Supplementary Figure Legends - PDF files 57K, Figure Legends

PDF file - 36K, Sensitivity and specificity of sCD163 and sTARC as biomarkers in cHL. (A-D) Receiver Operating Characteristic (ROC) plots demonstrate high sensitivity and specificity of (A) sCD163 pre-therapy cHL versus healthy participant; (B) sCD163 pre-therapy cHL versus CR six months post-therapy; (C) sTARC pre-therapy cHL versus healthy participants; (D) sTARC pre-therapy cHL versus CR six months post-therapy. AUC, area under the curve.

Supplementary Table 1 - PDF files 69K, Patient Data

Supplementary Figure 2 - PDF files 805K, Correlation of CD62L antibody responses and clinical parameters

Supplementary Table 2 - PDF files 77K, CD markers used in phenotype screen

Supplementary Figure 1 - PDF files 2472K, Expression of cell surface markers throughout CLL PBMC

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.

iLLUMINATE is a randomized, open-label phase III study of ibrutinib plus obinutuzumab (n=113) versus chlorambucil plus obinutuzumab (n=116) as first-line therapy for patients with chronic lymphocytic leukemia or small lymphocytic lymphoma. Eligible patients were aged ≥65 years, or <65 years with coexisting conditions. Patients received oral ibrutinib 420 mg once daily until disease progression or unacceptable toxicity or six cycles of oral chlorambucil, each in combination with six cycles of intravenous obinutuzumab. After a median follow-up of 45 months (range, 0.2-52), median progression-free survival continued to be significantly longer in the ibrutinib plus obinutuzumab arm than in the chlorambucil plus obinutuzumab arm (median not reached versus 22 months; hazard ratio=0.25; 95% confidence interval: 0.16-0.39; P<0.0001). The best overall rate of undetectable minimal residual disease (<0.01% by flow cytometry) remained higher with ibrutinib plus obinutuzumab (38%) than with chlorambucil plus obinutuzumab (25%). With a median treatment duration of 42 months, 13 months longer than the primary analysis, no new safety signals were identified for ibrutinib. As is typical for ibrutinib-based regimens, common grade ≥3 adverse events were most prevalent in the first 6 months of ibrutinib plus obinutuzumab treatment and generally decreased over time, except for hypertension. In this final analysis with up to 52 months of follow-up (median 45 months), ibrutinib plus obinutuzumab showed sustained clinical benefit, in terms of progression- free survival, in first-line treatment of chronic lymphocytic leukemia, including in patients with high-risk features. ClinicalTrials.gov identifier: NCT02264574.

Targeted antibody therapies improve outcomes for chronic lymphocytic leukemia (CLL) patients. However, resistance often develops. We have previously shown that resistance to therapeutic antibodies, by monocyte derived macrophages (referred to as nurse like cells, NLCs), from CLL patients is characterized by suppression of antibody dependent phagocytosis (ADP). The mechanism(s) contributing to the muted ADP responses remain unresolved. In this regard, an innate immune checkpoint was recently described that uses the CD47:SIRPα axis to suppress phagocytic responses by macrophages. In this study we examine whether the SIRPα axis regulates ADP responses to the anti-CD20 antibody, obinutuzumab, by NLCs. Using siRNA depletion strategies we show that SIRPα is a suppressor of ADP responses. Moreover, we show that this innate immune checkpoint contributes to the resistance phenotype in NLCs derived from CLL patients. Finally, we show that SIRPα suppression is mediated via the phosphatase, Shp1, which in turn suppresses SYK-dependent activation of ADP. Thus, we identify a druggable pathway that could be exploited to enhance sensitivity to existing therapeutic antibodies used in CLL. This is the first study to show that activation of the CD47:SIRPα innate immune checkpoint contributes to ADP resistance in NLCs from CLL patients.

