Cerebrospinal Fluid Flow Cytometry Analysis in Newly Diagnosed Aggressive Non-Hodgkin Lymphomas at High Risk for Leptomeningeal Disease: Result of a Multicentric Prospective Italian Study.

Flow cytometry (FCM) assessment of cerebrospinal fluid (CSF) has recently been known to increase the rate of positivity of occult leptomeningeal disease (LD) in comparison to conventional cytologic examination (CC). However it's still unknown its prognostic value. Patients and methods: The aim of this study was to compare CC vs FCM in a large cohort of patients with newly diagnosed aggressive NHL at high risk for LD (diffuse large B-cell lymphoma (DLBCL) IPI 2-3 and elevated LDH with at least two extranodal sites or with bone marrow, testis, paranasal sinuses, orbit or paravertebral involvement; Burkitt lymphoma (BL); blastoid variant of mantle cell lymphoma (B-MCL); B-cell precursor lymphoblastic lymphoma (B-LL); HIV+ aggressive lymphoma patients). All patients were required to have no evidence or signs of neurological disease. All patients received intrathecal standard prophylactic therapy with 12 mg of methothrexate except for BL that were given prophylaxis with 50 mg of liposomial aracytin for a total of 4 doses. CFS samples were analysed both with CC and FCM. The incidence of positive test for occult LD with FCM and CC was compared using the McNemar test for paired data.

Between August 2004 and June 2008, a total of 159 consecutive patients were enrolled in 11 Italian centres and underwent evaluation of CSF. Out of these, 128 patients (80%) were considered at high risk of occult LD. Clinical characteristics were: median age 53 years (IQR:43-62); DLBCL 96 patients (75%); BL 21 pts (16%); B-MCL 6 pts (5%); B-LL 5 pts (4%); 26 pts (20%) were HIV positive. FCM was able to detect a clonal population in 17 out of 128 patients (13%) whereas CC detected abnormal cells only among 7 pts (5%)(p= 0.0002). Therefore, 10 patients (8%) were discordant: FCM+/CC-. Among the 128 patients, there was no association between the CFS total protein, glucose level and the presence of positive analysis of FCM, whereas the difference between the number of WBC cells in CSF was significantly higher in patients with positive versus negative FCM with a median value of 12 cells/ul (IQR: 3.5;40) versus 1.0 cells/ul (IQR: 0.0;3.0) (p=0.0120). Univariate and multivariate analyses, using logistic models, showed that abnormal LDH (OR 3.98, 95%CI: 1-15.92)(p=0.05) and number of WBC cells in CSF ≥5 (OR 4.57, 95%CI:1.37-15.33)(p=0.014) were the only predictive factors of a positive test performed by FCM. From date of diagnosis, overall median follow up of survivors was 14 months (IQR:8-22). We observed 39 (30%) systemic progressions, 6 (5%) CNS progressions (in 5 cases an isolated CNS progression whereas 1 pts experienced a CNS along with systemic progression). Thirty-two (25%) patients died and causes of deaths were as follows: 27 progressive disease, 1 infection, 1 treatment related toxicity, 1 hepatitis, 2 unknown. PFS at 1 year was 71% (95%CI:62-78) in the whole group of patients. The progression risk was significantly higher in patients both FCM+/CC+ compared with patients both FCM-/CC- (1-yr PFS 43% vs 74%) (HR 3.8 95%CI:1.6-9.0) (p=0.003). An higher but not significant risk of progression was found in pts discordant (FCM+/CC-) with respect to patients both FCM-/CC- (1-yr PFS 65% vs 74%) (HR 1.61, 95%CI:0.63-4.11) (p=0.315). In the univariate and multivariate analyses performed with Cox models, we found that the presence of ECOG PS≥2 (HR 2.14, 95%CI: 1.14-4)(p=0.018) and level of protein in CSF >40/ul (HR 1.83 95%CI: 1.01-3.29)(p=0.045) were prognostic factor of PFS.

FCM assessment of CSF increase the rate of positivity of occult LD compare with CC but it's clinical relevance is still to be clearly defined. Our preliminary data suggest that patients both FCM+/CC+ have an higher risk of progression compared with those both negative, whereas discordant cases may have an intermediate prognosis.

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No relevant conflicts of interest to declare.