ALBERT

Description: [Background] Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma. We and others reported that the G17V RHOA mutation is frequent and highly specific to AITL and AITL-like peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Thus, detection of this mutation serves as a novel diagnostic strategy for AITL and AITL-like PTCL-NOS. [Aim] Analysis of disease-specific mutations in serum DNA from AITL and PTCL-NOS patients. [Method] Genomic DNA was collected from tumors and sera from 16 AITL and PTCL-NOS patients and 11 B-cell malignancy patients. Seven out of 16 T-cell lymphoma and all B-cell malignancy patients did not receive prior treatment. Amplicon-based sequencing by Ion Torrent PGM was performed to analyze the G17V RHOA mutation as well as TET2 and DNMT3A mutations in these samples. [Result] Three out of 7 tumor samples of T-cell lymphoma patients whose serum was preserved prior to treatment were positive for the G17V RHOA mutation. In the serum of these 3 patients, allele frequencies of the G17V RHOA mutation were 0.006 - 0.072 (mean 0.015, SDV 0.026), while 0 (mean 0, SDV 0) in the serum of the remaining 4 T-cell lymphoma patients and all the 11 B-cell malignancy patients whose tumor samples did not show RHOA mutation. Because we set cut off value as 0.002 (0.2%) based on the ROC curve, RHOA mutation was segregated into positive and negative faithfully according to whether the mutation was identified in the tumor samples. Then, we analyzed TET2 and DNMT3A mutations in sera of 5 patients whose tumor samples were positive for TET2 (n=2; a single mutation in each sample) and DNMT3A (n=3; 5 mutations all together), respectively. TET2 and DNMT3A mutations were detected in all the 5 sera, although 2 out of the 5 DNMT3A mutations were undetectable. In the clinical courses, RHOA mutations became undetectable after the treatment in sera of all the 3 patients whose pre-treatment sera were positive, while TET2 mutations were detected even after the treatment in sera of both patients whose pre-treatment sera were positive. [Conclusion] Biopsy is sometimes difficult in lymphoma patients with progressed diseases. Identification of the disease-specific G17V RHOA mutation in serum may provide a non-invasive method to diagnose AITL and AITL-like PTCL-NOS, although inapplicable to detect minimum residual disease. Stable detection of TET2 mutations regardless of chemotherapy might indicate the presence of the mutations in the 'pre-cancerous cells' which are resistant to the chemotherapy. Disclosures No relevant conflicts of interest to declare.

Description: [Backgrounds] Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of peripheral T-cell lymphoma (PTCL), characterized by generalized lymphadenopathy and autoimmune-like manifestations. Regarding genetic lesions of AITL, frequent mutations in TET2, IDH2, DNMT3A and RHOA have been identified. In some PTCL cases, TET2 and DNMT3A mutations were identified in cell populations beyond the CD4+ T-lymphocytes, in which the tumor cells are contained, suggesting that TET2 and DNMT3A mutations occurred earlier than the commitment to CD4+ T lymphocytes. [Objective] We performed this study to identify the cell-type-specific mutations and further explore mutational profiles in AITL and AITL-related cancer. [Methods] The dataset of targeted sequencing was analyzed for 76 genes in 79 PTCL samples. Mutational origin was analyzed by cell sorting and laser microdissection. [Results] Targeted sequencing identified 168 mutations in 33 genes. Recurrent mutations, in addition to the already known frequent mutations in RHOA/TET2/IDH2/DNMT3A, were found in ODZ1 [4/79 (5%)], Notch1, NAV2, and MTERFD3 [3/79 (4%) for each], MLL2, TET3, FAT2, and LAMA2 [2/79 (3%) for each]. TET2/DMNT3A mutations showed statistically higher allelic burden than the newly identified mutations, suggesting precedence of TET2/DNMT3A mutations. Cell sorting and laser microdissection, followed by amplicon sequencing, revealed that TET2/DNMT3A mutations were identified in both tumor cell-enriched and –depleted populations while RHOA and IDH2 mutations were confined to tumor cell-enriched populations. Most of the newly identified mutations were similarly classified into the above-mentioned two types. It is noteworthy that we found some mutations only in T-cell lymphoma cell-depleted CD20-positive population but not in the tumor-cell-enriched PD-1-positive population. [Conclusion and discussion] Differentiation stages that mutational events arise are likely to be multiple in AITL and AITL-related lymphoma. Moreover, in AITL, Epstein-Bar virus-infected B cells often grow in an oligoclonal manner, sometimes resulting in monoclonal proliferation with fully malignant features. Detection of B-cell specific mutations might suggest premalignant status of B cells in these cases. Disclosures No relevant conflicts of interest to declare.

