The specific niche adaptations that facilitate primary disease and Acute Lymphoblastic Leukaemia (ALL) survival after induction chemotherapy remain unclear. Here, we show that Bone Marrow (BM) adipocytes dynamically evolve during ALL pathogenesis and therapy, transitioning from cellular depletion in the primary leukaemia niche to a fully reconstituted state upon remission induction. Functionally, adipocyte niches elicit a fate switch in ALL cells towards slow-proliferation and cellular quiescence, highlighting the critical contribution of the adipocyte dynamic to disease establishment and chemotherapy resistance. Mechanistically, adipocyte niche interaction targets posttranscriptional networks and suppresses protein biosynthesis in ALL cells. Treatment with general control nonderepressible 2 inhibitor (GCN2ib) alleviates adipocyte-mediated translational repression and rescues ALL cell quiescence thereby significantly reducing the cytoprotective effect of adipocytes against chemotherapy and other extrinsic stressors. These data establish how adipocyte driven restrictions of the ALL proteome benefit ALL tumours, preventing their elimination, and suggest ways to manipulate adipocyte-mediated ALL resistance.

NF-κB and MYC are found co-deregulated in human B and plasma-cell cancers. In physiology, NF-κB is necessary for terminal B-to-plasma cell differentiation, whereas MYC repression is required. It is thus unclear if NF-κB/MYC co-deregulation is developmentally compatible in carcinogenesis and/or impacts cancer cell differentiation state, possibly uncovering unique sensitivities. Using a mouse system to trace cell lineage and oncogene activation we found that NF-κB/MYC co-deregulation originated cancers with a plasmablast-like phenotype, alike human plasmablastic-lymphoma and was linked to t(8;14)[MYC-IGH] multiple myeloma. Notably, in contrast to NF-κB or MYC activation alone, co-deregulation rendered cells addicted to IL6 for survival and phenotypic stability. We propose that conflicting oncogene-driven differentiation pressures can be accommodated at a cost in poorly-differentiated cancers.

Introduction: Although diffuse large B cell lymphoma (DLBCL) can be cured using immuno-chemotherapy, 40% of patients experience relapse or refractory disease. Large-scale profiling studies have mainly focused on DLBCL at diagnosis with a limited number of longitudinal studies and no compelling biomarkers linked to relapse identified. To address this, we utilized a multifaceted approach integrating transcriptomic and intratumoral T-cell repertoire analyses in paired diagnostic/relapse tumors to enable identification of signaling pathways and microenvironmental changes underlying disease relapse. Methods: We retrospectively collected archival paired diagnostic/relapse tumor biopsies from 38 de novo DLBCL patients (stage I-IV, 38-89 years old) treated with rituximab-based immuno-chemotherapy. We performed gene expression profiling (GEP) and T-cell repertoire analysis using the Ion AmpliSeq Transcriptome Kit and TCR-β sequencing (immunoSEQ), respectively. Cell-of-origin (COO) classification was performed by the Lymph2Cx assay on NanoString to distinguish activated B-cell-like (ABC) and germinal center B-cell-like (GCB) subtypes. Results: COO remained stable from diagnosis to relapse in >90% of pairs. In examples where we observed a switch in COO between diagnosis/relapse, targeted-seq analysis revealed some shared mutations suggesting that relapse tumors originated from a common ancestral clone. Our global GEP of 17 ABC-ABC and 11 GCB-GCB pairs identified 163 and 136 genes that were differentially expressed in ABC and GCB relapse tumors relative to their matched diagnostic biopsies respectively, with minimal overlap. Gene set enrichment analysis showed that ABC and GCB relapses are potentially mediated via different mechanisms, with tumor growth and proliferation signatures enriched in ABC relapse, compared with adaptive immunity-related signatures accompanying GCB progression. In parallel, we assessed the dynamics of the T-cell repertoire in paired biopsies observing a reduction in T-cell fraction upon relapse that was most pronounced in ABC pairs and was positively correlated with changes in CD8+ T cells. Furthermore, we noted a decrease in T-cell clonal diversity that was independent of COO at relapse with evidence for significant T-cell specific clonal expansion. Conclusions: The nature of the biological mechanisms responsible for DLBCL relapse has remained fairly elusive that may be inherent to the diagnostic tumor or acquired/enriched at disease relapse. Gene expression profiling of a series of DLBCL tumor pairs, resolved changes in gene expression that support distinct mechanisms of lymphoma relapse, based on a patient's COO, that parallel changes in the overall T-cell composition of the tumor microenvironment. Keywords: diffuse large B-cell lymphoma (DLBCL); gene expression profile (GEP); T-cells. Disclosures: Korfi, K: Consultant Advisory Role: Roche. Rule, S: Consultant Advisory Role: Celgene, Sunesis, Astrazeneca, Napp, Pharmacyclics, TG Therapeutics, Kite, Gilead, Roche, Janssen; Honoraria: Celgene, Sunesis, Astrazeneca, Pharmacyclics, TG Therapeutics, Gilead, Roche, Janssen; Research Funding: Roche, Janssen; Other Remuneration: Roche, Janssen. Davies, A: Consultant Advisory Role: Roche, Kite, Celgene, Acerta Pharma, MorphoSys, BioInvent; Honoraria: Roche, Celgene, Kite, Janssen; Research Funding: Roche, Acerta Pharma, Celgene, Gilead, Karyopharm, GSK; Other Remuneration: Roche, Celgene. Gribben, J: Honoraria: Janssen, Acerta, Celgene; Research Funding: Janssen, Acerta, Celgene. Johnson, P: Consultant Advisory Role: Janssen; Honoraria: Bristol-Myers Squibb, Takeda, Novartis, Celgene, Janssen, Epizyme, Boeringher Ingelheim, Kite, Genmab, Incyte; Research Funding: Janssen, Epizyme. Fitzgibbon, J: Consultant Advisory Role: Epizyme; Honoraria: Gilead; Research Funding: Epizyme.

