Early detection of T-cell lymphoma with T follicular helper phenotype by RHOA mutation analysis.

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma with T follicular helper phenotype (PTCL-TFH) are a group of complex clinicopathological entities that originate from T follicular helper cells and share a similar mutation profile. Their diagnosis is often a challenge, particularly at an early stage, because of a lack of specific histological and immunophenotypic features, paucity of neoplastic T cells and prominent polymorphous infiltrate. We investigated whether the lymphoma-associated RHOA Gly17Val (c.50G>T) mutation, occurring in 60% of cases, is present in the early "reactive" lesions, and whether mutation analysis could help to advance the early diagnosis of lymphoma. The RHOA mutation was detected by quantitative polymerase chain reaction with a locked nucleic acid probe specific to the mutation, and a further peptide nucleic acid clamp oligonucleotide to suppress the amplification of the wild-type allele. The quantitative polymerase chain reaction assay was highly sensitive and specific, detecting RHOA Gly17Val at an allele frequency of 0.03%, but not other changes in Gly17, nor in 61 controls. Among the 37 cases of AITL and PTCL-TFH investigated, RHOA Gly17Val was detected in 62.2% (23/37) of which 19 had multiple biopsies including preceding biopsies in ten and follow-up biopsies in 11 cases. RHOA Gly17Val was present in each of these preceding or follow-up biopsies including 18 specimens that showed no evidence of lymphoma by combined histological, immunophenotypic and clonality analyses. The mutation was seen in biopsies 0-26.5 months (mean 7.87 months) prior to the lymphoma diagnosis. Our results show that RHOA Gly17Val mutation analysis is valuable in the early detection of AITL and PTCL-TFH.

Angioimmunoblastic T-cell lymphoma contains multiple clonal T-cell populations derived from a common TET2 mutant progenitor cell.

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 ß (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.

Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B-cell neoplasms.

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.

Non-IG::MYC in diffuse large B-cell lymphoma confers variable genomic configurations and MYC transactivation potential.

MYC translocation occurs in 8-14% of diffuse large B-cell lymphoma (DLBCL), and may concur with BCL2 and/or BCL6 translocation, known as double-hit (DH) or triple-hit (TH). DLBCL-MYC/BCL2-DH/TH are largely germinal centre B-cell like subtype, but show variable clinical outcome, with IG::MYC fusion significantly associated with inferior survival. While DLBCL-MYC/BCL6-DH are variable in their cell-of-origin subtypes and clinical outcome. Intriguingly, only 40-50% of DLBCL with MYC translocation show high MYC protein expression (>70%). We studied 186 DLBCLs with MYC translocation including 32 MYC/BCL2/BCL6-TH, 75 MYC/BCL2-DH and 26 MYC/BCL6-DH. FISH revealed a MYC/BCL6 fusion in 59% of DLBCL-MYC/BCL2/BCL6-TH and 27% of DLBCL-MYC/BCL6-DH. Targeted NGS showed a similar mutation profile and LymphGen genetic subtype between DLBCL-MYC/BCL2/BCL6-TH and DLBCL-MYC/BCL2-DH, but variable LymphGen subtypes among DLBCL-MYC/BCL6-DH. MYC protein expression is uniformly high in DLBCL with IG::MYC, but variable in those with non-IG::MYC including MYC/BCL6-fusion. Translocation breakpoint analyses of 8 cases by TLC-based NGS showed no obvious genomic configuration that enables MYC transactivation in 3 of the 4 cases with non-IG::MYC, while a typical promoter substitution or IGH super enhancer juxtaposition in the remaining cases. The findings potentially explain variable MYC expression in DLBCL with MYC translocation, and also bear practical implications in its routine assessment.

Characteristics of CD44 alternative splice pattern in the course of human colorectal adenocarcinoma progression.

BACKGROUND: CD44 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. METHODS: We 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. RESULTS: We 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. CONCLUSION: Particular 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.

Notch2 controls non-autonomous Wnt-signalling in chronic lymphocytic leukaemia.

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-ß mediated degradation of ß-catenin in CLL cells. Additionally, stromal Notch2 activity regulates N-cadherin expression in CLL cells, which interacts with and further stabilises ß-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.

Demonstration of a melanoma-specific CD44 alternative splicing pattern that remains qualitatively stable, but shows quantitative changes during tumour progression.

The role of CD44 in the progression of human melanoma has mostly been characterised by qualitative changes in expression of its individual variable exons. These exons however, may be expressed to form a number of molecules, the alternative splice variants of CD44, which may be structurally and functionally different. Using real-time PCR measurements with variable exon specific primers we have determined that all are expressed in human melanoma. To permit comparison between different tumours we identified a stable CD44 variable exon (CD44v) expression pattern, or CD44 'fingerprint'. This was found to remain unchanged in melanoma cell lines cultured in different matrix environments. To evaluate evolution of this fingerprint during tumour progression we established a scid mouse model, in which the pure expression pattern of metastatic primary tumours, circulating cells and metastases, non-metastatic primary tumours and lung colonies could be studied. Our analyses demonstrated, that although the melanoma CD44 fingerprint is qualitatively stable, quantitative changes are observed suggesting a possible role in tumour progression.