Our assays

Haem-onc Next Generation Sequencing (NGS) panel

The Twist Haem-onc NGS panel is a targeted next-generation sequencing (NGS) panel in which full coding exons of 75 genes relevant to haemato-oncology are sequenced. It can detect single nucleotide substitutions and small insertion/deletion variants with a formal limit of detection of 5% variant allele frequency (VAF, e.g. 10% of cells in a sample harbouring a heterozygous mutation). It is therefore not adequately sensitive for minimal residual disease monitoring. Variants with a lower VAF will be reported with a caveat that they are low level and these should be interpreted with caution.

The full gene list is shown below, but subpanels of genes are analysed and reported depending on the clinical indication. The genes analysed in each sample will be detailed on the methodology section at the bottom of the report. If information regarding variants in additional genes, that have been sequenced by the panel but not analysed or reported, is required this can be performed upon request by contacting the laboratory.

Variant reporting

Variants are reported as pathogenic, likely pathogenic or a variant of uncertain significance. Reports will also give a comment about the prognostic significance of variants, where the diagnosis is known (either from specific clinical details on the request form or from CUH HODS laboratory reports) and where prognostic associations are clearly established in the literature.

Annotations of whether variants are pathogenic are made by searching several databases of variants in cancer, together with databases of genomic variation from large population sequencing studies. Pathogenic variants are generally considered to be those that have been reported recurrently in cancer but are absent or very rare in population studies, or alternatively have a very clear functional effect (e.g. a truncating mutation in a known tumour suppressor gene). Note that standardised criteria for annotation of pathogenicity are not available in cancer so at present in-house consensus guidance is used.

Variants of uncertain significance are variants for which there is currently inadequate evidence to confidently support a pathogenic effect. Some such variants might represent rare germline variants that are of no consequence (Single Nucleotide Polymorphisms, SNPs), or acquired passenger mutations that have occurred in the cancer clone but do not contribute to disease to the disease process. Others may be pathogenic, but there is not adequate information (e.g. reported recurrence in other cancers) to state this currently. Note that such information could change in future, but we will not review historical reports routinely unless requested to do so.

Some genes on the panel can be mutated in germline predispositions to haematological malignancies (e.g. RUNX1, CEBPA, DDX41). Where a potential germline variant is identified, reports may recommend consideration of further testing on a remission sample and/or germline sample. This should be performed after appropriate consent and some patients will require referral to clinical genetics.

Where variant annotation is particularly challenging or a possible germline variant is identified, cases may be referred to the haem-onc genomics tumour advisory board (GTAB) for further discussion, either before or after report verification.

The reporting team is always happy to discuss any variants if the interpretation is not clear from the report.

Indications and subpanels

1. Acute leukaemia, chronic myeloid disorders, bone marrow failure or cytopenias

2. Chronic lymphycytic leukaemia (CLL)

1. Other lymphoid malignancies

TP53 alone is analysed as standard. In individual cases, specific additional targets or, after clinical discussion, an extended panel can be analysed on request.

STAT3 / STAT5B analysis can be requested in cases of possible T-large granulocytic leukaemia.

Please note that MYD88 and BRAF standalone tests for lymphoplasmacytic lymphoma and hairy cell leukaemia are currently performed using different assays.