Primary cold agglutinin-associated lymphoproliferative disease: a B-cell lymphoma of the bone marrow distinct from lymphoplasmacytic lymphoma

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nCAD diagnosed in the time period between 1995 and 2012 were studied. There were 36 women and 18 men with an age range of 40-92 (median 73) years. All patients had a clinical history of CAD with a variable degree of anemia, a positive C3d-specific direct antiglobulin test and a cold agglutinin titer in excess of 64. Monoclonal IgM had been detected in the serum of all patients by agarose electrophoresis and immunofixation. None of the patients had lymphadenopathy or splenomegaly. Clinical follow-up ranged from 3 to 152 months, with a median follow-up of 72 months. The study was approved by the institutional and regional ethical committees. Biopsy materialArchival hematoxylin and eosin-stained sections of bone marrow trephine biopsies from the 54 patients, obtained at diagnosis, were reviewed. Fourteen biopsies were fixed in 4% formaldehyde, 18 in Bplus fixative and 22 in B5 fixative. In addition, part of the diagnostic trephine biopsy of eight patients had been snap-frozen in liquid nitrogen. Two of the patients had undergone splenectomy in an attempt to reduce hemolysis. Hematoxylin and eosin-stained archival sections of formalin-fixed splenic tissue of these patients were reviewed. ImmunohistochemistryImmunohistochemical analysis was extended or repeated whenever archival sections were not available or of bad quality. The primary antibodies and the method used for immunohistochemical analysis of the bone marrow trephine biopsies are described in the Online Supplementary Methods. Flow cytometryFlow cytometry was performed on samples from 25 patients and included a total of 46 bone marrow and 10 paired peripheral blood samples, anticoagulated with heparin and EDTA, respectively. On samples analyzed before 2011, a four-color analysis 18 and from 2011 onwards, an eight-color analysis 19 was performed with antibody combinations as described in the Online Supplementary Methods.Immunoglobulin heavy chain gene sequencing, and BCL6 and MYD88 mutation analyses DNA was extracted from frozen bone marrow trephine samples, obtained from eight patients, using the EZ1 tissue kit (Qiagen, Hilden, Germany) and from formaldehyde-fixed paraffin embedded-tissue sections from another nine patients using the QIAamp DNA FFPE tissue kit (Qiagen). Immunoglobulin heavy chain gene analysis and BCL6 intron 1 mutation analysis was only performed on DNA from frozen tissue samples whereas MYD88 mutation analysis, requiring less intact DNA, was performed on frozen and formaldehyde-fixed tissue samples.BCL6 intron 1 was amplified using three sets of overlapping polymerase chain reaction (PCR) primers covering nucleotides 24 to 790 in GenBank sequence AF191831. The primer pairs and PCR conditions are described in the Online Supplementary Methods. PCR products were sequenced and analyzed using the BLAST database.The MYD88 L265P mutation (NM_002468) was analyzed using PCR and a SNaPshot multiplex kit (Applied Biosystems). PCR primers and conditions are described in the Online Supplementary Me...

Immunoglobulin heavy and light chain gene features are correlated with primary cold agglutinin disease onset and activity

“…Previously, we demonstrated that CAD is caused by a low-grade clonal B-cell lymphoproliferative disease of the bone marrow with a typical histology that is different from lymphoplasmacytic lymphoma and, accordingly, does not display the MYD88 L265P mutation. 1 Almost all patients display circulating monoclonal antibodies encoded by the IGH gene V4-34. The framework region 1 (FR1) of IGHV4-34 encodes Gln 6 -Trp 7 (QW) and Ala 24 -Val 25 -Tyr 26 (AVY) sequences that determine I antigen-binding.…”

Acrocyanosis revealing chronic lymphocytic leukemia

“…Autoimmune disorders may precede or follow the diagnosis of CLL 2. In our case, the cold agglutinin and the increased lymphocytes were discovered at the time of the diagnosis.…”

MYD88 L265P mutation analysis helps define nodal lymphoplasmacytic lymphoma

“…The findings are similar to those of a recent study showing that cold agglutinin associated B-cell lymphoproliferative disorders lack the MYD88 L265P mutation. 55 These results suggest that lymphomas associated with cold agglutinin disease arise through a different pathogenetic mechanism than typical lymphoplasmacytic lymphoma, and that these may be considered to represent distinct entities. However, this must be further clarified as cold agglutinins have been reported in one study in 27% of MYD88 L2565P mutated lymphoplasmacytic lymphoma.…”