Cold Agglutinin Disease Shows Highly Recurrent Gains of Chromosome 3, 12 and 18
Authors: Malecka Agnieszka , Delabie Jan , Ostlie Ingunn , Tierens Anne , Randen Ulla , Berentsen Sigbjorn , Tjønnfjord Geir Erland , Trøen Gunhild 
Published: November 2019

Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia, caused by a distinct type of B-cell lymphoproliferative disease of the bone marrow. Anemia is mediated by binding of monoclonal cold agglutinin antibodies to the erythrocyte surface I antigen at temperatures below central body temperature, followed by agglutination and complement activation. These antibodies are almost exclusively encoded by IGHV4-34. We recently found recurrent KMT2D and CARD11 gene mutations in CAD (Malecka, et al 2017).
We analyzed 12 CAD samples from the CAD-5 study (NCT02689986) (Berentsen, et al 2017) using Affymetrix OncoScan CNV Plus Assay and exome sequencing data in order to detect chromosomal aberrations. All samples were sorted using fluorescence activated cell sorting prior to analysis.
Complete or partial gain of chromosome 3 (+3 or +3q) was detected in all samples (Table 1). Additionally, most cases showed either gain of chromosome 12 or 18 (9/12) (Table 1). Additional small regions of recurrent gain or loss were also detected in other chromosomes. The recurrent changes in least 4 samples are: +1p36.31-p36.13, -8p21.3-p21.2, +9q34.2-q34.3, +11q13.1-q13.2, +17q25.3, +21q22.3, +22q13.31-q13.33. Gains and losses of large parts of chromosomes are clearly visible in both Affymetrix OncoScan CNV Plus Assay and exome sequencing data (Figure 1). In contrast smaller changes are often not visible in exome sequencing data and therefore could not be fully validated. Further validation will be required for smaller chromosomal changes.
Gain of chromosome 3/3q was characteristic for all CAD cases analyzed. Gain of chromosome 3 in CAD has previously been reported in 9 of 26 patients (Gordon, et al 1990, Michaux, et al 1995). Gain of chromosome 3 is not a highly recurrent finding in most B-cell lymphomas with the exception of marginal zone lymphoma (MZL). Of interest, gain of chromosome 12 and 18, demonstrated in our study, is also a feature of MZL. These findings, together with our previous findings (Malecka, et al 2017), suggest that the CAD-associated lymphoproliferative disease, although exclusively present in the bone marrow, might be related to MZL.
The CAD-5 study assessed the efficacy of bendamustine plus rituximab in CAD patients. We explored whether a correlation existed between presence or absence of trisomy 12 and 18 with response to therapy. Although the series is small, we found a trend towards poorer response in patients with trisomy 18 compared to patients with trisomy 12. Furthermore, both patients without trisomy 12 or 18 had the best responses (Table 1). Despite the limited number of cases the Pearson correlation was statistically significant (p=0.02). However, more samples need to be analyzed to confirm these results. A correlation between response to therapy and presence of trisomy 12 or 18 has previously been demonstrated for other B-cell lymphomas. Trisomy 12 is frequent in patients with small lymphocytic lymphoma (28-36%) and in chronic lymphocytic leukemia (10-25%) and in the latter +12 is associated with intermediate prognosis (Autore, et al 2018). Trisomy 3 and 18 are found to be correlated with advanced stage extranodal marginal zone lymphoma (Krugmann, et al 2005). There is evidence from previous studies that trisomy 18 may be associated with upregulation of BCL2, an anti-apoptotic gene located on chromosome 18. Whether BCL2 is upregulated in CAD with trisomy 18 and whether it might be the cause of therapy resistance, needs to be investigated. Such a study is of importance since BCL2 inhibitors might be considered to improve treatment responses.
We conclude that gain of chromosome 3 is a highly recurrent finding in CAD-associated lymphoproliferative disease. Further gain of chromosome 12 and 18 might be predictors of therapy outcome. These genetic findings are similar to what has previously been demonstrated in nodal and extranodal MZL, suggesting that CAD-associated lymphoproliferative disease, a disease of the bone marrow, might be related to MZL.

Disclosures
Berentsen: Mundipharma: Research Funding; Apellis, Bioverativ (a Sanofi company), Momenta Pharmaceuticals, and True North Therapeutics: Consultancy; Alexion, Apellis, and Janssen-Cilag: Honoraria. Tjønnfjord:Mundipharma: Research Funding.