Background:With an expanding array of novel treatment options in follicular lymphoma (FL), we largely lack effective biomarkers to predict response in this molecularly heterogeneous disease. Activating mutations focused on components of the nutrient‐sensing arm of the mTORC1 pathway (RRAGC, ATP6V1B2, ATP6AP1), are particularly enriched in FL, occurring in ∼30% of cases. Meanwhile, clinical mTOR inhibitors (mTORi) have shown some promise, with response rates of 50–60% in relapsed/refractory FL.Aims:We therefore sought to determine whether gene mutation status ‐ particularly in genes regulating the mTOR pathway – may represent biomarkers of mTORi‐response in FL.Methods:We retrospectively analyzed pre‐treatment tumor samples from relapsed/refractory FL patients treated on two phase II clinical trials with single agent mTOR inhibitors, everolimus (NCT00436618) or temsirolimus (NCT00290472), with well characterized clinical outcomes. In total, 21 patients had available biopsies, consisting of 15 everolimus‐ and 6 temsirolimus‐treated cases.Tumor DNA extracted from formalin fixed paraffin embedded (FFPE) tissue was subjected to targeted sequencing using a 22 gene panel comprising genes recurrently mutated in FL, and PI3K/AKT/mTOR pathway genes mutated in germinal center lymphomas. Libraries were prepared using capture‐based target enrichment (Nonacus), and sequenced on the NextSeq550 (Illumina) with a mean coverage depth of x700. A stringent bioinformatic pipeline was adopted with variants called with VarScan2, and a 10% variant allele frequency (VAF) threshold was employed to filter out sequencing artefacts due to the FFPE material. 100% (15/15) of a selection of variants were validated by Sanger sequencing. Staining for pS6 was undertaken on tissue slides to examine downstream mTOR activation by immunohistochemistry.Results:Overall response rate (CR and PR) was 48% (10/21). Mutational analysis showed no significant association between clinical response and mTORC1‐pathway mutations (mut) taken individually or collectively; although, absolute numbers of mutations in these genes were low, including RRAGC (responder: 0/10 mut vs non‐responder: 1/11 mut), ATP6AP1 (responder: 1/10 vs non‐responder: 1/11), and ATP6V1B2 (responder: 1/10 vs non‐responder 3/11). Notably, no difference in pS6 staining was seen between responders and non‐responders.However, we found mutations in the epigenetic regulator CREBBP to be enriched in the responder group (100% (10/10) responders vs 54.5% (6/11) non‐responders, p = 0.04). This effect was more pronounced when considering only mutations within the catalytic histone acetyltransferase (HAT) domain of CREBBP, with HAT mutations seen in 100% (10/10) responders, and 27.3% (3/11) non‐responders (p = 0.001).Summary/Conclusion:Mutations in the mTOR pathway did not predict for response to mTOR inhibition in our study. The enrichment of CREBBP mutations in responders, and particularly those affecting the catalytic HAT domain, points to CREBBP as a potential biomarker of response which warrants validation in trials of drugs modulating the PI3K/AKT/mTOR axis. More broadly our data may lend support to a distinct clinical and biological behavior of CREBBP HAT domain‐mutant disease that also warrants further evaluation. Taken together, our findings highlight the potential opportunities afforded by undertaking correlative studies, and underlines the need to incorporate prospective biomarker discovery into clinical trials in order to help focus future therapeutic strategies more rationally.

The leukocyte adhesion cascade is important in chronic lymphocytic leukemia (CLL), as it controls migration of malignant cells into the pro-survival lymph node microenvironment. Circulating trisomy 12 CLL cells have increased expression of the integrins CD11a and CD49d, as well as CD38, but the tissue expression of these and other molecules, and the functional and clinical sequelae of these changes have not been described. Here, we demonstrate that circulating trisomy 12 CLL cells also have increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and the adhesion molecule CD323. Notably, there was reduced expression of CD11a, CD11b, and CD18 in trisomy 12 cases with NOTCH1 mutations compared with wild type. Trisomy 12 cells also exhibit upregulation of intracellular integrin signaling molecules CALDAG-GEFI, RAP1B, and Ras-related protein ligand, resulting in enhanced very late antigen-4 [VLA-4] directed adhesion and motility. CD38 expression in CLL has prognostic significance, but the increased CD38 expression in trisomy 12 CLL cells must be taken into account in this subgroup, and the threshold of CD38 positivity should be raised to 40% for this marker to retain its prognostic value. In conclusion, trisomy 12 CLL cells exhibit functional upregulation of integrin signaling, with β2-integrin expression being modulated by NOTCH1 mutation status.

