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Mantle Cell Lymphoma (MCL) is, despite recent advances, still not considered curable. The aim of this thesis is to increase the understanding of MCL tumour biology by describing the impact of the immune microenvironment and tumour genetics on the outcome for the patient.

In a first study (paper I) we described the composition of the tumour immune microenvironment in MCL tumour tissue. We found that CD3+ T-cells were the most abundant cell type in the tumour tissue, and that high numbers of FOXP3+ regulatory T-cells and CD163+ macrophages were associated with shorter survival.

The negative prognostic impact of macrophages in MCL was confirmed in a second study (paper II) where we investigated the soluble macrophage marker sCD163 and found that high serum levels correlated with worse prognosis in both newly diagnosed and relapsed MCL patients. 

The third study (paper III) further underscores the importance of macrophages in MCL. In this study, we analysed differences in the plasma proteome between patients that did or did not relapse before 24 months (POD24). Among 1463 proteins, the most differentially expressed were two macrophage markers, sCD169 and sVSIG4. 

In the fourth study (paper IV) we focused on tumour genetics, using sequential tumour biopsies. We showed that MCL tumours accumulate high-risk genetic alterations over time, and that the type of treatment the patient received affected the risk of developing such alterations.

Treatment is also the focus of the last study (paper V), where we described a higher risk of acute haematologic side effects in patients with clonal haematopoiesis. 

In summary, this thesis has provided new insights about the tumour microenvironment in MCL and advanced the understanding of how anti-tumour treatments interact with tumour biology as well as with patient intrinsic factors.

Objectives

Patients with primary Sj & ouml;gren's disease (SjD) have an increased risk of B cell lymphoma. The aim of this study was to determine serum protein biomarkers for lymphoma development and to advance our understanding of the functional mechanisms underlying lymphomagenesis in SjD.

Methods

Patients with SjD and incident, current lymphoma (n=18) with serum sampled before treatment and at 6, 12 and 24 months of follow-up, and four patients sampled 1-5 years before lymphoma diagnosis (pre-lymphoma) were included. SjD without lymphoma (n=21), SjD with historical lymphoma (n=6) and healthy blood donors (n=39) served as controls. Differentially expressed proteins between groups were analysed using the Olink Target 96 Immuno-Oncology panel applying a false discovery rate (FDR) adjusted p value of 0.05. Protein-derived interferon activation scores (pIFN scores) were calculated.

Results

We determined 18 differentially expressed proteins in SjD with incident lymphoma compared with both SjD without lymphoma and healthy controls. Among the top upregulated proteins were TNFSF14, FGF2, IL8, CD40 and CXCL13, where CXCL13 was the only protein with decreased levels at follow-up. We also observed upregulated expression of CD40 in the SjD pre-lymphoma group compared with SjD without lymphoma and healthy controls. All SjD patient groups presented elevated pIFN scores compared with healthy controls, where SjD sampled pre-lymphoma showed the most distinct IFN activation.

Conclusions

We identified altered protein expression and an increased IFN system activation in SjD with incident lymphoma and pre-lymphoma. This knowledge may contribute to earlier detection of high-risk patients, identification of therapeutic targets and may ultimately improve SjD patient outcomes.

The use of Bruton's tyrosine kinase inhibitors (BTKi) is rapidly increasing for patients with mantle cell lymphoma (MCL). Side effects reported so far are usually manageable. However, here we present two cases of life-threatening aplastic anemia (AA) upon treatment with the BTKi acalabrutinib for MCL. The first patient died of neutropenic infection secondary to AA. The second patient was successfully treated with immunosuppressive treatment but the MCL relapsed shortly thereafter. AA is a potentially fatal complication that should be considered when patients present with pancytopenia during treatment with BTKi.

Mantle cell lymphoma (MCL) is dependent on a supportive tumor immune microenvironment (TIME) in which infiltration of CD163⁺ macrophages has a negative prognostic impact. This study explores how abundance and spatial localization of CD163⁺ cells are associated with the biology of MCL, using spatial multiomic investigations of tumor and infiltrating CD163⁺ and CD3⁺ cells. A total of 63 proteins were measured using GeoMx digital spatial profiling in tissue microarrays from 100 diagnostic MCL tissues. Regions of interest were selected in tumor-rich and tumor-sparse tissue regions. Molecular profiling of CD163⁺ macrophages, CD20⁺ MCL cells, and CD3⁺ T-cells was performed. To validate protein profiles, 1811 messenger RNAs were measured in CD20⁺ cells and 2 subsets of T cells. Image analysis was used to extract the phenotype and position of each targeted cell, thereby allowing the exploration of cell frequencies and cellular neighborhoods. Proteomic investigations revealed that CD163⁺ cells modulate their immune profile depending on their localization and that the immune inhibitory molecules, V-domain immunoglobulin suppressor of T-cell activation and B7 homolog 3, have higher expression in tumor-sparse than in tumor-rich tissue regions and that targeting should be explored. We showed that MCL tissues with more abundant infiltration of CD163⁺ cells have a higher proteomic and transcriptional expression of key components of the MAPK pathway. Thus, the MAPK pathway may be a feasible therapeutic target in patients with MCL with CD163⁺ cell infiltration. We further showed the independent and combined prognostic values of CD11c and CD163 beyond established risk factors.

