ALBERT

Description: Key Points Expansion of PD-1+ CD3−CD56hiCD16-ve NK cells and PD-L1+ monocytes/macrophages is more prominent in cHL than DLBCL. PD-1 blockade reverses the immune evasion mediated by the interaction of PD-1+ NK cells and PD-L1+ monocytes/macrophages.

Description: Background: We have recently demonstrated that an 'immune score' is strongly and independently prognostic in de novo DLBCL treated with R-CHOP immuno-chemotherapy. The score quantifies the relative composition of immune effectors (T cells) and checkpoints (e.g. PD-1 axis molecules and M2 macrophages), as a measure of net anti-tumoral immunity within the TME. It is also known that a diverse TCR repertoire is a hallmark of a robust anti-HIV T cell immune response; conversely in metastatic melanoma treated with anti-PD-1 checkpoint blockade, narrow more clonal TCR repertoires are associated with favorable response. The relationship between the intra-tumoral TCR repertoire and the TME in DLBCL following R-CHOP immuno-chemotherapy is unknown. Methods High-throughput unbiased TCR β chain sequencing was performed on 116 nodal tissues (101 de novo DLBCL patients treated with R-CHOP with long-term follow-up including 8 EBV+DLBCL; and 15 age/gender matched healthy lymph nodes). Outcomes included measurement of productive uniques (a measure of the number of functional T cells with a distinct TCR rearrangement or 'richness'); entropy (a measure of TCR 'diversity'), 'clonality' (a measure of clonal expansions) and the 'maximal frequency' of the most highly expressed clone within tumor biopsies. Results were compared to digital quantification (by nanoString) of key immune effector and checkpoint genes within the TME, the immune score, malignant cell-of-origin (COO), R-IPI and patient survival. Results: First we compared the TCR repertoire in lymphomatous and healthy nodes. There was a marked increase in clonality, reduced diversity and high maximal frequency within DLBCL nodes relative to healthy nodal tissue (both p

Description: LAG3 is an immune checkpoint expressed on a variety of immune cells including a sub-population of 'exhausted' effector T cells and TREGs. Early-phase studies of anti-LAG3 mAb show promise in solid and haematological cancers. We have previously demonstrated LAG3 is enriched within the tumor microenvironment in Hodgkin Lymphoma (Gandhi et al. Blood 2006). Data in the aggressive B-cell lymphoma DLBCL is lacking. We used a conventional discovery/ validation approach in two population based Australian cohorts (discovery: Brisbane/Canberra; validation: Sydney) totalling 250 patients treated with R-CHOP with 〉4 year follow-up. Digital gene expression (NanoString) using a consistent LAG3 cut-off showed inferior 5-year overall survival (OS) in both cohorts (discovery: 54% vs. 82%, HR 3.13, p=0.003; validation: 63% vs. 86%, HR 2.95, p=0.025 respectively). In a multivariate model, LAG3HI(p=0.001) was a predictor of OS independent of R-IPI and Cell-of-Origin (by NanoString LST assay). PD-L1 expression was also a predictor of survival though to a lesser degree than LAG3. Notably, LAG3 expression stratified PD-L1HIpatients into two sub-groups with differential survival, with dual LAG3 and PD-L1 positivity conferring particularly poor OS (PD-L1HI/LAG3HI39% vs. 81% PD-L1HI/LAG3LO, HR 3.65, p=0.023). Next, the discovery/validation cohorts were combined with 129 additional DLBCL cases from the ALLG biobank (in whom tissue but no outcome data was available), to test for biological associations and correlations. In these 379 cases, LAG3HIwas enriched in the ABC/UC (66%) subtype vs. LAG3LO(p=0.003). LAG3 was positively correlated with numerous immune checkpoints/effectors including CD4, CD8, PD-1, PD-L1, PD-L2, TIM-3 and CD163 (all p

