The Mutational Landscape of Cold Agglutinin Disease

Primary cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia mediated by monoclonal IgM autoantibodies directed against the I antigen on erythrocytes. CAD is due to a B-cell lymphoproliferative disorder of the bone marrow. We have previously reported recurrentKMT2DandCARD11mutations and trisomy 3 and 12 or 18 in CAD patients (Malecka et al., 2018 & 2020). In addition, we have revealed that the cold-reactive antibody is highly stereotyped (Malecka et al., 2016). In this study, we present new and detailed data on the mutation landscape of CAD patients.

We have analyzed the bone marrow from 14 patients with CAD, included in a recent clinical trial (Berentsen et al., 2017), by whole exome sequencing. Clonal B cells and normal T cells (normal control) were acquired using fluorescence-activated cell sorting. The data from whole exome sequencing were aligned to the human genome hg38 and analyzed as described before (Malecka et al., 2020). Variant discovery and functional annotation were performed with the use of GATK4 tools: Mutect2 and Funcotator. Detected mutations were validated manually using IGV browser.

The total amount of non-synonymous mutations detected in each CAD sample ranged from 13 to 62, while the total amount of all mutations in the exome regions ranged from 36 to 163 (Figure 1A). Four genes showed non-synonymous mutations in 3 or more cases, these are:KMT2D(8/14; 57%),IGLL5(5/14; 36%),CARD11(3/14; 21%),CXCR4(3/14; 21%) (Figure 1B and Table 1). Additionally, several genes showed non-synonymous mutations in 2 of the 14 cases (14%):PHLDB1, HIST1H1E, LPIN3, LTB, MACF1, NBEA, NEFH, PCNX2, RRAGC, TMPRSS7, ZNF618(Figure 1B). A majority of these genes are also found mutated in lymphoma.

All patients with eitherCARD11orCXCR4mutations have concurrentKMT2Dmutations. One patient has bothCARD11andCXCR4mutations together withKMT2Dmutation. Recurrent mutations seem to cluster in some of the cases (Figure 1C). Almost all patients with aKMT2Dmutation (7/8, 87%) have at least 3 other recurrent mutations. Patients with aCARD11(3 cases) or aCXCR4(3 cases) mutation have at least 3 other recurrent mutations.

KMT2Dmutations are found in many lymphomas and are associated with Kabuki syndrome. It is suggested thatKMT2Dis a tumor suppressor gene, andKMT2Dmutations might act as driver mutations in many lymphomas. In addition,CARD11mutations in our cases are located in coiled-coil domain in exon 6, and therefore it is expected to result in NF-kB activation (Malecka et al. 2018).IGLL5is located in the IG lambda locus. The function of theIGLL5gene is unknown, but it is homologues to theIGLL1gene known to be important in B cell development.IGLL5mutations are seen in lymphoma as well as other malignancies and might have some prognostic value in diseases like multiple myeloma.CXCR4has been implicated in the migration and trafficking of malignant B cells in several hematological malignancies.CXCR4mutations are frequently seen in lymphoplasmacytic lymphoma, and almost always in combination with aMYD88mutation. In our series, aCXCR4mutation always occurs in combination with aKMT2Dmutation.MYD88was not found to be mutated. Detected mutations in theCXCR4gene are of potential therapeutic interest, sinceCXCR4inhibitors exist.

CAD patients with aKMT2Dmutation associated with other recurrent mutations (Figure 1C), show a trend to lower hemoglobin levels at diagnosis compared to patients with noKMT2Dmutation or patients withKMT2Dmutation without other recurrent mutations. We are currently working on sequencing clonal B cells from additional patients in order to confirm these findings.

In conclusion, CAD-associated lymphoproliferative disease shows a relatively low mutation burden. but with recurrent gene mutations. The most common recurrent mutations are in genes known to be involved in lymphoma development. Our preliminary data suggest thatKMT2Dmutation associated with other recurrent mutations might determine severity of hemolysis.

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