AmpliVar: mutation detection in high-throughput sequence from amplicon-based libraries.

Conventional means of identifying variants in high-throughput sequencing align each read against a reference sequence, and then call variants at each position. Here, we demonstrate an orthogonal means of identifying sequence variation by grouping the reads as amplicons prior to any alignment. We used AmpliVar to make key-value hashes of sequence reads and group reads as individual amplicons using a table of flanking sequences. Low-abundance reads were removed according to a selectable threshold, and reads above this threshold were aligned as groups, rather than as individual reads, permitting the use of sensitive alignment tools. We show that this approach is more sensitive, more specific, and more computationally efficient than comparable methods for the analysis of amplicon-based high-throughput sequencing data. The method can be extended to enable alignment-free confirmation of variants seen in hybridization capture target-enrichment data.

Genomic characterisation of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a genomically heterogenous disease comprised of many subtypes that display significantly different clinical outcomes, in the context of treatment with conventional immunochemotherapy. Poor clinical outcomes in some subtypes, and imperfect identification of high risk individuals in otherwise low risk subgroups, demonstrate there is room for improvement in the subclassification and risk stratification of DLBCL. In addition, more comprehensive profiling may lead to improved molecular testing guided treatment selection. Existing characterisation and risk stratification strategies, such as division of DLBCL into activated B-cell (ABC) and germinal centre B-cell (GCB) subtypes, although prognostically useful, may oversimplify the underlying biology and have proven to be less useful in improving therapy selection. Several groups have proposed more predictive molecular testing based prognostic models with potentially more relevance to therapy choice. These alternative approaches use more resource intensive comprehensive genomic profiling strategies which present practical challenges to implement in diagnostic laboratories. The addition of genomic testing to the subclassification of DLBCL shows promise, but laboratories must identify testing strategies relevant to clinical practice. A consensus on optimal molecular profiling techniques is yet to be achieved. In this article we review various next generation sequencing-based analytical techniques and molecular classification models proposed recently. Emerging therapeutics where molecular profiling may guide patient selection are also reviewed. The potential utility of genomic testing in DLBCL is discussed, in addition to practical considerations when considering introducing genomics into the diagnostic laboratory.

Laboratory quality assessment of candidate gene panel testing for acute myeloid leukaemia: a joint ALLG / RCPAQAP initiative.

Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories. This study compared the results obtained from MPS testing of ten diagnostic AML bone marrow aspirate samples sent to eight participating laboratories across Australasia. A reassuringly high concordance of 94% was observed with regard to variant detection and characterisation of pathogenicity. The level of discordance observed, although low, demonstrates the need for ongoing assessment of concordance between diagnostic testing laboratories through quality assurance programs.

Assessing the safety of cytotoxic T lymphocytes transduced with a dominant negative transforming growth factor-beta receptor.

Transforming growth factor (TGF)-beta, a pleiotropic cytokine that regulates cell growth, is secreted by many human tumors and markedly inhibits tumor-specific cellular immunity. It has previously been shown by our group that transduction of cytotoxic T lymphocytes (CTLs) with a retroviral vector expressing the dominant-negative TGFbeta type II receptor (DNR) overcomes this tumor evasion in a model of Epstein-Barr virus (EBV)-positive Hodgkin disease. TGFbeta is an important physiologic regulator of T-cell growth and survival, however, abrogation of this regulatory signal in genetically modified cells is potentially problematic. To ensure that unresponsiveness to TGFbeta did not lead to the unregulated growth of genetically modified CTLs, the characteristics of DNR-transduced CTLs in vivo were studied. Donor C57BL6 mice were vaccinated with human papillomavirus-E7 plasmid DNA to induce production of E7-specific CTLs. The E7-specific CTLs were genetically modified to express enhanced green fluorescent protein (GFP) or DNR and administered to syngeneic mice. All mice received monthly boosts with E7 DNA for 9 months, and during this time, transduced CTLs were detected in the peripheral blood of most of the mice using a quantitative real-time polymerase chain reaction. By 12 months, 3 months after cessation of vaccination, no DNR-transduced CTLs or GFP-transduced CTLs were detected in the peripheral blood. There were 4 cases of lymphoma (2 DNR-transduced mice and 2 control mice): all tumors were CD3-/CD8- and were also negative for the DNR transgene. Hence, mature antigen-specific cytotoxic T cells can be genetically modified to resist the antiproliferative effects of TGFbeta without undergoing spontaneous lymphoproliferation in vivo. They may be of value for treating human cancers, which use TGFbeta as a powerful immune evasion mechanism.