The Wnt signalling pathway, one of the core de-regulated pathways in chronic lymphocytic leukaemia (CLL), is activated in only a subset of patients through somatic mutations. Here we describe alternative, microenvironment-dependent mechanisms of Wnt activation in malignant B cells. We show that tumour cells specifically induce Notch2 activity in mesenchymal stromal cells (MSCs) required for the transcription of the complement factor C1q. MSC-derived C1q in turn inhibits Gsk3-β mediated degradation of β-catenin in CLL cells. Additionally, stromal Notch2 activity regulates N-cadherin expression in CLL cells, which interacts with and further stabilises β-catenin. Together, these stroma Notch2-dependent mechanisms induce strong activation of canonical Wnt signalling in CLL cells. Pharmacological inhibition of the Wnt pathway impairs microenvironment-mediated survival of tumour cells. Similarly, inhibition of Notch signalling diminishes survival of stroma-protected CLL cells in vitro and disease engraftment in vivo. Notch2 activation in the microenvironment is a pre-requisite for the activation of canonical Wnt signalling in tumour cells.

BackgroundCD44 is considered as ‘a’ metastasis associated gene, despite the fact that it is an umbrella term for a group of molecules produced from a single gene by alternative splicing. However, little consideration is given to the above in the literature of colorectal carcinomas as well as other tumour types, leading to confusion and contradictory results about its possible role in tumour progression.MethodsWe compared the CD44 alternative splice pattern (ASP) of three genetically different human colorectal cancer cell lines (HT25, HT29, HCT116) using a series of PCR reactions and next- generation sequencing method, as well as identified a colorectal adenocarcinoma specific CD44 ASP. This ASP was further investigated in terms of its qualitative and quantitative stability in our experimental iso- and xenograft mouse models for colorectal cancer progression. A complex preclinical experimental set-up was established to separately test the different steps of tumour progression and the role of tumour microenvironment, respectively, focusing on the role of ‘CD44’ in this process.ResultsWe managed to present a colorectal cancer-specific CD44 ASP, which remained unchanged from cell lines throughout primary tumour formation and metastatic progression. Furthermore, we report a unique roster of all expressed CD44 variant isoforms characteristic to colorectal cancer. Finally, on quantitative assessment of the variable exons v3 and v6, higher co-expression levels were found to be characteristic to metastatically potent tumour cells.ConclusionParticular CD44 variant isoforms seem to act as “metastasis genes” via tumour microenvironment-driven shifts in v3 and v6 expressions. However, this function may just affect a minority of tumour subclones. This fact and the huge potential number of different CD44 splice variants that can contain v3 and v6 domains can explain incoherence of clinical studies regarding functional asessment of CD44 variants, as well as diminish the chances of using CD44 variants for predictive purpose.

Angioimmunoblastic T‐cell lymphoma (AITL) is a neoplastic proliferation of T follicular helper cells with clinical and histological presentations suggesting a role of antigenic drive in its development. Genetically, it is characterized by a stepwise acquisition of somatic mutations, with early mutations involving epigenetic regulators (TET2, DNMT3A) and occurring in haematopoietic stem cells, with subsequent changes involving signaling molecules (RHOA, VAV1, PLCG1, CD28) critical for T‐cell biology. To search for evidence of potential oncogenic cooperation between genetic changes and intrinsic T cell receptor (TCR) signaling, we investigated somatic mutations and T‐cell receptor β (TRB) rearrangement in 119 AITL, 11 peripheral T‐cell lymphomas with T follicular helper phenotype (PTCL‐TFH), and 25 PTCL‐NOS using Fluidigm polymerase chain reaction (PCR) and Illumina MiSeq sequencing. We confirmed frequent TET2, DNMT3A, and RHOA mutations in AITL (72%, 34%, 61%) and PTCL‐TFH (73%, 36%, 45%) and showed multiple TET2 mutations (2 or 3) in 57% of the involved AITL and PTCL‐TFH. Clonal TRB rearrangement was seen in 76 cases with multiple functional rearrangements (2–4) in 18 cases (24%). In selected cases, we confirmed bi‐clonal T‐cell populations and further demonstrated that these independent T‐cell populations harboured identical TET2 mutations by using BaseScope in situ hybridization, suggesting their derivation from a common TET2 mutant progenitor cell population. Furthermore, both T‐cell populations expressed CD4. Finally, in comparison with tonsillar TFH cells, both AITL and PTCL‐TFH showed a significant overrepresentation of several TRB variable family members, particularly TRBV19*01. Our findings suggest the presence of parallel neoplastic evolutions from a common TET2 mutant haematopoietic progenitor pool in AITL and PTCL‐TFH, albeit to be confirmed in a large series of cases. The biased TRBV usage in these lymphomas suggests that antigenic stimulation may play an important role in predilection of T cells to clonal expansion and malignant transformation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.