Characterisation of a New Clinical Presentation of Chronic Lymphocytic Leukemia with Symptomatic Nasopharyngeal Mucosa Involvement

Patients with chronic lymphocytic leukemia (CLL) are prone to infectious complications, including Ears, Nose and Throat (ENT) infections due to the humoral immunodepression and/or to the immunosuppression related to the therapy. However, specific CLL infiltration in non-lymphoid regions of the head and neck causing ENT symptoms but unrelated to an infection is not well described. Extra-nodal localizations of CLL cells, including involvement of the mucosa of the rhinopharynx is uncommon and poorly reported. Here, we retrospectively analyzed the clinical, histopathologic and molecular features of 25 CLL patients with specific head and neck involvement. To date, this is the largest cohort reporting this new entity of CLL also named Nasal Associated Lymphoid Tissue (NALT) CLL.

All patients had proven CLL prior to symptoms. Median time between ENT manifestation and CLL was 3 years [1-11 years]. Symptoms included chronic coughing (44%), antero-posterior nasal discharge (44%), nasal congestion (33%) and pharyngitidis (33%). ENT examination evidenced cervical lymphadenopathies in 68 % of cases, a granular aspect of the mucosa of the pharynx in 56%, enlarged tonsil (37%) or adenoids (37%). All patients underwent a biopsy of the mucosa of the nose or the throat. Histology and immunochemistry analysis demonstrated an infiltration of small lymphocytes CD20+, CD5+ and CD23+, consistent with a phenotype of CLL. The infiltration was diffuse in 50% of biopsies and perivascular in 38%.

Patients with NALT-CLL had a poor prognosis: the majority was IGHV unmutated (n=18/25, 72%). Furthermore, they all required a treatment according to IWCLL criteria few time after the first ENT symptoms (median time 2 years), indicating that NALT-CLL is associated with a more progressive disease.

To characterize the genetic background of NALT-CLL, we analyzed the cytogenetic data and NGS sequencing of 13 CLL-associated mutations from peripheral CLL cells. The karyotype was normal in only 2/24 cases (8%) and complex (>3 abnormalities) in 5/24 cases (20%). Half of the patients had a trisomy 12(12/24; 50%), while 13q, 17p and 11q deletions were found in 29%, 8% and 4% respectively. Eleven patients harbored one mutation (44%) while 9 patients had 2 to 5. Mutation of the NOTCH1 pathway was found in half of the cases (11/25 NOTCH1 and 2/25 FBXW7 mutated cases). TP53 and SF3B1 mutations occurred respectively in 20% (5/25 cases) and in 12% (3/25 cases).

To gain insight into the molecular mechanism associated with involvement of the rhinopahrynx, we studied the expression of 70 genes related to cell migration and cell adhesion pathways using a qPCR array in the peripheral CLL cells of patients with NALT (n=4) compare to no NALT-CLL (n=4). Genes significantly up regulated with a fold change >2 included the chemokine receptors CCR7 (p=0.05) and CCR5 (p=0.04), the receptor involved in leukocyte trafficking CXCR3 (p=0.01) or the chemoattractant chemokine-like factor CKLF. By targeted qPCR, we confirmed the up regulation of CCR7 (p=0.002), CXCR3 (p=0.04) and CCR5 (p=0.03) in the cohort NALT-CLL patient (n=25) as compared to 20 age-matched CLL patients without head and neck symptoms (77% with unmutated IGHV, 30% with NOTCH1 mutation and 30% with trisomy 12). Up regulation of those targets was independent of the presence of NOTCH1/trisomy12 aberration or of the IGHV mutation status. These results suggest an increased migratory capacity of the leukemic CLL cells into the rhynopharynx mucosa related to a higher expression of these receptors involved in cell trafficking and migration. In line with these results, immunohistochemistry analysis of 5 patients with nasal involvement showed a strong staining of the CCR7 marker on the membrane of CLL cells infiltrating the mucosa. Interestingly, the staining of CCL21, the cognate ligand of CCR7, was positive in the vessels of the mucosa, suggesting that the recruitment and the transendothelial migration of CLL cells into the mucosa occur through a local secretion of CCL21 by the vessels.

In summary, we report here a new presentation of CLL associated with symptomatic and specific ENT localization. CLL cells are predominantly IGHV unmutated, harbor NOTCH1 mutation and/or trisomy12 and show a higher expression of the chemokine receptors CCR7, CCR5 and CXCR3. We are currently studying the expression of those receptors by flow cytometry and the enhanced migratory capacity toward CCL21 through an in vitro chemotaxis assay.