Alexandra Clipson

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.

Significant association between TNFAIP3 inactivation and biased immunoglobulin heavy chain variable region 4‐34 usage in mucosa‐associated lymphoid tissue lymphoma

Both antigenic drive and genetic change play critical roles in the development of mucosa‐associated lymphoid tissue (MALT) lymphoma, but neither alone is sufficient for malignant transformation, and lymphoma development critically depends on their cooperation. However, which of these different events concur and how they cooperate in MALT lymphomagenesis is totally unknown. To explore this, we investigated somatic mutations of 17 genes and immunoglobulin heavy chain variable region (IGHV) usage in 179 MALT lymphomas from various sites. We showed that: (1) there was a significant association between the biased usage of IGHV4‐34 (binds to the carbohydrate I/i antigens) and inactivating mutation of TNFAIP3 [encoding a global negative regulator of the canonical nuclear factor‐κB (NF‐κB) pathway] in ocular adnexal MALT lymphoma; (2) IGHV1‐69 was significantly overrepresented (54%) in MALT lymphoma of the salivary gland, but was not associated with mutation in any of the 17 genes investigated; and (3) MALT lymphoma lacked mutations that are frequently seen in other B‐cell lymphomas characterized by constitutive NF‐κB activities, including mutations in CD79B, CARD11, MYD88, TNFRSF11A, and TRAF3. Our findings show, for the first time, a significant association between biased usage of autoreactive IGHV and somatic mutation of NF‐κB regulators in MALT lymphoma, arguing for their cooperation in sustaining chronic B‐cell receptor signalling and driving oncogenesis in lymphoma development. Copyright

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

Significant association between TNFAIP3 inactivation and biased IGHV4-34 usage in MALT lymphoma

Both antigenic drive and genetic change play critical roles in the development of mucosa‐associated lymphoid tissue (MALT) lymphoma, but neither alone is sufficient for malignant transformation, and lymphoma development critically depends on their cooperation. However, which of these different events concur and how they cooperate in MALT lymphomagenesis is totally unknown. To explore this, we investigated somatic mutations of 17 genes and immunoglobulin heavy chain variable region (IGHV) usage in 179 MALT lymphomas from various sites. We showed that: (1) there was a significant association between the biased usage of IGHV4‐34 (binds to the carbohydrate I/i antigens) and inactivating mutation of TNFAIP3 [encoding a global negative regulator of the canonical nuclear factor‐κB (NF‐κB) pathway] in ocular adnexal MALT lymphoma; (2) IGHV1‐69 was significantly overrepresented (54%) in MALT lymphoma of the salivary gland, but was not associated with mutation in any of the 17 genes investigated; and (3) MALT lymphoma lacked mutations that are frequently seen in other B‐cell lymphomas characterized by constitutive NF‐κB activities, including mutations in CD79B, CARD11, MYD88, TNFRSF11A, and TRAF3. Our findings show, for the first time, a significant association between biased usage of autoreactive IGHV and somatic mutation of NF‐κB regulators in MALT lymphoma, arguing for their cooperation in sustaining chronic B‐cell receptor signalling and driving oncogenesis in lymphoma development. Copyright

Prognostic significance and correlation to gene expression profile of EZH2 mutations in diffuse large B‐cell lymphoma (DLBL) in 2 large prospective studies

EZH2, a histone methyl transferase subunit of Polycomb repressor complex 2, is frequently mutated in DLBL. Inhibitors of EZH2 have demonstrated promising responses in early clinical trials. We examined the frequency of EZH2 mutation in 2 large prospective series of DLBL and correlated this to clinical outcomes in relation to other biological features.

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.

Mutation screening using formalin-fixed paraffin-embedded tissues: a stratified approach according to DNA quality

