Turning the Heat up a Notch in Biclonal Lymphoproliferative Disorders
Authors: Fogarty Helen , Dowling Anita , O'Brien David , Bacon Christopher Laurence , Thornton Patrick , Hennessy Brian , O'Leary Hilary M , Crotty Gerard , Henderson Robert , Nolan James , Vandenberghe Elisabeth A. , Quinn Fiona M 
Published: October 2019

Abstract
Biclonal lymphoid disorders (BLD) are a rare (1.4%) but distinct entity in which Chronic lymphocytic leukaemia (CLL) co-exists with a second lymphoproliferative disorder (LPD) and are poorly understood in terms of pathogenesis and clinical impact. The NOTCH1 pathway is involved in cell proliferation, cell cycle progression and is anti-apoptotic, with mutations of the regulatory PEST domain causing constitutive up-regulation of the pathway. NOTCH1 mutations occur early in CLL leukaemogenesis and are found in 10% of poor prognosis CLL at diagnosis as well as being associated with 50% of Richter's transformation (both Hodgkin's and non-Hodgkin's subtype). We hypothesised that NOTCH1 mutations may be implicated in BLD pathogenesis both because they occur early in leukaemogenesis when clonal divergence is possible and because of its association with Hodgkin's Richter's, which is biologically distinct from CLL.
We identified 19 patients with BLD at initial presentation, confirmed by flow cytometry and/or molecular analysis of the Immunoglobulin heavy (IgH) and light (IgL) chain genes between 2008 and 2015. This cohort consisted of 14 males and 5 females with a mean age of 72 (range 47-90) years. BLD was defined as follows: (1) co-existence of cells with different immunophenotypes such as a CD5+/CD19+ and CD5-/CD10- population confirming the co-existence of 2 diagnoses, (2) demonstration of both kappa (κ) and lambda (λ) light chain restriction on a CD5+/CD19+ population and (3) CD5+/CD19+ population without demonstrable surface light chains but with >2 clonal gene rearrangements of their IgH and IgL loci. All 19 cases were tested for a c.7544-7545delCT resulting in truncation of the regulatory C-terminal PEST domain of the NOTCH1 gene by PCR.
Eleven of 19 patients had CLL co-existing with a different LPD as follows: Mantle Cell Lymphoma (MCL) (2), Hairy Cell Leukaemia (HCL) (2) and Marginal Zone Lymphoma (7). Of this subgroup 5 of 11 cases (45%) had a NOTCH1 mutation. Four samples in our cohort displayed dual κ and λ light chain expression detected by flow cytometry and confirmed molecularly by identifying 2 clonally distinct populations of LPD and 3 samples (75%) had a NOTCH1 mutation. Four samples were identified with >90% CD5+/CD19+ expression but no monoclonality by flow cytometry, but with 2 distinct populations identified by IgH and IgL rearrangements and of this subgroup 2 (50%) had NOTCH1 mutations. Clinical data was available on 13/19 patients, of whom 11 patients had Binet A and 2 Binet B disease. No patient has required CLL-directed treatment, 2 patients have died of other causes and 11 remain under surveillance. Three of 9 patients with a second LPD have required treatment: both patients with HCL received cladribine for pancytopaenia and a patient with MCL was treated with Rituximab and Bendamustine.
Overall, 10 of the 19 (52%) patients with BLD had a NOTCH1 mutation at diagnosis, which is higher than the 10% reported in CLL literature. The absence of any germinal centre cell derived lymphoma in the cohort of patients with additional LPD is intriguing and needs confirmation in other series. The prognostic implications of BLD and NOTCH1 mutations are not possible to assess in this small cohort, but accurate characterization of BLD aids therapeutic decision-making as in the case of concurrent CLL and HCL. Light chain restriction by flow cytometry in a mature B-cell population confirms monoclonality and suggests malignancy, however dual κ and λ expressing CLL may give a polyclonal pattern resulting in failure to make a CLL diagnosis; therefore awareness of this rare subtype of CLL is important.
In summary we have added a further association between NOTCH1 mutations and CLL associated disorders which currently includes: poor prognosis CLL at diagnosis, Hodgkin's and non Hodgkin's Richter's transformation and most recently BLD. Increased sensitivity of NOTCH1 detection by next generation sequencing techniques may provide a further insight into the evolutionary origin and pathogenesis of BLD and further elucidate the enigmatic role of NOTCH1.

Disclosures
Crotty: BMS, Takeda, Novartis, Janssen, Roche: Honoraria.