Description: Backgrounds Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of T-cell lymphoma, characterized by generalized lymphadenopathy and frequent autoimmune-like manifestations. The diagnosis of AITL is sometimes challenging for hematopathologists, because the tumor cell content is generally low and relatively large reactive lymphocytes are confused as tumor cells. Possibly because of the low tumor cell frequency, clonal rearrangement of T-cell receptor gene is undetectable in 30% of the cases. We identified recurrent mutations in RHOA at c.G50T, predicting to generate p.G17V in 70% of AITL and PTCL-NOS harboring AITL features, which implies diagnostic properties for AITL (M S-Y and SC, unpublished). Purpose To establish a novel cost-effective method to diagnose AITL, we performed allele-specific realtime PCR to detect RHOA G17V mutation. Methods Genomic DNA was extracted from 119 AITL and PTCL-NOS samples, which include 47 periodate-lysine-paraformaldehyde (PLP)-fixed, 12 formalin-fixed-paraffin- embedded (FFPE) and 60 frozen tissues. Forty-one out of 60 genomic DNA samples, purified from frozen tissue, were amplified by RepliG kit (QIAGEN). Allele-specific primers for RHOA G17V mutant and wild-type sequences were designed by Wangkumhang's algorithm. The [mut] and [WT] values were individually measured by realtime PCR using each primer set, and the [mut]/([mut]+[WT]) values were calculated. Mutant allele frequencies were determined by amplicon-based deep sequencing using MiSeq. Results The [mut] values were distributed from 1.5×10-7 to 7.6×10-2, and the [WT] values were from 7.9×10-5 to 1.3×10-2. The [mut]/[mut]+[WT] values were distributed from 1.9×10-4 to 8.5×10-1. We set a cut-off value to determine existence of mutation as 2% for MiSeq, and 1.3×10-2 for the [mut]/([mut]+[WT]) value. Then we compared these two methods to detect RHOA G17V mutation. Forty-three cases were positive for the RHOA mutation in this study cohort by MiSeq, including 32 AITL and 11 PTCL-NOS cases. The [mut]/([mut]+[WT]) values of DNA from FFPE samples tend to be lower than those from other samples, and 4 out of 12 FFPE samples determined as mutation-positive by MiSeq were not detected by our allele-specific realtime PCR. We therefore excluded the FFPE samples and analyzed the data of 107 DNA samples, purified from PLP-fixed and frozen tissues. Rank correlation coefficient was 0.753. Sensitivity was 97.4%, and specificity was 97.1%. Positive concordance rate was 94.9%, and negative concordance rate was 98.6%. Conclusions We established a method to detect RHOA G17V hotspot mutation for AITL. It is expected to be highly accurate and cost effective. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin's lymphoma. Although most cases (~95%) show histology of diffuse large B-cell lymphomas (DLBCLs), PCNSL shows very different biological and clinical characteristics from systemic DLBCL. Nevertheless, our knowledge about the molecular pathogenesis of PCNSL and genetic differences between both lymphomas are still incomplete. Method To obtain a comprehensive view of the genetic alterations, including mutations in non-coding regions as well as structural variants (SVs), we performed whole-genome sequencing (WGS) of 22 PCNSL cases. Subsequently, to unravel the genetic differences between PCNSL and systemic DLBCL, we re-analyzed WGS data from systemic DLBCL cases (N = 47) generated by the Cancer Genome Atlas Network (TCGA) and Cancer Genome Characterization Initiative (CGCI) using our in-house pipeline. The mean depth of WGS for tumor samples were 49X and 37X for PCNSL and DLBCL cases, respectively. Whole-exome sequencing (WES) was also performed for an additional 37 PCNSL cases to reliably capture driver alterations and also to analyze mutational signatures in PCNSL, which were compared to those obtained from the WES data for DLBCL from TCGA (N = 49). Results WGS identified 10.5 and 5.6 mutations per mega-base on average in PCNSL and DLBCL, respectively. We first explored the density of somatic mutations and identified 64 and 33 genomic loci showing significantly high mutation densities in PCNSL and DLBCL, respectively. In PCNSL, most of these loci corresponded to known targets of somatic hypermutations (SHMs) induced by activation-induced cytidine deaminase (AID), including those for IG genes (IGK, IGH and IGL), BCL6, and PIM1, as well as those for known driver genes, such as MYD88 and CD79B. Although most of the hypermutated regions were overlapped between PCNSL and DLBCL, some regions were differentially affected by hypermutations between both lymphoma types. For