Old DAT / New Data : Positive Direct Antiglobulin Test Identifies a Specific Subgroup Of CLL Patients
Authors: Quinquenel Anne , Al Nawakil Chadi , Baran-Marszak Fanny , Eclache Virginie , Letestu Remi , Le Roy Christine , Varin-Blank Nadine , Delmer Alain , Levy Vincent , Cymbalista Florence 
Published: October 2019

Abstract
Autoimmune cytopenias represent a well-known complication of chronic lymphocytic leukemia (CLL). The most frequent one is autoimmune haemolytic anemia (AIHA), occurring in about 5% of the patients during the course of the disease. The prognostic significance of autoimmune cytopenias remains uncertain and controversial. In previous studies, a positive direct antiglobulin test (DAT) was found in up to 35% of CLL cases, however only a minority of the patients would develop AIHA at any time of the disease. The aim of this monocentric study was to explore the clinical and biological characteristics and the outcome of CLL patients showing a positive direct antiglobulin test at any time during the course of the disease. We reviewed all CLL patients seen at our institution who had at least one positive DAT between January 2007 and May 2013.
Fifty-four patients were found, representing about 10% of our CLL cohort. The sex ratio was 1,8 (35M/19F). Median age at diagnosis was 66,2 years (range 44,7 to 87,3). According to the Binet classification, 41 (76 %) patients were in stage A, 7 in stage B, and 6 in stage C. Study of usual prognostic parameters evidenced that these patients represented a very high risk group: 82% of cases harbored unmutated IGHV, and CD38 was expressed in 60% cases. Cytogenetic alterations were studied by FISH analysis: 11q deletion, trisomy 12 and 17p deletion were found in 12,5%, 22,9% and 14,3% cases respectively.
In this cohort, with a median follow up of 85 months, 41 (76 %) patients required treatment. The first line of treatment was initiated for CLL progression in 33 cases, for symptomatic AIHA along with CLL progression in 5, and for symptomatic AIHA (without CLL progression) in 3. The median time to first treatment was very short (16 months), and the median overall survival of this cohort was 84 months. In 22 cases, the DAT was positive from the time of diagnosis, while in 34 patients it became positive later during the course of CLL. Only 19 patients (35%) developed symptomatic AIHA. DAT specificity was IgG, IgG+ C or C for respectively 36, 10 and 8 patients. Interestingly, there was no impact of the time of first positive DAT during the course of the disease (at diagnosis or later). Moreover, occurrence of a symptomatic AIHA had no impact on treatment free survival or overall survival when compared with cases with positive DAT only.
Among these cases, 41 were in stage A at diagnosis and up to 78% harbored unmutated IGHV (as compared with 30 % in our institutional stage cohort). In order to evaluate the impact of a positive DAT on a homogenous population, we focused on the 31 IGHV unmutated stage A cases and compared them with our cohort of IGHV unmutated stage A with consistently negative DAT. We found a significant prevalence of VH1-69 and VH 3-21 use (43 %) and a high percentage belonging to a stereotyped subset (34%). The majority of the cases (19/28 tested) were responders to in vitro B-cell receptor stimulation by anti IgM. In this population, time to first treatment (26 months) was slightly shorter but median overall survival (55 months) was significantly reduced highlighting the poor response to treatment.
In conclusion, occurrence of a positive DAT at any time of the course of CLL is associated with poor outcome and has the same adverse prognostic impact as the onset of a symptomatic AIHA. Moreover, study of B-Cell receptor (BCR stimulation by anti-IgM, CDR3 stereotypy) show that these patients may represent a pathophysiological distinct subgroup in which antigen stimulation is likely to play a major role.

Disclosures:
No relevant conflicts of interest to declare.