Identification of genetic subtypes in follicular lymphoma.

Follicular lymphoma (FL) exhibits considerable variability in biological features and clinical trajectories across patients. To dissect the diversity of FL, we utilized a Bernoulli mixture model to identify genetic subtypes in 713 pre-treatment tumor tissue samples. Our analysis revealed the existence of five subtypes with unique genetic profiles that correlated with clinicopathological characteristics. The clusters were enriched in specific mutations as follows: CS (CREBBP and STAT6), TT (TNFAIP3 and TP53), GM (GNA13 and MEF2B), Q (quiescent, for low mutation burden), and AR (mutations of mTOR pathway-related genes). The subtype Q was enriched for patients with stage I disease and associated with a lower proliferative history than the other subtypes. The AR subtype was unique in its enrichment for IgM-expressing FL cases and was associated with advanced-stage and more than 4 nodal sites. The existence of subtypes was validated in an independent cohort of 418 samples from the GALLIUM trial. Notably, patients assigned to the TT subtype consistently experienced inferior progression-free survival when treated with immunochemotherapy. Our findings offer insight into core pathways distinctly linked with each FL cluster and are expected to be informative in the era of targeted therapies.

Resolution of Melanoma to Programmed Death-1 Blockade but Simultaneous Rapid Progression of Concomitant Chronic Lymphocytic Leukemia.

Here, we present a novel case of a patient with chronic lymphocytic leukemia (CLL) who received CTLA-4 and then PD-1 immune-checkpoint blockade (ICB) as treatment for concomitant metastatic melanoma. Whereas the metastatic melanoma was responsive to ICB, the CLL rapidly progressed (but responded to ICB cessation and ibrutinib). There were no new genetic mutational drivers to explain the altered clinical course. PD-1/PD-L1/PD-L2 and CTLA-4/CD80/CD86 expression was not increased in CLL B cells, CD8+ or CD4+ T-cell subsets, or monocytes. The patient's CLL B cells demonstrated strikingly prolonged in vitro survival during PD-1 blockade, which was not observed in samples taken before or after ICB, or with other patients. To our knowledge, a discordant clinical course to ICB coupled with these biological features has not been reported in a patient with dual malignancies.

Immune evasion via PD-1/PD-L1 on NK cells and monocyte/macrophages is more prominent in Hodgkin lymphoma than DLBCL.

Much focus has been on the interaction of programmed cell death ligand 1 (PD-L1) on malignant B cells with programmed cell death 1 (PD-1) on effector T cells in inhibiting antilymphoma immunity. We sought to establish the contribution of natural killer (NK) cells and inhibitory CD163+ monocytes/macrophages in Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL). Levels of PD-1 on NK cells were elevated in cHL relative to DLBCL. Notably, CD3-CD56hiCD16-ve NK cells had substantially higher PD-1 expression relative to CD3-CD56dimCD16+ cells and were expanded in blood and tissue, more marked in patients with cHL than patients with DLBCL. There was also a raised population of PD-L1-expressing CD163+ monocytes that was more marked in patients with cHL compared with patients with DLBCL. The phenotype of NK cells and monocytes reverted back to normal once therapy (ABVD [doxorubicin 25 mg/m2, bleomycin 10 000 IU/m2, vinblastine 6 mg/m2, dacarbazine 375 mg/m2, all given days 1 and 15, repeated every 28 days] or R-CHOP [rituximab 375 mg/m2, cyclophosphamide 750 mg/m2 IV, doxorubicin 50 mg/m2 IV, vincristine 1.4 mg/m2 (2 mg maximum) IV, prednisone 100 mg/day by mouth days 1-5, pegfilgrastim 6 mg subcutaneously day 4, on a 14-day cycle]) had commenced. Tumor-associated macrophages (TAMs) expressed high levels of PD-L1/PD-L2 within diseased lymph nodes. Consistent with this, CD163/PD-L1/PD-L2 gene expression was also elevated in cHL relative to DLBCL tissues. An in vitro functional model of TAM-like monocytes suppressed activation of PD-1hi NK cells, which was reversed by PD-1 blockade. In line with these findings, depletion of circulating monocytes from the blood of pretherapy patients with cHL and patients with DLBCL enhanced CD3-CD56hiCD16-ve NK-cell activation. We describe a hitherto unrecognized immune evasion strategy mediated via skewing toward an exhausted PD-1-enriched CD3-CD56hiCD16-ve NK-cell phenotype. In addition to direct inhibition of NK cells by the malignant B cell, suppression of NK cells can occur indirectly by PD-L1/PD-L2-expressing TAMs. The mechanism is more prominent in cHL than DLBCL, which may contribute to the clinical sensitivity of cHL to PD-1 blockade.

