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.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity.

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL.

A20 inactivation in ocular adnexal MALT lymphoma.

Recent studies showed A20 inactivation by deletion, mutation and promoter methylation in ocular adnexal mucosa-associated lymphoid tissue lymphoma. However, the incidences of A20 abnormalities and their clinical impact remain for the most part unknown. It is also unknown whether ABIN-1 and ABIN-2, the components of the A20 NF-κB inhibitor complex, are inactivated by genetic changes in ocular adnexal mucosa-associated lymphoid tissue lymphoma. A total of 105 cases were investigated for A20 mutation/deletion, ABIN-1/2 mutation, MALT1 and IGH involved translocation. Somatic mutation was seen frequently in A20 (28.6%) but rarely in ABIN-1 (1%) and ABIN-2 (1%). A20 mutations were significantly associated with A20 heterozygous deletion, and both were mutually exclusive from the MALT1 or IGH involved translocations. A20 mutation/deletion was also significantly associated with increased expression of the NF-κB target genes CCR2, TLR6 and BCL2. The cases with A20 mutation/deletion required significantly higher radiation dosages to achieve complete remission than those without these abnormalities.

BCR and TLR signaling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas.

The genetics and pathogenesis of splenic marginal zone lymphoma are poorly understood. The lymphoma lacks chromosome translocation, and approximately 30% of cases are featured by 7q deletion, but the gene targeted by the deletion is unknown. A recent study showed inactivation of A20, a "global" NF-κB negative regulator, in 1 of 12 splenic marginal zone lymphomas. To investigate further whether deregulation of the NF-κB pathway plays a role in the pathogenesis of splenic marginal zone lymphoma, we screened several NF-κB regulators for genetic changes by PCR and sequencing. Somatic mutations were found in A20 (6/46=13%), MYD88 (6/46=13%), CARD11 (3/34=8.8%), but not in CD79A, CD79B and ABIN1. Interestingly, these genetic changes are largely mutually exclusive from each other and MYD88 mutation was also mutually exclusive from 7q deletion. These results strongly suggest that deregulation of the TLR (toll like receptor) and BCR (B-cell receptor) signaling pathway may play an important role in the pathogenesis of splenic marginal zone lymphoma.

BIOMED-2 PCR assays for IGK gene rearrangements are essential for B-cell clonality analysis in follicular lymphoma.

B-cell clonality analysis is commonly performed by polymerase chain reaction (PCR) targeting the IGH genes although a high false-negative rate is recognized for germinal centre/post-germinal centre B-cell malignancies, especially follicular lymphoma. We assessed the diagnostic value of BIOMED-2 IGK assays and investigated the cause of IGH PCR failure in 77 patients with follicular lymphoma. Using the full set of BIOMED-2 reactions, clonal immunoglobulin gene rearrangements were detected in 74 (96%) cases. The clonality detection rate was 86% by two IGK reactions but only 68% by five IGH reactions (P < 0·001). Sequencing of the clonal PCR products showed significantly fewer somatic mutations in the rearranged IGKV (9/27 cases, 33%, mean mutation rate 0·5%) than IGHV (17/17 cases, 100%, rate 11·0%) (P < 0·01). All IGHV-IGHJ PCR failures occurred in cases with at least one mutation at the corresponding IGHV primer binding sites. t(14:18)(q32:q21)/IGH-BCL2 was detected in 50 of 71 (70%) cases and the presence of the translocation was not associated with the poor performance of IGH assays. Our results showed that BIOMED-2 IGK assays are significantly more sensitive than IGH assays in follicular lymphoma due to the fact that the rearranged IGKV is less frequently targeted by somatic hypermutation than IGHV, and therefore, are essential in routine clonality analysis of these lymphomas.

Splenic marginal zone lymphoma: characterization of 7q deletion and its value in diagnosis.