example, BCL2 and SGK1 loci were frequently affected by SHMs in germinal center B-cell (GCB) DLBCL, while not in PCNSL. In terms of non-coding driver mutations, we identified frequent mutations in a PAX5 enhancer region in 8/22 (36%) of PCNSL and 18/47 (38%) of DLBCL cases. SVs were common in both lymphoma types, where 104 (PCNSL) and 57 (DLBCL) SVs were detected per sample. SV clusters were identified in 34 (PCNSL) and 13 (DLBCL) regions, of which several clusters were commonly seen in both PCNSL and DLBCL, and included IG loci, BCL6, FHIT, TOX and CDKN2A. In PCNSL, SVs were clustered within the loci for known targets of SHMs, such as BCL6, BTG2 and PIM1. As was the case with somatic mutations, the SV cluster corresponding to BCL2 was only seen in DLBCL. We then analyzed these clustered breakpoints for their proximity to known sequence motifs targeted by AID (CpG and WGCW). Breakpoints of SVs found in the targets of SHMs, including PIM1, BCL6, BTG2 and BCL2, showed an enrichment at or near the CpG, supporting the involvement of AID in the generation of these SVs. By analyzing these SV clusters, we identified several novel driver genes in PCNSL. For example, WGS and WES identified an enrichment of breakpoints of deletions (7/22) and loss-of-function mutations (6/37) in GRB2, strongly indicating its tumor suppressor role in PCNSL. We also analyzed pentanucleotide signatures of mutations in coding sequences detected by WES of PCNSL and DLBCL, taking into consideration the two adjacent bases 3' and 5' of the substitutions as well as transcription strand biases. Two predominant mutational signatures were identified in PCNSL: the AID signature characterized by C〉T mutations within the WRCY motif targeted by SHMs and the age-related signature involving C〉T transition at CpG dinucleotides. For DLBCL, an additional signature (signature 17 according to Alexandrov et al.) was detected as well, which had been reported in DLBCL with an unknown mechanistic basis. Conclusions Comprehensive genomic analyses of a large cohort of PCNSL and DLBCL cases have revealed the major targets of somatic mutations and SVs, including novel driver genes. In both PCNSL and systemic DLBCL, an enhanced AID activity is thought to be associated with generation of both SHMs and SVs, although the activity and targets of AID seem to substantially differ between both lymphoma types, suggesting distinct pathogenesis therein. Disclosures Kataoka: Boehringer Ingelheim: Honoraria; Yakult: Honoraria; Kyowa Hakko Kirin: Honoraria. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Description: Background Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of peripheral T-cell lymphoma (PTCL) characterized by generalized lymphadenopathy, hyperglobulinemia, and autoimmune-like manifestations. Frequent mutations in TET2, IDH2, and DNMT3A have been described in AITL, which are commonly found in myeloid malignancies. However, the molecular pathogenesis specific to AITL is still unknown. Methods To clarify the molecular pathogenesis of AITL, we performed comprehensive gene-mutation analysis. Somatic mutations in 3 AITL and 3 PTCL-NOS specimens were explored using whole-exome sequencing (WES). Targeted resequencing for genes identified by WES was also performed in a cohort of 157 patients with AITL/PTCL-NOS. Results We identified a novel recurrent mutation in RHOA (c.G50T/p.G17V) in 3 AITL and one PTCL-NOS samples by WES. Validation in an extended cohort revealed an extremely high frequency of the identical G17V RHOA mutation in AITL (50/72 [69.4%]), together with mutations in TET2 (39/47 [83.0%]), IDH2 (14/47 [29.8%]), and DNMT3A(12/47 [25.5%]). The G17V RHOA mutation was also found in PTCL-NOS samples at a lower frequency (14/85 [16.5%]), especially in those harboring AITL features (PTCL-NOS with AITL features vs PTCL-NOS w/o AITL features: 13/21 [61.9%] vs 0/38 [0%]). Remarkably, mutations in RHOA, TET2, and IDH2 showed striking correlations. All RHOA-mutated samples were accompanied by TET2 mutations. IDH2 mutations were confined to the samples having simultaneous mutations of RHOA and TET2. Mutations in DNMT3A largely overlapped to TET2 mutations, but its correlation with RHOA or IDH2 mutations was much less clear. TET2 mutations showed a consistently higher allelic burden than RHOA mutations. Gene-mutation analysis of tumor cells and infiltrated cells demonstrated that the G17V RHOA mutation specifically existed in tumor cells, but not in non-tumor cells, while TET2 mutations were identified both in tumor and non-tumor cells. RHOA encodes a small GTPase, which operates as a molecular switch that regulates a wide variety of biological processes through cycling between an active (GTP-bound) and an inactive (GDP-bound) state. We demonstrated that the G17V RHOA mutant did not bind GTP and also inhibited GTP-binding of the wild-type RHOA protein. Accordingly, unlike wild-type RHOA, the G17V mutant was not able to activate transcription from the serum response factor-responsive element (SRF-RE). Conclusions Our data suggests that combination of preceding mutations in TET2 and subsequent tumor-specific G17V RHOA mutation determines distinct disease properties of AITL. