thompson, A., Vanderplas, A., Zelenetz, A.D., Abel, G.A., Rodriguez, M.A., Nademanee, A., Kaminski, M.S., Czuczman, M.S., Millenson, M., Niland, J., Gascoyne, R.D., Connors, J.M., Friedberg, J.W. & Winter, J.N. (2014) An enhanced International Prognostic Index (NCCN-IPI) for patients with diffuse large B-cell lymphoma treated in the rituximab era. Blood, 123, 837–842.

ASXL1 c.1934dup;p.Gly646Trpfs*12—a true somatic alteration requiring a new approach

The additional sex combs-like 1 (ASXL1) gene has a central role in the epigenetic regulation of chromatin remodelling and subsequent gene transcription via multiple mechanisms. These include the regulation of histone H2A deubiquitination as well as polycomb group repressor complex 2 mediated homeobox (HOX) gene transcription. ASXL1mutations are a recurrent finding in myeloid malignancies, where they are typically heterozygous in keeping with a haploinsufficiency effect. Mutated ASXL1 status has been associated with an inferior overall survival in acute myeloid leukaemia (AML), myelodysplastic syndromes (MDS), chronic myelomonocytic leukaemia (CMML), myelofibrosis, aplastic anaemia and age-related clonal haematopoiesis. The majority of ASXL1 exon 12 mutations are frameshift or nonsense and result in a C-terminal truncation of the resulting gene product. Missense mutations are also detected but these appear not to have an effect on clinical outcome and are of uncertain significance. The most commonly detected ASXL1 mutation is ASXL1 NM_015338.5:c.1934dup;p.Gly646Trpfs*12 (ASXL1 c.1934dupG), accounting for approximately half of somatic truncating mutations. This duplication of a single guanine occurs within an eight base-pair mononucleotide guanine repeat sequence (8G repeat) that extends from c.1927 to c.1934. Areas of repetitive sequence may be prone to accelerated mutagenesis due to replication slippage. This occurs when DNA polymerase pauses and dissociates from repeated areas of sequence allowing the terminal portion of the newly synthesised strand to anneal to a different yet still complimentary location on the template. Resumption of DNA replication completes the slippage event, which may result in duplications or deletions. This process, however, has also been described as a source of polymerase chain reaction (PCR) sequencing artefact. This fact, coupled with the detection by Sanger sequencing and mass spectrometry of ASXL1 c.1934dupG within the buccal DNA of individuals with myeloid malignancies and by Sanger sequencing in the granulocyte DNA of those without, has led some to assert that this variant is not a real somatic alteration. In addition, ASXL1 c.1934dupG has been reported at a frequency of between 0.001634% (Exome Aggregation Consortium) and 2.58% (Exome Sequencing Project) in the general population by whole-exome sequencing. Despite the fact that ASXL1 may be mutated in otherwise well individuals with agerelated clonal haematopoiesis, these detection frequencies may be overestimated due to artefact-related false-positive ASXL1 c.1934dupG detection. Various evidences in support of ASXL1 c.1934dupG being a true somatic alteration have been put forward. These include an inability to reproduce ASXL1 c.1934dupG detection consistently in samples known not to contain a myeloid malignancy (likely due to the use of high fidelity polymerases) and a failure to differentiate patients harbouring ASXL1 c.1934dupG and those with other truncating ASXL1 mutations by clinical outcome or gene expression profiling. However, these lines of evidence either rely on sequencing of the ASXL1 8G repeat or are circumstantial in nature. We aimed to evaluate the performance of various methodologies for the detection of ASXL1 c.1934dupG and to assess whether it is a true somatic alteration utilising a mutation-specific assay.

Clinicopathological differences exist between CALR- and JAK2 -mutated myeloproliferative neoplasms despite a similar molecular landscape: data from targeted next-generation sequencing in the diagnostic laboratory

Mutations in CALR have recently been detected in JAK2-negative myeloproliferative neoplasms (MPNs) and are key pathological drivers in these diseases. CALR-mutated MPNs are shown to have numerous clinicopathological differences to JAK2-mutated MPNs. The basis of these differences is poorly understood. It is unknown whether these differences result directly from any differences in intracellular signalling abnormalities induced by JAK2/CALR mutations or whether they relate to other phenomena such as a differing spectrum of genetic lesions between the two groups. We aimed to review the clinicopathological and molecular features of CALR- and JAK2-mutated MPNs from samples referred for diagnostic testing using a custom-designed targeted next-generation sequencing (NGS) panel. Eighty-nine CALR-mutated cases were compared with 70 JAK2-mutated cases. CALR-mutated MPNs showed higher platelet counts and a female predominance as compared to JAK2-mutated MPNs in our cohort. We have also observed differences between CALR mutation subtypes in terms of disease phenotype, mutational frequency and allelic burden. Type 1 CALR mutations were found to be more common in myelofibrosis, associated with a higher frequency and number of additional mutations and a higher mutant allelic burden as compared to type 2 CALR mutations. Despite these biological differences, our molecular characterisation suggests that CALR- and JAK2-mutated MPNs are broadly similar in terms of the quantity, frequency and spectrum of co-occurring mutations and therefore observed biological differences are likely to not be heavily influenced by the nature and quantity of co-mutated genes.

Sensitive NPM1 Mutation Quantitation in Acute Myeloid Leukemia Using Ultradeep Next-Generation Sequencing in the Diagnostic Laboratory

Context Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. Objective To investigate the feasibility of using next-generation sequencing (NGS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000×-500 000×) sequencing (NGS-q NPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. Design A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-house-designed bioinformatic pipeline. NGS-q NPM1 was compared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparameter flow cytometry). Results The NGS-q NPM1 assay had a sensitivity of between 10-4 and 10-5 and showed high concordance and correlation with reference methodologies. Moreover, the NGS-q NPM1 assay was able to be integrated into the laboratorys existing, targeted amplicon-based sequencing workflow. Conclusions An NGS-based, quantitative NPM1-mutation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities.

Mutation analysis for systemic mastocytosis

Mast cells from over 80% of adult systemic mastocytosis (SM) patients carry an activating mutation at codon 816 of the KIT gene. In fact, a KIT mutation constitutes one of the minor WHO 2008 classification criteria for SM. The most common is the c.2447A>T: p.Arg816Val (D816V) mutation. The KIT gene encodes a type III transmembrane receptor tyrosine kinase whose ligand is stem cell factor. Activation of KIT mediates proliferation and maturation signalling. D816 V causes constitutive activation of the KIT tyrosine kinase activity, which also results in resistance to the tyrosine kinase inhibitor imatinib mesylate. Therefore knowing the mutational status of KIT codon 816 is important for diagnosis and treatment strategy. The population of mutant mast cells is often low in relation to normal cells and so a sensitive molecular detection technique is required. Several have been reported in the literature, the most sensitive include RT-PCR + RFLP, PNA-mediated PCR and allele-specific PCR. Within the diagnostic setting the Peter MacCallum uses the rapid and highly sensitive allele-specific competitive blocker PCR (ACB-PCR) assay with a sensitivity level of between 1 and 0.1%. Between Jan 2008 and Oct 2010, 338 samples were tested, of which 58% were positive.