Anti-CD20 antibody treatments, such as obinutuzumab, have been associated with infusion-related reactions (IRRs). In the phase 3 iLLUMINATE study of ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab in first-line chronic lymphocytic leukemia/small lymphocytic lymphoma, IRRs were substantially reduced with ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab. We prospectively analyzed inflammatory cytokines to evaluate the impact of ibrutinib on circulating cytokine levels following obinutuzumab infusion. In iLLUMINATE, ibrutinib or chlorambucil was given approximately 30–120 min before the first obinutuzumab infusion. Cytokines evaluated were IFNγ, IL-6, IL-8, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, and TNFα. Changes in peak cytokine levels from baseline (immediately before obinutuzumab) to post-obinutuzumab infusion were compared between arms and between patients with versus without IRRs using Wilcoxon rank sum test. Of 228 treated patients, 95 on ibrutinib-obinutuzumab (15 with IRRs, 80 without) and 88 on chlorambucil-obinutuzumab (45 with IRRs, 43 without) with cytokine data were included. Irrespective of IRR occurrence, median increase in cytokines was lower with ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab for all cytokines ( P < 0.01) except MIP-1β. Across treatment arms, post-obinutuzumab median increase in all cytokines except MIP-1β was greater in patients with versus without IRRs ( P < 0.001). IL-6 and IL-8 elevations were associated with IRRs in both treatment arms. Among patients with IRRs, those receiving ibrutinib-obinutuzumab had lower post-obinutuzumab increases in IL-6, IL-8, IL-10, and MCP-1 ( P < 0.04) than patients receiving chlorambucil-obinutuzumab. For patients in the ibrutinib-treatment arm, we observed a reduction in both the rate of clinically apparent IRRs and the levels of IRR-related cytokines and chemokines. This observation supports an immunomodulatory mechanism of action for ibrutinib. Clinical Trial Registration: NCT02264574

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Resistance, to therapeutic antibodies used to treat chronic lymphocytic leukemia (CLL) patients is common. Monocyte-derived macrophages (MDMs) are a major effector of antitumour responses to therapeutic antibodies and we have previously reported that resistance to therapeutic antibodies, by MDMs, increases as CLL disease progresses. In this study, we examine the effect of a Class IIa-selective HDAC inhibitor (TMP195) on the phagocytic response to opsonised tumor cells or non-opsonised targets by MDMs derived from CLL patients. We report that TMP195 enhances phagocytic responses to antibody-opsonised CLL cells and E. coli within 30 min of treatment. The enhanced response is phenocopied by knockdown of the Class IIa HDAC, HDAC7, or by low concentrations of the pan-HDAC inhibitor, vorinostat. HDAC7 knockdown and inhibition induces hyperacetylation and hyperphosphorylation of Bruton’s tyrosine kinase (BTK). Moreover, BTK inhibitors abrogated the enhanced response to HDAC7 inhibition. Our data show that HDAC7 is an actionable driver of resistance to therapeutic antibodies by MDMs derived from CLL patients.

n amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antibody therapies are important treatment options for Chronic lymphocytic leukemia (CLL). Monocyte-derived-macrophages (MDMs) are thought to be a major immune effector that clears leukaemic cells ...

