In a substantial number of patients with systemic mastocytosis (SM), an associated clonal haematological non-mast cell lineage disease (AHNMD) is detectable. Although most of these patients display KIT mutations, especially KIT(D816V), little is known about their exact frequency and their distribution in AHNMD subtypes. We examined 48 patients with SM-AHNMD for the presence of mutant KIT in the SM and AHNMD components of the disease. Mast cells and AHNMD cells were obtained from immunostained bone marrow sections by laser microdissection and examined by melting point analysis of nested-PCR products. KIT(D816V) was found in AHNMD cells in the vast majority of patients with SM-chronic myelomonocytic leukaemia (CMML, 89%). Unexpectedly, KIT(D816V) was far less frequently detectable in AHNMD cells in patients with SM-myeloproliferative neoplasm (MPN, 20%) and SM-acute myeloid leukaemia (AML, 30%). None of the patients with lymphoproliferative AHNMDs displayed KIT codon 816 mutations in AHNMD cells (0/8). In FIP1L1/PDGFRA-positive chronic eosinophilic leukaemia (CEL), neither the SM nor the CEL component of the disease exhibited the KIT mutation. Our findings demonstrate that KIT codon 816 mutations are variably present in AHNMD cells in patients with SM-AHNMD, depending on the subtype of AHNMD. The high frequency of KIT(D816V) in neoplastic mast cells and leukaemic myelomonocytic cells in SM-CMML may point to a common precursor in these patients, and may have implications for the biology of the disease and the development of KIT-targeting therapies.

Follicular lymphoma (FL) is characterized genetically by a significant intraclonal diversity of rearranged immunoglobulin heavy chain (IGH) genes and a substantial cell migration activity (follicular trafficking). Recently, in situ follicular neoplasia (ISFN), characterized by accumulations of immunohistochemically strongly BCL2-positive, t(14;18)+ clonal B cells confined to germinal centers in reactive lymph nodes, has been identified as a precursor lesion of FL with low risk of progression to manifest FL. The extent of ongoing somatic hypermutation of rearranged IGH genes and interfollicular trafficking in ISFN is not known. In this study we performed an in depth analysis of clonal evolution and cell migration patterns in a case of pure ISFN involving multiple lymph nodes. Using laser microdissection and next generation sequencing (NGS) we documented significant intraclonal diversity of the rearranged IGH gene and extensive interfollicular migration between germinal centers of the same lymph node as well as between different lymph nodes. Furthermore, we identified N-glycosylation motifs characteristic for FL in the CDR3 region.

Analysis of T cell receptor (TCR) gene rearrangements is an important tool for the diagnosis of T cell non-Hodgkin lymphomas (NHL). A number of PCR-based T cell clonality protocols with increasing complexity in primer design have been published in the last decades. The multiplex TCRγ and TCRβ assays developed by the BIOMED-2 consortium have shown superior sensitivity for the detection of clonality in T-NHL. However, they have mainly been tested on fresh frozen tissues and may be difficult to interpret due to their complex design with multiple product size ranges. In this study, two relatively simple, first-generation TCRγ PCR protocols published by McCarthy et al. (Diagn Mol Pathol 1:173-179, 1992) and Trainor et al. (Blood 78:192-196, 1991) were compared with the BIOMED-2 TCRγ and TCRβ assays, using fluorescence-labeled primers and GeneScan analysis. FFPE samples of 52 peripheral T-NHL and 55 controls, including 20 B-NHL, were included. A 50-case subset including all samples with false negative or false positive results with the two TCRγ protocols was analysed additionally with BIOMED-2 TCRγ and TCRβ assays. With the combined BIOMED-2 assays, clonality was detected in four out of six previously false negative T-NHL and increased sensitivity for this selected subgroup to 92%, as compared to 64% (McCarthy) and 72% (Trainor). The overall specificity of 80% for BIOMED-2 assays was comparable to Trainor (84%) and McCarthy (88%) protocols, but incomplete TCRβ DJ rearrangements were identified in four out of ten B-NHL cases. In conclusion, BIOMED-2 TCRγ and TCRβ assays show superior sensitivity for the detection of T cell clonality. However, the complexity of BIOMED-2 protocols requires stringent quality control and experience in interpreting GeneScan patterns.