The T-cell Receptor Repertoire Influences the Tumor Microenvironment and Is Associated with Survival in Aggressive B-cell Lymphoma.

Purpose: To investigate the relationship between the intra-tumoral T-cell receptor (TCR) repertoire and the tumor microenvironment (TME) in de novo diffuse large B-cell lymphoma (DLBCL) and the impact of TCR on survival.Experimental Design: We performed high-throughput unbiased TCRß sequencing on a population-based cohort of 92 patients with DLBCL treated with conventional (i.e., non-checkpoint blockade) frontline "R-CHOP" therapy. Key immune checkpoint genes within the TME were digitally quantified by nanoString. The primary endpoints were 4-year overall survival (OS) and progression-free survival (PFS).Results: The TCR repertoire within DLBCL nodes was abnormally narrow relative to non-diseased nodal tissues (P < 0.0001). In DLBCL, a highly dominant single T-cell clone was associated with inferior 4-year OS rate of 60.0% [95% confidence interval (CI), 31.7%-79.6%], compared with 79.8% in patients with a low dominant clone (95% CI, 66.7%-88.5%; P = 0.005). A highly dominant clone also predicted inferior 4-year PFS rate of 46.6% (95% CI, 22.5%-76.6%) versus 72.6% (95% CI, 58.8%-82.4%, P = 0.008) for a low dominant clone. In keeping, clonal expansions were most pronounced in the EBV+ DLBCL subtype that is known to express immunogenic viral antigens and is associated with particularly poor outcome. Increased T-cell diversity was associated with significantly elevated PD-1, PD-L1, and PD-L2 immune checkpoint molecules.Conclusions: Put together, these findings suggest that the TCR repertoire is a key determinant of the TME. Highly dominant T-cell clonal expansions within the TME are associated with poor outcome in DLBCL treated with conventional frontline therapy. Clin Cancer Res; 23(7); 1820-8. ©2016 AACR.

Ratios of T-cell immune effectors and checkpoint molecules as prognostic biomarkers in diffuse large B-cell lymphoma: a population-based study.

BACKGROUND: Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured, despite initial treatment with R-CHOP. The prognostic importance of the revised International Prognostic Index (R-IPI) and cell of origin of the malignant B cell are established in DLBCL. We aimed to develop a novel, easily applicable, tissue-based prognostic biomarker based on quantification of the tumour microenvironment that is independent of and additive to the R-IPI and cell of origin. METHODS: We performed digital hybridisation on the NanoString platform to assess the relation between immune effector and inhibitory (checkpoint) genes in 252 formalin-fixed, paraffin-embedded DLBCL tissue specimens obtained from patients treated with R-CHOP. We used a tree-based survival model to quantify net antitumoral immunity (using ratios of immune effector to checkpoint genes) and to generate a cutoff as an outcome predictor in 158 of the 252 patients. We validated this model in tissue (n=233) and blood (n=140) samples from two independent cohorts treated with R-CHOP. FINDINGS: T-cell and NK-cell immune effector molecule expression correlated with tumour-associated macrophage and PD-1/PD-L1 axis markers, consistent with malignant B cells triggering a dynamic checkpoint response to adapt to and evade immune surveillance. The ratio of CD4*CD8 to (CD163:CD68[M2])*PD-L1 was better able to stratify overall survival than was any one immune marker or combination, distinguishing groups with disparate 4-year overall survival. 94 (59%) of 158 patients had a score above the cutoff and 4-year overall survival of 92·1% (95% CI 82·9-96·7), and the remaining 64 (41%) patients had a score below the cutoff and 4-year overall survival of 47·0% (32·8-60·5; hazard ratio [HR] 8·3, 95% CI 4·3-17·3; p<0·0001). The CD4*CD8:M2*PD-L1 immune ratio was independent of and added to the R-IPI and cell of origin. Tissue findings in the independent tissue cohort accorded with those in our initial tissue cohort. 139 (60%) of 233 patients had a score above the cutoff and 4-year overall survival of 75·6% (95% CI 64·6-83·6), with the remaining 94 (40%) patients having a score below the cutoff (63·5% [52·5-72·7]; HR 1·9, 95% CI 1·1-3·3; p=0·0067). INTERPRETATION: Ratios of immune effectors to checkpoints augment the cell of origin and R-IPI in DLBCL and are applicable to paraffin-embedded biopsy specimens. These findings might have potential implications for selection of patients for checkpoint blockade within clinical trials. FUNDING: Leukaemia Foundation of Queensland, Kasey-Anne Oklobdzijato Memorial Fund, the Australasian Leukaemia and Lymphoma Group (Malcolm Broomhead Bequest), the Australian Cancer Research Foundation, and the Cancer Council of Queensland.