S. Fogazzi, L. Arcaini, R. Zanotti, C. Bottelli, M. Gotti, M. Paulli, C. Sissa, A. Tucci, M. Ungari, A. Zamò, F. Facchetti, G. Rossi Ematologia, Spedali Civili di Brescia, Brescia, Italy, Ematologia, Fondazione IRCCS Policlinico S.Matteo, Università di Pavia, Pavia, Italy, Ematologia, Azienda Ospedaliera Universitaria Integrata, Verona, Italy, I Anatomia Patologica, Università di Brescia, Brescia, Italy

The role of TP53 mutation in transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (t-FL) was examined in a panel of 91 lymph node biopsies derived from 29 patients pre- and post-transformation. The entire TP53 coding sequence was screened and immunocytochemistry performed to determine expression of p53 and its key regulator MDM2. A total of 10 mutations were detected in eight patients (28%), although none were present at FL diagnosis. Mutations were not detected solely at the time of transformation; in three patients, mutated TP53 arose in at least one antecedent FL sample ( 6 months, 2.5 years and 4 years prior to transformation). Loss of heterozygosity at the TP53 locus occurred in 2/20 informative patients ( only in t-FL samples). p53 staining was positive in 82% (9/11) of available biopsies with a missense mutation, and negative in 71% (45/63) with wtTP53. MDM2 expression was significantly higher in t-FL samples ( mean 72% positive; 95% confidence interval ( 95% CI) 68-76%) than FL ( mean 58% positive; 95% CI 54-62%) (P < 0.001) but did not correlate with TP53 status. TP53 mutation has only a limited role in the transformation of FL, exerting a heterogeneous influence upon phenotypic change. In contrast, dysregulation of MDM2 is frequent and may provide a more rational therapeutic target.

Transformation of follicular lymphoma (FL) to a more aggressive clinical and histological phenotype, typically diffuse large B-cell lymphoma (DLBCL), occurs frequently. It is associated with a number of recurrent genomic insults, including the acquisition of TP53 mutations in a subset of patients (pts). The use of novel agents targeting p53 and mdm2 appears attractive given the resistance of transformed DLBCL to conventional therapies. Tailoring these therapies will require precise characterisation of mutation status and functional consequence in transformation. The frequency and temporal relationship of TP53 mutation gain to transformation was analysed in DNA from sequentially collated lymph node biopsies taken pre and post transformation (n=91) obtained from 29 pts. A median of 3 samples (range 2–5) was available from each pt (13 taken at FL presentation). Transformation was documented a median of 3.8 years (range 0.2 to 15.2) from diagnosis, and median follow up from diagnosis for all pts at the time of analysis was 6.7 years (range 2 to 19.1). The entire coding sequence of TP53 was screened by PCR, fluorescent-SSCP and sequencing. Loss of heterozygosity (LOH) was examined at 5 common polymorphic sites with in TP53. Immunocytochemistry for p53, mdm2 and p21 was performed on slides obtained from 77 available paraffin blocks. Ten mutations were detected in 8 pts (28%), of which 5 were missense. The remaining was accounted for by two nonsense mutations, a splice mutation, a branch site mutation and a single base insertion. All mutations were within the genomic region covered by primer sets exon 5–7 inclusive. Mutated TP53 was first documented only at the time of histologic transformation in 4 pts, in the remainder latency between documentation in FL sample and transformation was variable (0.5–6 years). For pts with mutations, time from documentation to death ranged from 1 month-12 years (median 37 months), with 2 pts alive 8.5 and 13.5 years following initial documentation. LOH occurred in 2 pts, both at the time of transformation and was associated with short survival (1 and 17 months). Overall survival from diagnosis or histological transformation was not significantly different between pts with mutated TP53 and wtTP53. Five TP53 mutated pts. recurred post transformation (either with FL or DLBCL); in 4 pts the identical mutation was detected at this time. p53 staining was positive in 82% (9/11) of biopsies with missense mutations, and negative in 71% (45/63) with wtTP53. Mdm2 expression was predominantly centroblastic in FL and was correspondingly higher in DLBCL samples (mean 72%; 95% CI 68–76%) compared to FL (mean 58%; 95% confidence interval: 54–62%) (p<0.001). Mdm2 expression did not correlate with TP53 mutation status. Expression of p21 antigen was positive in 19/71 (27%) cases and did not correlate with histology. Absence of p21 occurred in both wtTP53 (66%) and mutated TP53 (94%) samples. TP53 mutations were associated with transformation in only a subset of pts; the potential of individual mutations to induce phenotypic change was variable and thus may influence the potential success of novel TP53 directed therapies.