Description: Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma with ~30% of cases failing treatment or relapsing after R-CHOP chemotherapy. Treatment options for refractory/ relapsed DLBCL are limited and often have a poor outcome. Tumour evolution and selection of a chemo resistant clone are likely explanations making it important to understand clonal heterogeneity at diagnosis and relapse. Green et al have noted a hierarchical acquisition of mutations with disease progression in follicular lymphoma (FL), with BCL2 and CREBBP mutations seen in earlier precursors and MLL2 and TNFRSF14 mutations proposed to be later events. Notably, 1 case analysed at diagnosis and at relapse had variable IgVH indicating origin in a more immature precursors. [Green, M.R., Blood, 2013]. More recent studies indicate that the dominant clone of FL and transformed FL arise by either convergent or divergent evolution from a common mutated precursor through the acquisition of additional genetic events [Pasqualucci, L., Cell Rep, 2014]. Defining the deregulated pathways and oncogenic mutations at diagnosis and later at relapse could help identify novel therapeutic targets for treatment at relapse. We aimed to map clonal evolution at relapse using immunophenotyping, sequencing for the Immunoglobulin variable heavy chain gene (IGHV) and genotyping for known somatic mutations in key lymphoma genes, within matched diagnostic and relapsed DLBCL pairs. Methods: Six matched diagnostic and relapsed DLBCL cryopreserved samples were subjected to comprehensive immunophenotyping using 10-colour B-cell flow cytometry panels based on normal ontogeny. PCR on initial and relapsed samples was performed to amplify clonally rearranged IgHV genes from fresh/paraffin embedded primary diagnostic samples. Sanger sequencing was done for IgHV mutation status by targeting the conserved framework regions (FR) FR1 [IGHA: VHFR1-JH] or FR3 [IGHC: VHFR3-JH] using the Invivoscribe kit based on the Biomed-2 protocols. A custom Haloplex library preparation kit designed and ordered from Agilent was used to capture the exons of ~40 genes commonly mutated in DLBCL. Targeted fragments were PCR amplified and target enriched samples sequenced on an Illumina MiSeq sequencer Results: Table 1. Immunophenotypic diversity was noted among cases but the phenotype did not change at relapse. IGHV sequencing showed that 3 cases with two distinct lymphoma clones at diagnosis evolved to a single detectable IGHV clonal population at relapse. Lymphoma-associated somatic mutations detected at diagnosis were preserved at relapse and in 4 cases, acquisition of additional mutations was observed. Table 1: Immunophenotype, IgHV sequence and somatic lymphoma mutations in paired DLBCL samples at diagnosis and relapse Patient number Disease course Immunophenotype IgHV sequence Lymphoma mutations DL157 Diagnosis IgM+, IgD+++, IgG-, CD27-, CD21+++, CD38++, CD10- N/A (not available) EP300 I-〉V EZH2 D-〉H NOTCH2 L-〉H TRAF2 A-〉T DL157 Relapse Same as diagnosis IgHV3-64 IgHV3-30 EZH2 D-〉H FOXO1G-〉D NOTCH2L-〉HTRAF2 A-〉T DL214 Diagnosis IgM+++,IgD++, IgG-, CD27-, CD21-, CD38+++, CD10+ IgHV4-30 IgHV4-39 N/A DL214 Relapse Same as diagnosis IgHV4-39 EP300 I-〉V MYD88 L-〉P NOTCH1 R-〉W PRDM1 C-〉Y SPEN D-〉E TP53 Y-〉N DL215 Diagnosis IgM-,IgD- IgG++, CD27++,CD21++, CD38-, CD10- IGHV3-23 IGHV3-23D KMT2D R-〉C MYD88 L-〉P NOTCH1 E-〉K DL215 Relapse Same as diagnosis IGHV3-23 BTG1 H-〉Y BTG1 Q-〉Stop BTG2 S-〉N FOXO1 V-〉E GNA13 R-〉H KMT2D R-〉C MYD88 L-〉P NOTCH1 E-〉K PIM1 L-〉F PIM1 K-〉N PIM1 V-〉L DL245 Diagnosis N/A IGHV3-11 IGHV3-33 EP300,I-〉V,997 NOTCH2,L-〉V,1559 DL245 Relapse N/A IGHV3-11 EP300,I-〉V,997 NOTCH2,L-〉V,1559 DL213 Diagnosis IgM+,IgD-, IgG-, CD27-,CD21+++, CD38+++, CD10++ IGHV3-15 ETS1 A-〉T EZH2 D-〉H PRDM1 L-〉F TNFRSF14 C-〉S DL213 Relapse Same as diagnosis IGHV3-15 ETS1 A-〉T EZH2 D-〉H KMT2D R-〉Stop PRDM1 L-〉FSTAT3 R-〉W DL252 Diagnosis IgM-,IgD++, IgG-, CD27-,CD21-, CD38+, CD10- IGHV1-2 NOTCH2,P-〉A,2359 TNFRSF14,V-〉I,241 DL252 Relapse Same as diagnosis IGHV1-2 N/A Conclusions: While multiple IGHV sub-clones are present at diagnosis of DLBCL, evolution of a single clone is noted at relapse (convergent model) with acquisition of additional somatic mutations. Immunophenotyping using B-cell maturation flow cytometry panels demonstrates heterogeneity of differentiation between cases but no change in immunophenotype at relapse. Disclosures No relevant conflicts of interest to declare.