DNA samples from formalin-fixed paraffin-embedded tissues are highly degraded with variable quality, and this imposes a big challenge for targeted sequencing due to false positives, largely caused by PCR errors and cytosine deamination. To eliminate false positives, a common practice is to validate the detected variants by Sanger sequencing or perform targeted sequencing in duplicate. Technically, PCR errors could be removed by molecular barcoding of template DNA prior to amplification as in the HaloPlexHS design. Nonetheless, it is uncertain to what extent variants detected using this approach should be further validated. Here, we addressed this question by correlating variant reproducibility with DNA quality using HaloPlexHS target enrichment and Illumina HiSeq4000, together with an in-house validated variant calling algorithm. The overall sequencing coverage, as shown by analyses of 70 genes in 266 cases of large B-cell lymphoma, was excellent (98%) in DNA samples amenable for PCR of ≥400 bp, but suboptimal (92%) and poor (80%) in those amenable for PCR of 300 bp and 200 bp respectively. By mutation analysis in duplicate in 93 cases, we demonstrated that 20 alternative allele depth (AAD) was an optimal cut-off value for separating reproducible from non-reproducible variants in DNA samples amenable for PCR of ≥300 bp, with 97% sensitivity and 100% specificity. By cross validation with a previously established targeted sequencing protocol by Fluidigm-PCR and Illumina MiSeq, the HaloPlexHS protocol was shown to be highly sensitive and specific in mutation screening. To conclude, we proposed a stratified approach for mutation screening by HaloplexHS and Illumina HiSeq4000 according to DNA quality. DNA samples with good quality (≥400 bp) are amenable for mutation analysis with a single replicate, with only variants at 15–20 AAD requiring for further validation, while those with suboptimal quality (300 bp) are better analysed in duplicate with reproducible variants at >15 AAD regarded as true genetic changes.HaloPlexHS target enrichment incorporates molecular barcodes in hybridization probes in order to detect data resulting from PCR errors. The authors combined HaloPlexHS technology with Illumina HiSeq sequencing and developed a new validated variant-calling algorithm. Thus, they were able to establish a targeted sequencing protocol that allows reliable mutation detection with a single replicate using DNA samples from formalin-fixed paraffin-embedded tissues.

CROSS-PLATFORM VALIDATION OF GENE EXPRESSION PROFILING (GEP) BASED CELL OF ORIGIN (COO) CLASSIFICATION IN a CLINICAL LABORATORY SETTING

cating regions of the genome. 166 Kataegis‐ROIs were identified, 42/64/17/4 of which were known targets of aberrant somatic hypermutation (SHM) / were located within the immunoglobulin (IG) loci / overlapped with lymphoma‐associated genes / overlapped with cancer genes known from other entities, respectively. Kataegis‐ROIs were enriched in early replicating regions of the genome. In an analysis of mutational signatures, 11 known signatures including clocklike signatures (e.g. spontaneous deamination), DNA repair defect signatures, an APOBEC signature and the B‐cell specific signature AC9 (attributed to AID and polymerase η) were found. Furthermore, we discovered three new signatures (L1 – L3). L1 was enriched at the IG loci. L2 was specifically enriched in the constant domains of the IGH locus. Conclusions: L1 and L2 may be interpreted as an imprint of the action of AID on the genome, L2 with a high amount of modulation by altered repair pathways, L1 with a lower amount of modulation. Both L1 and L2 contribute to SHM, whereas class switch recombination (CSR) may be explained with only L1. Kataegis clusters may be classified into two groups, one of which is attributable to aberrant SHM, the other to dysregulated CSR.

Identifying somatic mutations in cell-free DNA of aggressive lymphoma patients: First cell-free DNA results from the molecular profiling for lymphoma (MaPLe) study

B‐cell development. Somatic mutations of KMT2D are predominantly found in follicular lymphoma (FL), resulting in the inability of KMT2D to activate gene transcription through H3K4 methylation. ID3 is a negative regulator of TCF3, which activates the prosurvival phosphatidylinositol‐3‐OH kinase pathway by enhancing B‐cell receptor signaling in B‐cell lymphomas. Specific pathways important in lymphomagenesis that likely were altered in this cohort of patients include apoptosis/cell cycle (CCND3, TP53, BCL2 family genes at 13.33%, FOXO1 4%, GNA13 4%, MFHAS1 4%, MYC 5.33%, XPO1 0.67%); immunity (B2M, CD58 7.33%, CIITA); NOTCH (NOTCH2 9.33%, DTX1, SPEN); NFkB (PIM1, TNFAIP3, CARD11 6%, IRF4 6.67%, MYD88 7.33%); epigenetic regulation (CREBBP 8%, EP300 8.67%, EZH2 2%, KMT2C, KMT2D, MEF2B 4.67%); MAP kinase (BRAF at 2%, MAP2K1 2.67%); and BCR (CD79A 2%, CD79B, ID3, ITPKB, TCF3 2%). PTEN (4%), TET2 (2%), and transcription factors ARID1A and ARID3A/B (4%), which are members of the SWI/SNF family, were also detected in this cohort of patients. Conclusions: Our study demonstrates that B‐cell lymphoma is characterized by remarkable genetic heterogeneity. The frequently mutated genes involved in different pathways identified by NGS may be further exploited for improving diagnosis and personalized therapies for patients with B‐cell lymphomas.