The diagnosis of splenic marginal zone lymphoma (SMZL) is frequently a challenge, due to its lack of specific histological features and immunophenotypic markers, and the existence of other poorly characterized splenic lymphomas defying classification. Moreover, the clinical outcome of SMZL is variable, with 30% of cases pursuing an aggressive clinical course, the prediction of which remains problematic. Thus, there is a real need for biomarkers in the diagnosis and prognostication of SMZL. To search for genetic markers, we comprehensively investigated the genomic profile, TP53 abnormalities, and immunoglobulin heavy gene (IGH) mutation in a large cohort of SMZLs. 1 Mb resolution array comparative genomic hybridization (aCGH) on 25 SMZLs identified 7q32 deletion (44%) as the most frequent copy number change, followed by gains of 3q (32%), 8q (20%), 9q34 (20%), 12q23-24 (8%), and chromosome 18 (12%), and losses of 6q (16%), 8p (12%), and 17p (8%). High-resolution chromosome 7 tile-path aCGH on 17 SMZLs with 7q32 deletion identified by 1 Mb aCGH or interphase FISH screening mapped the minimal common deletion to a 3 Mb region at 7q32.1-32.2. Although it is not yet possible to identify the genes targeted by the deletion, interphase FISH screening showed that the deletion was seen in SMZL (19/56 = 34%) and splenic B-cell lymphoma/leukaemia unclassifiable (3/9 = 33%), but not in 39 cases of other splenic lymphomas including chronic lymphocytic leukaemia (n = 14), hairy cell leukaemia (4), mantle cell lymphoma (12), follicular lymphoma (6), and others. In SMZL, 7q32 deletion was inversely correlated with trisomy 18, but not associated with other copy number changes, TP53 abnormalities, or IGH mutation status. None of the genetic parameters examined showed significant and independent association with overall or event-free survival. In conclusion, 7q32 deletion is a characteristic feature of SMZL, albeit seen in isolated cases of splenic B-cell lymphoma/leukaemia unclassifiable, and its detection may help the differential diagnosis of splenic B-cell lymphomas.

Primary effusion lymphoma: genomic profiling revealed amplification of SELPLG and CORO1C encoding for proteins important for cell migration.

Primary effusion lymphoma (PEL) is associated with Kaposi sarcoma herpesvirus (KSHV) but its pathogenesis is poorly understood. Many KSHV-associated products can deregulate cellular pathways commonly targeted in cancer. However, KSHV infection alone is insufficient for malignant transformation. PEL also lacks the chromosomal translocations seen in other lymphoma subtypes. We investigated 28 PELs and ten PEL cell lines by 1 Mb resolution array comparative genomic hybridization (CGH) and found frequent gains of 1q21-41 (47%), 4q28.3-35 (29%), 7q (58%), 8q (63%), 11 (32%), 12 (61%), 17q (29%), 19p (34%), and 20q (34%), and losses of 4q (32%), 11q25 (29%), and 14q32 (63%). Recurrent focal amplification was seen at several regions on chromosomes 7, 8, and 12. High-resolution chromosome-specific tile-path array CGH confirmed these findings, and identified selectin-P ligand (SELPLG) and coronin-1C (CORO1C) as the targets of a cryptic amplification at 12q24.11. Interphase FISH and quantitative PCR showed SELPLG/CORO1C amplification (>4 extra copies) and low levels of copy number gain (1-4 extra copies) in 23% of PELs, respectively. Immunohistochemistry revealed strong expression of both SELPLG and coronin-1C in the majority of PELs, irrespective of their gene dosage. SELPLG is critical for cell migration and chemotaxis, while CORO1C regulates actin-dependent processes, thus important for cell motility. Their overexpression in PEL is expected to play an important role in its pathogenesis.

The lymphoma-associated fusion tyrosine kinase ITK-SYK requires pleckstrin homology domain-mediated membrane localization for activation and cellular transformation.

ITK-SYK, a novel fusion tyrosine kinase (FTK) resulting from a recurrent t(5;9)(q33;q22), was recently identified in a poorly understood subset of peripheral T-cell lymphomas. However, the biochemical and functional properties of ITK-SYK are unknown. Here we demonstrate that ITK-SYK is a catalytically active tyrosine kinase that is sensitive to an established inhibitor of SYK. The expression of ITK-SYK, but not SYK, transformed NIH3T3 cells, inducing loss of contact inhibition and formation of anchorage-independent colonies in soft agar, in a kinase activity-dependent manner. ITK-SYK is unusual among FTKs in having an N-terminal phosphatidylinositol 3,4,5-trisphosphate-binding pleckstrin homology (PH) domain. Introduction of a well characterized loss-of-function mutation (R29C) into the PH domain of ITK-SYK inhibited its phosphorylation, markedly reduced its catalytic activity, and abrogated its ability to activate the ERK signaling pathway and to transform NIH3T3 cells. Although ITK-SYK was membrane-associated, ITK-SYK-R29C was not. However, each of these properties could be recovered by retargeting ITK-SYK-R29C back to the plasma membrane by the addition of an N-terminal myristylation sequence. Consistent with a model in which ITK-SYK requires PH domain-mediated binding to phosphatidylinositol 3,4,5-trisphosphate generated by phosphatidylinositol 3-kinase (PI3K), ITK-SYK activity was reduced by pharmacological inhibition of PI3K and increased by co-expression with a constitutively active form of PI3K. Together, these findings identify ITK-SYK as an active, transforming FTK dependent upon PH domain-mediated membrane localization, identify a novel mechanism for activation of an oncogenic FTK, and suggest ITK-SYK as a rational therapeutic target for t(5;9)(q33;q22)-positive lymphomas.