Description: There is increasing evidence for antigen-driven B-cell receptor (BCR) signalling in diffuse large B-cell lymphoma (DLBCL) and other lymphoid malignancies. This includes antigens from infections e.g.Helicobacter pyloriand Hepatitis C virus, but it is theorised that self-antigens may play a major role in some cases of lymphoid malignancy. IgVH4-34 demonstrates intrinsic autoreactivity to self-antigens on red cells, which appears to be largely mediated by two motifs within the first framework region (FR1); Q6W7 and A24V25Y26. These motifs work together to for a hydrophobic patch which determines red cell antigen binding and are frequently mutated away from self-reactivity in normal B cells. IgVH4-34 has been reported to be over-represented in DLBCL compared with expression in normal B cells. We therefore sought to identify IgVH4-34 DLBCL cases from a local cohort and to screen them for Q6W7 and A24V25Y26 motifs expecting them to be less frequently mutated in DLBCL compared with normal B cells.We also aimed to screen V4-34 cases for associated somatic mutations in other genes using high-throughput sequencing. DLBCL patient samples were obtained via the Haematology Research Tissue Bank (HRTB) in Canberra, Australia, and the Victoria Cancer BioBank. Forty-eight Formalin-Fixed, Paraffin-Embedded (FFPE) samples and 26 fresh frozen samples were screened. All samples were collected at the time of diagnosis. Patients were treated with standard chemoimmunotherapy approaches. IgVH 4-34 gene sequences were determined using an IgVH4 family-specific leader primer in combination with a JH consensus reverse primer. The IgVH region was then sequenced using Sanger sequencing. Sequences were analyzed using the IgBLAST database (National Centre for Biotechnology Information). DNA extracted from FFPE samples generally proved to have low concentration and fragmented DNA. Only 1 IgVH4-34 sequence was obtained from FFPE tissue. Five samples sequenced from fresh tissue were identified as using IgVH4-34. Using Hans criteria, it was possible to classify 3 of the 6 cases as germinal center (GC) and 1 as non-GC origin. Using fresh samples, we estimated the frequency of IgVH4-34 cases at 23%. Within FR1, Q6W7 was unmutated in all 6 samples. One sample had mutations in the A24V25Y26 motif resulting in a change to A24V25F26. The other 5 samples (83.3%) had unmutated AVY motifs. We extracted genomic DNA from and performed next generation sequencing on the 5 samples with unmutated Q6W7 and A24V25Y26 motifs using a customized capture library (SureSelectXT Target Enrichment System, Aqilent Technologies) covering genes involved in lymphomagenesis. The purified libraries were sequenced on the Illumina NextSeq500 platform at AGRF (Australian Genome Research Facility, Australia). Several genes (FCGR3A,NOTCH2andNOTCH2NLR) had mutations in all 5 samples.FCGR3Ais an IgG Fc receptor gene, and mutations inFCGR3Ahave previously been linked to systemic lupus erythematosus (SLE).NOTCH2pathway genes are frequently mutated in DLBCL.CREBBPwas mutated in four of the five samples. Mutations inCREBBPhave previously been linked with DLBCL development and regulation of immune responses. We identified high rates of IgVH4-34 (23%) in our cohort of fresh samples as previously reported. Further, we noted preservation of the Q6W7 and A24V25Y26 motifs in IgVH4-34-expressing DLBCL. This over-representation of unmutated FR1 motifs suggests that the ability to recognise self-antigens likely provides important ongoing BCR signalling that promotes survival in DLBCL. This study also highlights the difficulties in conducting DNA-based research on FFPE clinical samples which have not been collected for research purposes and the importance of tissue banking fresh samples. Studies are currently being conducted into the efficacy of BCR pathway inhibitors e.g. ibrutinib in the treatment of DLBCL and testing for unmutated IgVH4-34 FR1 motifs may present a method to predict patients who are more likely to respond. Mutations in genes such as FCGR3A,NOTCH2andCREBBPmay work in conjunction with the preserved QW and AVY motifs to promote lymphomagenesis in IgVH4-34-expressing B cells and may present targets for future research into treatment therapies. Figure Disclosures Talaulikar: Roche:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Janssen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Amgen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Takeda:Research Funding.