Introduction: Infusion-related reactions (IRRs) are potentially serious complications resulting from release of inflammatory cytokines in response to administration of certain drugs, including obinutuzumab (G), an anti-CD20 antibody (Freeman, Blood2015). In the phase 3 iLLUMINATE study of first-line ibrutinib-G (ibr-G) vs chlorambucil-G (clb-G) in patients (pts) with chronic lymphocytic leukemia (CLL), IRRs were decreased with ibr-G vs clb-G (any grade: 25% vs 58%; grade ≥3 or serious: 3% vs 9%) (Moreno, Lancet Oncol 2019). As IRR results from the release of inflammatory cytokines, we prospectively analyzed cytokines thought to be associated with IRRs in pts from iLLUMINATE to evaluate the impact of ibr on secretion of cytokines following G infusion. Methods: Pts with previously untreated CLL/small lymphocytic lymphoma were randomized to ibr-G or clb-G. Ibr or clb was given approximately 30-120 min before the first G infusion. Plasma samples were collected at 4 timepoints (before ibr/clb, immediately before G infusion, and 2h and 4h post-G infusion) on day 1. Cytokines evaluated were IFNγ, IL6, IL8, IL10, IL18, MCP1, MIP1α, MIP1β, and TNFα. Changes from baseline (immediately before G infusion) to post-G infusion peak cytokine levels were compared between arms and between pts with vs without IRRs using Wilcoxon rank sum test. A 2-sided P value of <0.05 was considered significant with no adjustments for multiplicity. Results: Of 229 randomized pts, 95 pts on ibr-G (15 with IRR; 80 without IRR) and 88 pts on clb-G (45 with IRR; 43 without IRR) had cytokine data and were included in the analysis population. Baseline characteristics were similar between arms, except for lower platelets in ibr-G pts and higher baseline MCP1 levels in clb-G pts. As expected, all cytokine levels increased after infusion of G. Analysis of cytokine levels by treatment arm (ibr-G vs clb-G), irrespective of IRR occurrence, revealed that the median increase in cytokines was lower in ibr-G vs clb-G pts for all cytokines (P<0.01) except MIP1β (Figure). When analyzing pts with and without IRR, the median increase in post-G peak from baseline was greater in pts with vs without IRR for all cytokines (P<0.001) except MIP1β. Analysis of cytokine levels in pts with and without IRR within each treatment arm showed that IL6 and IL8 elevations were associated with IRRs in both treatment arms. In ibr-G arm, pts with IRR had increases in post-G levels of IL6, IL8, IL18, MCP1, MIP1α, and TNFα (P<0.04); in clb-G arm, pts with IRR had increases in IFNγ, IL6, IL8, and IL10 (P<0.03). Among the pts with IRR, ibr-G pts had lower post-G increases in IL6, IL8, IL10, and MCP1 levels (P<0.04) than clb-G pts. Keywords: cytokines; ibrutinib; obinutuzumab. Disclosures: Greil, R: Employment Leadership Position: Celgene, Roche, Merck, AstraZeneca, Novartis, Amgen, BMS, MSD, Takeda, Sandoz; Consultant Advisory Role: Celgene, Novartis, Roche, BMS, Abbvie, AstraZeneca, Janssen, Takeda; Research Funding: Celgene, Roche, Merck, AstraZeneca, Novartis, Amgen, BMS, MSD, Takeda, Sandoz; Other Remuneration: Travel, Accomodations, Expenses: Roche, Amgen, Janssen, AstraZeneca. Tedeschi, A: Consultant Advisory Role: Janssen spa, Gilead, AbbVie, SUNESIS; Other Remuneration: Speaker's Bureau: Janssen. Moreno, C: Consultant Advisory Role: Janssen, Pharmacyclics, AbbVie. Larratt, L: Consultant Advisory Role: Abbvie, Janssen. Simkovic, M: Consultant Advisory Role: Abbvie, Roche, Gilead; Honoraria: Abbvie, Janssen-Cilag, Roche, Gilead; Other Remuneration: Speaker's Bureau: Abbvie, Roche, Janssen-Cilag, Gilead; Travel, Accomodations, Expenses: Roche, Janssen-Cilag, Gilead. Gill, D: Consultant Advisory Role: Janssen Cilag; Honoraria: Janssen-Cilag. Gribben, J: Consultant Advisory Role: Pharmacyclics, Janssen; Honoraria: Pharmacyclics LLC, an AbbVie Company, Janssen; Research Funding: Janssen. Flinn, I: Research Funding: Agios, ArQule, Beigene, Calithera, Celgene, Constellation, Curis, Forma, Forty Seven, Genentech, Gilead, Incyte, infinity, Janssen, KITE, Merck, Novartis, Pfizer, Pharmamcyclics, Portola, Seattle genetics, Takeda, TG Therapeutics, Trillium, Verastem. Wang, Z: Employment Leadership Position: Pharmacyclics LLC, an AbbVie Company; Stock Ownership: Abbvie, Anaptysbio. Cheung, L: Employment Leadership Position: Pharmacyclics LLC, an AbbVie Company; Stock Ownership: AbbVie & Eli Lilly and Company; Other Remuneration: Patents, Royalties: Pharmacyclics LLC, an AbbVie Company. Nguyen, A: Employment Leadership Position: Pharmacyclics, an AbbVie Company; Stock Ownership: Abbvie; Other Remuneration: Travel, Accomodations, Expenses: AbbVie. Zhou, C: Employment Leadership Position: Pharmacyclics LLC, an AbbVie Company; Stock Ownership: Abbvie. Styles, L: Employment Leadership Position: Pharmacyclics LLC, an AbbVie Company; Stock Ownership: Abbvie. Demirkan, F: Consultant Advisory Role: Amgen, Abbvie; Research Funding: Janssen, Abbvie; Other Remuneration: Travel, Accomodations, or Other Expenses: Abbvie, Pfizer, Janssen, Amgen.

Background: Ibrutinib (ibr), a first-in-class, once-daily inhibitor of Bruton's tyrosine kinase, is approved in the US and EU for patients (pts) with CLL and allows for treatment without chemotherapy. Standard of care for first-line CLL in older pts or those with comorbidities includes single-agent ibr or chemoimmunotherapy (CIT) with chlorambucil (clb) plus anti-CD20 therapy. As no data were available from phase 3 studies directly comparing single-agent ibr with CIT, we performed a cross-trial analysis using data from two phase 3 studies to assess single-agent ibr vs clb plus obinutuzumab (G) in first-line CLL.