In Mantle Cell Lymphoma (MCL), the role of macrophages within the tumour microenvironment (TME) has recently gained attention due to their impact on prognosis and response to therapy. Despite their low absolute number in MCL tumour tissue, recent findings reveal an association between the levels of macrophages and prognosis, consistent with trends observed in other lymphoma subtypes. M2-like macrophages, identified by markers such as CD163, contribute to angiogenesis and suppression of the immune response. Clinical trials with MCL patients treated with chemoimmunotherapy and targeted treatments underscore the adverse impact of high levels of M2-like macrophages. Immunomodulatory drugs like lenalidomide reduce the levels of MCL-associated CD163⁺ macrophages and enhance macrophage phagocytic activity. Similarly, clinical approaches targeting the CD47 “don’t eat me” signalling, in combination with the anti-CD20-antibody rituximab, demonstrate increased macrophage activity and phagocytosis of MCL tumour cells. Cell-based therapies such as chimeric antigen receptor (CAR) T-cell have shown promise but various challenges persist, leading to a potential interest in CAR-macrophages (CAR-M). When macrophages are recruited to the TME, they offer advantages including phagocytic function and responsiveness to microenvironment alterations, suggesting their potential as a manipulable and inducible alternative when CAR T-cell therapies fails in the complex landscape of MCL treatment.

The outcome for patients with mantle cell lymphoma (MCL) has drastically improved with new treatments directed toward the tumor immune microenvironment, where macrophages play an important role. In MCL, the presence of M2 macrophages defined by CD163 expression in diagnostic biopsies has been associated with a worse prognosis. An alternative way to assess the abundance of M2 macrophages is by measuring the level of soluble CD163 in serum (sCD163). We aimed to investigate the prognostic value of sCD163 in 131 patients with MCL. We found that high sCD163 at diagnosis was associated with shorter progression-free survival (PFS) and shorter overall survival (OS) in 81 patients who were newly diagnosed and subsequently treated with chemoimmunotherapy. The same was seen in a cohort of 50 patients with relapsed MCL that were mainly treated within the phase 2 Philemon-trial with rituximab, ibrutinib, and lenalidomide. In patients who were newly diagnosed and had low levels of sCD163, 5-year survival was 97%. There was a moderate correlation between sCD163 and tissue CD163. The association with a poor prognosis was independent of MCL international prognostic index, Ki67, p53 status, and blastoid morphology, as assessed in a multivariable Cox proportional hazards model. In this study, high sCD163 was associated with both shorter PFS and shorter OS, showing that high levels of the M2 macrophage marker sCD163 is an independent negative prognostic factor in MCL, both in the chemoimmunotherapy and ibrutinib/lenalidomide era. In addition, low sCD163 levels identify patients with MCL with a very good prognosis.

We characterised patients with mantle cell lymphoma (MCL) with poor prognosis based on differences in immune infiltration. Different expressions of the tumour cell markers Cyclin D1 and sex-determining region Y-box transcription factor 11 (SOX11), and the immune markers cluster of differentiation 3 (CD3), CD4, CD8, CD25, forkhead box protein P3 (FoxP3), T-box transcription factor TBX21 (T-bet), programmed cell death protein 1 (PD-1), programmed-death ligand 1 (PD-L1) and CD163 were investigated for all-cause mortality in 282 patients with MCL and time-to-progression (TTP) in 106 clinical trial patients. With increasing age, a significantly lower infiltration of CD3(+) T lymphocytes was seen. T-cell infiltration was independent of cellular tumour antigen p53 (p53) expression, Ki-67, morphology and frequency of tumour cells. The all-cause mortality was higher in patients with PD-L1-expression above cut-off [hazard ratio (HR) 1 center dot 97, 95% confidence interval (CI) 1 center dot 18-3 center dot 25, adjusted for sex and MCL International Prognostic Index (MIPI)] and a higher frequency of CD163(+) cells (continuously, HR 1 center dot 51, 95% CI 1 center dot 03-2 center dot 23, adjusting for age, sex, morphology, Ki-67 and p53). In patients treated within the Nordic Lymphoma Group MCL2/3 trials, TTP was shorter in patients with a higher frequency of FoxP3(+) cells (HR 3 center dot 22, 95% CI 1 center dot 40-7 center dot 43) and CD163(+) cells (HR 6 center dot 09, 95% CI 1 center dot 84-20 center dot 21), independent of sex and MIPI. When combined a higher frequency of CD163(+) macrophages and PD-L1(+) cells or high CD163(+) macrophages and FoxP3(+) regulatory T cells indicated worse outcome independent of established risk factors. The T-cell infiltrate was in turn independent of molecular characteristics of the malignant cells and decreased with age.