To enhance the diagnosis of unclassifiable, non-CLL B-LPDs using next-generation sequencing technology.

In recent years, next generation sequencing has revealed the genomic landscape of lymphoid disorders and identified mutations that have improved our understanding of their pathogenesis. It has also revealed new targets for drug development. While some of these mutations, such as the BRAF V600E mutation in Hairy Cell Leukaemia (HCL)2, are now accepted as disease defining mutations, others such as MYD88 and NOTCH1/2 mutations are found in more than one subtype of B-LPD3. The overlapping nature of some of these molecular aberrations could have important implications for treatment of these disorders as we move towards targeted therapy. EZH2 inhibitors, which are currently in early phase trials, are one such example of targeted therapy for B-LPDs based on mutations identified by next generation sequencing (NGS)4. The genetic makeup of these tumours is also likely to influence future classification systems.

At present, an integrated approach incorporating morphology and immunophenotyping remains integral to the classification of B-LPDs. The Haemato-oncology department at the Royal Marsden Hospital has an international reputation in the development of immunophenotyping as a tool for the diagnosis of lymphoproliferative disorders. For example, the CLL score developed by the Haemato-Oncology department continues to be used in several centres around the world for the diagnosis of CLL5. A similar score proposed for HCL by our Haemato-Oncology department is also widely used (6). On a service evaluation, we found 100% concordance between a HCL score of 4 and presence of the BRAF mutation in samples referred to us (unpublished data).

Our plan therefore is to systematically study unclassifiable groups of B-LPD by creating a well-defined immunomorphology work flow for their identification. Samples thus identified will be screened using a next-generation sequencing (NGS) panel which is able to detect well established, B-LPD associated translocations and genetic mutations.

Inclusion Criteria:

• Chronic, mature clonal B-cell malignancy that is not assignable to a specific WHO category by current technology (see flowchart in section below).

Exclusion Criteria:

• Non-clonal B-cell proliferation.
• High grade and/or immature clonal B-cell malignancy.
• Bone marrow samples with less than 20% infiltration will be excluded
• Samples from patients with a known classifiable chronic B-LPD based on lymph node biopsy for e.g. staging bone marrow samples on a patient with marginal zone lymphoma. If a definitive diagnosis is established on a subsequent lymph node biopsy, patient will remain on study and this will be correlated with NGS findings.
• Patient unable to provide consent for tumour and germ line samples.

Patients suffering with an unclassifiable B-cell lymphoproliferative disorder