Fcγ receptor (FcγR) signalling in monocyte derived macrophages from chronic lymphocytic leukaemia (CLL) patients is poorly understood. This signalling pathway is the key determinant of the ability of the macrophages to respond to therapeutic antibodies in current clinical use for CLL. Muted FcγR signalling activity accompanies disease progression and results in resistance to therapeutic antibodies. The molecular mechanisms controlling FcγR signalling and resistance are unknown. Here, we demonstrate that the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110δ is essential for CLL-derived macrophages to respond to therapeutic antibodies. Inhibition of p110δ in the macrophages reduces FcγR-mediated antibody immune responses. Surprisingly, our studies indicated that FcγR downstream signalling is independent of SYK and BTK activity. Thus, we show that FcγR antibody responses occur via a previously unidentified p110δ-dependent pathway, which is independent of the previously described SYK/BTK activation pathway. These data provide novel insights into the effectors of antibody responses. Our data also provide mechanistic insights into therapy resistance in CLL.

Fusion genes are a major cause of cancer. Their rapid and accurate diagnosis can inform clinical action, but current molecular diagnostic assays are restricted in resolution and throughput. Here, we show that targeted RNA sequencing (RNAseq) can overcome these limitations. First, we establish that fusion gene detection with targeted RNAseq is both sensitive and quantitative by optimising laboratory and bioinformatic variables using spike-in standards and cell lines. Next, we analyse a clinical patient cohort and improve the overall fusion gene diagnostic rate from 63% with conventional approaches to 76% with targeted RNAseq while demonstrating high concordance for patient samples with previous diagnoses. Finally, we show that targeted RNAseq offers additional advantages by simultaneously measuring gene expression levels and profiling the immune-receptor repertoire. We anticipate that targeted RNAseq will improve clinical fusion gene detection, and its increasing use will provide a deeper understanding of fusion gene biology.

In classical Hodgkin lymphoma (CHL) the tumor microenvironment (TME) is enriched in T cells that modulate antitumor immunity. PD1 blockade partially restores anti-tumoral T cell function, to induce impressive responses in a proportion of patients with relapsed/refractory CHL (Chen et al JCO 2017). Further characterisation of T cell immune evasion mechanisms in CHL will permit the rational development of enhanced immunotherapeutic strategies. Lymphocyte-activation gene 3 (LAG3) is a cell surface molecule known to be expressed on a subset of immune effector T cells and intratumoral regulatory T cells (Tregs) in solid-organ tumors, with combination PD1/LAG3 mAb blockade showing early promise (Ascierto et al 2017 JCO abst 9520). In contrast, data in haematological malignancies is limited, although it is known that LAG3+ T cells suppress anti-tumoral immunity in CHL and B-CLL (Gandhi et al Blood 2006; Shapiro et al Haematologica 2017). Interestingly, in B-CLL LAG3 is found on both T cells and malignant B cells. Whether Hodgkin Reed-Sternberg (HRS) cells express LAG3 is unknown.

Chemoimmunotherapy (CIT) and targeted therapy with single-agent ibrutinib are both recommended first-line treatments for chronic lymphocytic leukemia (CLL), although their outcomes have not been directly compared. Using ibrutinib data from the RESONATE-2 (PCYC-1115/1116) study conducted in patients ≥65 years without del(17p), we performed a cross-trial comparison with CIT data from published phase 3 studies in first-line treatment of CLL. Progression-free survival (PFS), overall survival (OS), and safety data for ibrutinib (median follow-up 35.7 months) were evaluated alongside available CIT data. CIT regimens included: fludarabine + cyclophosphamide + rituximab (CLL8, CLL10), bendamustine + rituximab (CLL10), obinutuzumab + chlorambucil and rituximab + chlorambucil (CLL11), and ofatumumab + chlorambucil (COMPLEMENT-1). Median age across studies was 61-74 years, with older populations receiving ibrutinib, obinutuzumab + chlorambucil, or rituximab + chlorambucil. Median follow-up varied across studies/regimens (range 14.5-37.4 months). Among all patients, PFS appeared longer with ibrutinib relative to CIT and OS appeared comparable. Relative to CIT studies that similarly excluded patients with del(17p) (CLL10) or enrolled older/less-fit patients (CLL11), PFS appeared favorable for ibrutinib in high-risk subgroups, including advanced disease, bulky lymph nodes, unmutated IGHV status, and presence of del(11q). Grade ≥ 3 infections ranged from 9% (ofatumumab + chlorambucil) to 40% (fludarabine + cyclophosphamide + rituximab), and was 25% with ibrutinib. Grade ≥ 3 neutropenia was 12% for ibrutinib and 26%-84% for CIT. Although definitive conclusions cannot be made due to inherent limitations of cross-trial comparisons, this report suggests that ibrutinib has a favorable benefit/risk profile and may potentially eliminate the need for chemotherapy in some patients. Randomized, comparative studies are needed to support these findings.