Variable Responses of MYC Translocation Positive Lymphoma Cell Lines To Different Combinations of Novel Agents: Impact of BCL2 Family Protein Expression.

Several newly developed drugs including JQ1 (BET inhibitor), ABT199 (BCL2 inhibitor), and bortezomib (proteasome inhibitor) may offer novel therapeutic strategies for aggressive diffuse large B-cell lymphoma (DLBCL). We tested these drugs together with doxorubicin in a series of combinations in 16 DLBCL cell lines including 4 ABC-DLBCL (OCI-Ly3, OCI-Ly10, SUDHL2, RIVA) and 12 GCB-DLBCL lines (OCI-Ly4, OCI-Ly18, BJAB, SUDHL4, SUDHL6, SUDHL10, DB, PR1, VAL, SC1, Karpas-231, Karpas-422). Among these cell lines, ABT199 and doxorubicin, and to a lesser extent JQ1 and bortezomib, showed high variations in their ED50 values. Of the six cell lines showing high ABT199 ED50 values, four (SUDHL10, OCI-Ly4, SUDHL2, and BJAB) had no or little BCL2 expression, and SUDHL6 also displayed a low BCL2 expression. There was no association between the ED50 value of doxorubicin, JQ1 and bortezomib, and TP53/MYC/BCL2 genetic abnormalities or cell of origin subtype. A synergistic effect in all or the majority of drug combinations was seen in 11 cell lines, while an antagonistic effect in a high proportion of drug combinations was observed in the remaining 5 cell lines including the 3 (SUDHL10, OCI-Ly4, and SUDHL2) with little BCL2 expression, and additionally OCI-Ly18 and RIVA. Extensive Western blot analyses revealed high MCL1 expression in SUDHL10 and OCI-Ly4 but no apparent alterations in other cell lines. The molecular mechanism underlying the antagonistic effect of drug combinations in DLBCL is heterogeneous with the altered BCL2 family protein expression (absent BCL2, but high MCL1) in some cell lines.

Application of a multi-gene next-generation sequencing panel to a non-invasive oesophageal cell-sampling device to diagnose dysplastic Barrett's oesophagus.

The early detection and endoscopic treatment of patients with the dysplastic stage of Barrett's oesophagus is a key to preventing progression to oesophageal adenocarcinoma. However, endoscopic surveillance protocols are hampered by the invasiveness of repeat endoscopy, sampling bias, and a subjective histopathological diagnosis of dysplasia. In this case-control study, we investigated the use of a non-invasive, pan-oesophageal cell-sampling device, the Cytosponge™, coupled with a cancer hot-spot panel to identify patients with dysplastic Barrett's oesophagus. Formalin-fixed, paraffin-embedded (FFPE) Cytosponge™ samples from 31 patients with non-dysplastic and 28 with dysplastic Barrett's oesophagus with good available clinical annotation were selected for inclusion. Samples were microdissected and amplicon sequencing performed using a panel covering >2800 COSMIC hot-spot mutations in 50 oncogenes and tumour suppressor genes. Strict mutation criteria were determined and duplicates were run to confirm any mutations with an allele frequency <12%. When compared with endoscopy and biopsy as the gold standard the panel achieved a 71.4% sensitivity (95% CI 51.3-86.8) and 90.3% (95% CI 74.3-98.0) specificity for diagnosing dysplasia. TP53 had the highest rate of mutation in 14/28 dysplastic samples (50%). CDKN2A was mutated in 6/28 (21.4%), ERBB2 in 3/28 (10.7%), and 5 other genes at lower frequency. The only gene from this panel found to be mutated in the non-dysplastic cases was CDKN2A in 3/31 cases (9.7%) in keeping with its known loss early in the natural history of the disease. Hence, it is possible to apply a multi-gene cancer hot-spot panel and next-generation sequencing to microdissected, FFPE samples collected by the Cytosponge™, in order to distinguish non-dysplastic from dysplastic Barrett's oesophagus. Further work is required to maximize the panel sensitivity.