Description: Background: Approximately 40% of patients with diffuse large B-cell lymphoma (DLBCL) are 〉70 years old and have poor clinical outcomes attributable to treatment-related factors or to disease biology. Genotyping data in elderly DLBCL patients is limited because of lack of representation in clinical trials. R-mini-CHOP is better tolerated than R-CHOP in elderly DLBCL but with reduced efficacy. The addition of ibrutinib to R-CHOP has been shown to be effective in younger pts aged 〈 60 years but is associated with poor tolerability and outcomes in older patients. The ALLG Ibrutinib with R-mini-CHOP (IRiC) study, a prospective multicentre single-arm phase II study in elderly (≥ 75 years) DLBCL patients provided an opportunity to genotype this uncommon cohort. We therefore aimed to map the genetic landscape of mutations in elderly DLBCL and compare them to a non-trial younger cohort. Methods: Mutations in genes commonly associated with DLBCL were assessed in 55 IRIC study patients with a median age of 81 years (75-91 years) and a gender ratio of 1.0 (27 M/28 F). A cohort of 51 non-trial patients withde novoDLBCL treated with anthracycline-based regimens was also genotyped. The median age of the control group was 65 years (29-91 years) with a gender ratio of 1.2:1 (28 M/23 F). The cell of origin (COO) measured using gene expression profiling on 39/55 trial patients (non-GC=13, GC= 22, unclassified= 4), and Hans algorithm on 45/51 non-trial patients (non-GC=14, GC=31) was comparable (p=0.571). Outcome data was available at a median follow-up of 18 and 40 months for the trial and non-trial cohorts respectively. We extracted genomic DNA from and performed next generation sequencing on diagnostic formalin-fixed paraffin-embedded or fresh frozen tissue samples using a customized capture library (SureSelectXT Target Enrichment System, Aqilent Technologies) covering genes involved in lymphomagenesis. The purified libraries were sequenced on the Illumina NextSeq500 platform at AGRF (Australian Genome Research Facility, Australia). Mutations in the following genes were compared across the two cohorts: ARID1A, BCL2, BTG1, BTG2, CARD11, CCND3, DTX1, EP300, ETS1, EZH2, FOXO1, GNA13, HIST1H1C, IKBKB, IRF8, KDM2B, KLHL6, MYC, MYD88, NOTCH1, NOTCH2, PIK3CD, PIM1, PRDM1, PTEN, PTPN21, SGK1, SPEN, STAT3, TET2, TNFAIP3, TNFRSF11A, TNFRSF14, TP53 and TRAF5. Statistical analysis for nominal data was done using the chi-square test and for ordinal data using the Kruskal-Wallis test (p 〈 0.05= significant). Kaplan-Meier curves were calculated for patients with and without each mutation and the curves compared using a log-rank approach. Results: Patients were divided into 2 groups, IRiC trial cohort aged 〉75 years (n=55) + non-trial elderly patients 〉75 years (n=9) =elderly cohort(n=64) and non-trialnon-elderly cohortof patients 〈 75 years (n=42). As expected, elderly patients were more likely to have high-risk disease with higher IPI of ≥ 3 (n=42, n=17, p=0.009). There was no significant difference in gender or COO. The frequency of mutations in the elderly (n=64) was compared to the non-elderly cohort (n=42). NOTCH2 was the most common mutation irrespective of age (34 [53%]; 16 [38%], p=0.129). Notably, we found that mutations in MYC (8 [12.5%]; 0, p= 0.021), PTEN (17 [26.5%]; 4 [9%], p=0.045) and TET2 (28 [43.7%]); 7 [16.6%], p=0.004) were more frequent in the elderly. As expected, MYD88 and CD79B mutations were more frequently associated with non-GC subtype (p=0.001). No other associations with COO were identified. No clear prognostic individual genes or gene clusters could be identified in the trial or the elderly cohort. Ibrutinib-responsive (MYD88 [L265P n=7] and CD79B [n=11]) and ibrutinib-resistant mutations (CARD11 [n=6] or PIM1 [n=12]) did not show clear associations with response rate, overall survival or progression-free survival. Conclusions: Our study found that the mutational profile of elderly DLBCL patients aged ≥ 75 years is enriched for targetable mutations in MYC, PTEN and TET2 compared to those 〈 75 years. PTEN and TET2 are tumour suppressors and MYC is an oncogene with an important regulatory role in cell growth and proliferation. We hypothesize that hypo-methylating agents targeting TET2, BET inhibitors in MYC mutated tumours and PI3K inhibitors to target PTEN deficient lymphoma may help improve clinical outcomes in elderly DLBCL. No clear prognostic markers were identified in this small cohort. Disclosures Trotman: Celgene:Research Funding;BeiGene:Research Funding;Takeda:Research Funding;PCYC:Research Funding;F. Hoffmann-La Roche:Research Funding.Verner:Janssen Cilag Pty Ltd.:Research Funding.Gandhi:Celgene:Research Funding;Bristol-Myers Squibb:Research Funding;Mater Research:Current Employment;Janssen-Cilag:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Roche:Other: Travel, accommodation, expenses ;Genentech:Honoraria;Gilead Sciences:Honoraria;Amgen:Honoraria;Merck Sharp & Dohme:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.Gandhi:Integrated Sciences:Current Employment.Talaulikar:Takeda:Research Funding;Amgen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Janssen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Roche:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.