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin lymphoma, of which approximately 95% are diffuse large B-cell lymphomas (DLBCLs). Despite the substantial development of intensive chemotherapy during the past two decades, overall clinical outcome of PCNSL has been poorly improved especially in elderly and so has been our knowledge about the molecular pathogenesis of PCNSL, in terms of driver alterations that are relevant to the development of PCNSL. Method To delineate the genetic basis of PCNSL pathogenesis, we performed a comprehensive genetic study. We first analyzed paired tumor/normal DNA from 35 PCNSL cases by whole-exome sequencing (WES). Significantly mutated genes identified by WES and previously known mutational targets in PCNSL and systemic DLBCL were further screened for mutations using SureSelect-based targeted deep sequencing (Agilent) in an extended cohort of PCNSL cases (N = 90). Copy number alterations (CNAs) have been also investigated using SNP array-karyotyping (N =54). We also analyzed WES and SNP array data of systemic DLBCL cases (N = 49) generated by the Cancer Genome Atlas Network (TCGA) to unravel the genetic difference between PCNSL and systemic DLBCL. Results The mean number of nonsynonymous mutations identified by WES was 183 per sample, which was comparable to the figure in systemic DLBCL and characterized by frequent somatic hypermutations (SHMs) involving non-Ig genes. A higher representation of C〉T transition involving CpG dinucleotides and hotspot mutations within the WRCY motif targeted by SHM further suggested the involvement of activation-induced cytidine deaminase (AID) in the pathogenesis of PCNSL. We found 12 genes significantly mutated in PCNSL (q 〈 0.1), including MYD88, PIM1, HLA-A, TMEM30A, B2M, PRDM1, UBE2A, HIST1H1C, as well as several previously unreported mutational targets in systemic DLBCL or PCNSL, such as SETD1B, GRB2, ITPKB, EIF4A2. Copy number analysis identified recurrent genomic segments affected by focal deletions (N = 27) and amplifications (N = 10), most of which included driver genes targeted by recurrent somatic mutations or known targets of focal CNAs such as CDKN2A and FHIT. Subsequent targeted sequencing finally identified a total of 107 significantly mutated genes, of which 43 were thought to be targeted by SHM according to their mutational signature and genomic distribution. Most cases with PCNSL (98%) had mutations and CNAs involving genes that are relevant to constitutive NF-KB/Toll-like receptor (TLR)/BCR activity, including those in MYD88 (80%), CD79B/A (60%), CARD11 (18%), TNFAIP3 (26%), GRB2 (24%) and ITPKB (23%). Genetic alterations implicated in escape from immunosurveillance were also frequently identified in as many as 76% of cases. Mutations of HLA-B (64%), HLA-A (36%), HLA-C (28%), B2M (14%) and CD58 (12%) were commonly detected in addition to CNAs in 6p21.32 (HLA class II), 1p13.1 (CD58) and 15q15.2 (B2M), suggesting the importance of immune escape in the pathogenesis of PCNSL. SHMs were also seen in most cases (98%), which affected not only known targets of AID including PIM1, IGLL5 and BTG2 but also previously unreported genes involved in cell proliferation, apoptosis, or B cell development. The pattern of frequently mutated genes in PNCSL was more uniform compared with that in systemic DLBCL, and similar to that found in the activated B cell subtype of DLBCL (ABC-DLBCL), which was in accordance with the previous report of immunophenotypic analysis of PCNSL. On the other hand, mutations of HLA class I genes (HLA-B, HLA-A) were more frequently mutated in PCNSL compared with ABC-type DLBCL. Conclusion WES, SNP array karyotyping and follow-up targeted sequencing of a large cohort of PCNSL cases revealed the genetic landscape of PCNSL, which were more homogeneous than that of systemic DLBCL, and thought to be involved in activation of constitutive NF-KB/TLR/BCR signaling, escape from immunosurveillance, as well as highly frequent SHMs. Disclosures No relevant conflicts of interest to declare.