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues.

The prognosis of MYC translocation positive diffuse large B-cell lymphoma depends on the second hit.

A proportion of MYC translocation positive diffuse large B-cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double-hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double-hit DLBCL, and whether there is a difference in clinical outcome between the double-hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81 = 74%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R-CHOP (n = 67), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double-hits had the worst overall survival, followed by those with MYC/BCL2 double-hits. In MYC translocation negative DLBCL treated by R-CHOP (n = 101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double-hit DLBCLs from those with an isolated MYC translocation.

A comparison of methods for EGFR mutation testing in non-small cell lung cancer.

EGFR mutation testing of tumor samples is routinely performed to predict sensitivity to treatment with tyrosine kinase inhibitors for patients with non-small cell lung cancer. At least 9 different methodologies are employed in UK laboratories, and the aim of this study was to compare the sensitivity of different methods for the detection of EGFR mutations. Participating laboratories were sent coded samples with varying mutation loads (from 0% to 15%) to be tested for the p.Leu858Arg (p.L858R) missense mutation and c.2235_2249del exon 19 deletion. The p.L858R mutation and deletions within exon 19 of the EGFR gene account for ∼90% of mutation-positive cases. The 11 laboratories used their standard testing method(s) and submitted 15 sets of results for the p.L858R samples and 10 for the exon 19 deletion. The p.Leu858Arg (p.L858R) mutation was detected at levels between 1% and 7.5% by Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system, and capillary electrophoresis single-strand conformation analysis. The c.2235_2249del mutation was detected at 1% to 5% by fragment size analysis, Sanger sequencing or real-time PCR. A mutation was detected in 24/25 (96%) of the samples tested which contained 5% mutated DNA. The 1% sensitivity claimed for commercial real-time PCR-targeted EGFR tests was achieved and our results show greater sensitivity for the Sanger sequencing and pyrosequencing screening methods compared to the 10% to 20% detection levels cited on clinical diagnostic reports. We conclude that multiple methodologies are suitable for the detection of acquired EGFR mutations.

An integrated genomic and expression analysis of 7q deletion in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is an indolent B-cell lymphoproliferative disorder characterised by 7q32 deletion, but the target genes of this deletion remain unknown. In order to elucidate the genetic target of this deletion, we performed an integrative analysis of the genetic, epigenetic, transcriptomic and miRNomic data. High resolution array comparative genomic hybridization of 56 cases of SMZL delineated a minimally deleted region (2.8 Mb) at 7q32, but showed no evidence of any cryptic homozygous deletion or recurrent breakpoint in this region. Integrated transcriptomic analysis confirmed significant under-expression of a number of genes in this region in cases of SMZL with deletion, several of which showed hypermethylation. In addition, a cluster of 8 miRNA in this region showed under-expression in cases with the deletion, and three (miR-182/96/183) were also significantly under-expressed (P<0.05) in SMZL relative to other lymphomas. Genomic sequencing of these miRNA and IRF5, a strong candidate gene, did not show any evidence of somatic mutation in SMZL. These observations provide valuable guidance for further characterisation of 7q deletion.

Primary cerebral diffuse large B-cell lymphoma relapsed solely in the skin with the same clonal origin.

Diffuse large B-cell lymphoma is the most common type of primary central nervous system (CNS) lymphoma(PCNSL) with poor prognosis. Relapse occurs early and most commonly in CNS, whereas systemic relapse solely in skin is exceptional. We presented a unique case of PCNSL in an 82-year-old man with 2 consecutive skin relapses without concomitant local failure or other systemic involvement. The lymphoma cells of all 3 specimens were of the same histology and immunophenotype with expression of CD20, IgM, bcl-2, bcl-6, and MUM1 but not CD3, CD10, CD30, IgD, cyclin D1,or cutaneous lymphocyte-associated antigen. The skin tumors were in the dermis and subcutis with a spared grenz zone. The brain and skin tumors demonstrated the same clonal origin by B-cell clonality study followed by cloning and sequencing. To our knowledge, this is the first case of PCNSL relapsed solely in the skin with proven clonal identity.