MYC protein is a high-risk factor in mantle cell lymphoma and identifies cases beyond morphology, proliferation and TP53/p53 - a Nordic Lymphoma Group study.

The transcription factor MYC is a well-described oncogene with an important role in lymphomagenesis, but its significance for clinical outcome in mantle cell lymphoma (MCL) remains to be determined. We performed an investigation of the expression of MYC protein in a cohort of 251 MCL patients complemented by analyses of structural aberrations and mRNA, in a sub-cohort of patients. Fourteen percent (n=35) of patients showed high MYC protein expression with >20% positive cells (MYChigh), among whom only one translocation was identified, and 86% (n=216) of patients showed low MYC protein expression. Low copy number gains of MYC were detected in ten patients, but with no correlation to MYC protein levels. However, MYC mRNA levels correlated significantly to MYC protein levels with a R2 value of 0.76. Patients with a MYChigh tumor had both an independent inferior overall survival and an inferior progression-free survival (hazard ratio [HR]=2.03, 95% confidence interval [95% CI]: 1.2-3.4 and HR=2.2, 95% CI: 1.04-4.6, respectively) when adjusted for additional high-risk features. Patients with MYChigh tumors also tended to have additional high-risk features and to be older at diagnosis. A subgroup of 13 patients had concomitant MYChigh expression and TP53/p53 alterations and a substantially increased risk of progression (HR=16.9, 95% CI: 7.4-38.3) and death (HR=7.8, 95% CI: 4.4-14.1) with an average overall survival of only 0.9 years. In summary, we found that at diagnosis a subset of MCL patients (14%) overexpressed MYC protein, and had a poor prognosis but that MYC rearrangements were rare. Tumors with concurrent MYC overexpression and TP53/p53 alterations pinpointed MCL patients with a dismal prognosis with a median overall survival of less than 3 years. We propose that MYC needs to be assessed beyond the current high-risk factors in MCL in order to identify cases in need of alternative treatment.

Overexpression of the key metabolic protein CPT1A defines mantle cell lymphoma patients with poor response to standard high-dose chemotherapy independent of MIPI and complement established highrisk factors.

The variable outcome to standard immunochemotherapy for mantle cell lymphoma (MCL) patients is a clinical challenge. Established risk factors, including high MCL International Prognostic Index (MIPI), high proliferation (Ki-67), non-classic (blastoid/pleomorphic) morphology, and mutated TP53, only partly identify patients in need of alternative treatment. Deepened understanding of biological factors that influence time to progression and relapse would allow for an improved stratification, and identification of novel targets for high-risk patients. We performed gene expression analyses to identify pathways and genes associated with outcome in a cohort of homogeneously treated patients. In addition to deregulated proliferation, we show that thermogenesis, fatty acid degradation and oxidative phosphorylation are altered in patients with poor survival, and that high expression of carnitine palmitoyltransferase 1A (CPT1A), an enzyme involved in fatty acid degradation, can specifically identify high-risk patients independent of the established high-risk factors. We suggest that complementary investigations of metabolism may increase the accuracy of patient stratification and that immunohistochemistry- based assessment of CPT1A can contribute to defining high-risk MCL.

Infiltration of CD163-, PD-L1- and FoxP3-positive cells adversely affects outcome in patients with mantle cell lymphoma independent of established risk factors.

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) 1·97, 95% confidence interval (CI) 1·18-3·25, adjusted for sex and MCL International Prognostic Index (MIPI)] and a higher frequency of CD163+ cells (continuously, HR 1·51, 95% CI 1·03-2·23, 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 3·22, 95% CI 1·40-7·43) and CD163+ cells (HR 6·09, 95% CI 1·84-20·21), 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.

p53 is associated with high-risk and pinpoints TP53 missense mutations in mantle cell lymphoma.

Survival for patients diagnosed with mantle cell lymphoma (MCL) has improved drastically in recent years. However, patients carrying mutations in tumour protein p53 (TP53) do not benefit from modern chemotherapy-based treatments and have poor prognosis. Thus, there is a clinical need to identify missense mutations through routine analysis to enable patient stratification. Sequencing is not widely implemented in clinical practice for MCL, and immunohistochemistry (IHC) is a feasible alternative to identify high-risk patients. The aim of the present study was to investigate the accuracy of p53 as a tool to identify patients with TP53 missense mutations and the prognostic impact of overexpression and mutations in a Swedish population-based cohort. In total, 317 cases were investigated using IHC and 255 cases were sequenced, enabling analysis of p53 and TP53 status among 137 cases divided over the two-cohort investigated. The accuracy of predicting missense mutations from protein expression was 82%, with sensitivity at 82% and specificity at 100% in paired samples. We further show the impact of p53 expression and TP53 mutations on survival (hazard ratio of 3·1 in univariate analysis for both), and the association to risk factors, such as high MCL International Prognostic Index, blastoid morphology and proliferation, in a population-based setting.

Immune-related protein signature in serum stratify relapsed mantle cell lymphoma patients based on risk.

BACKGROUND: Response to modern treatment strategies, which combine cytotoxic compounds with immune stimulatory agents and targeted treatment is highly variable among MCL patients. Thus, providing prognostic and predictive markers for risk adapted therapy is warranted and molecular information that can help in patient stratification is a necessity. In relapsed MCL, biopsies are rarely available and molecular information from tumor tissue is often lacking. Today, the main tool to access risk is the MCL international prognostic index (MIPI), which does not include detailed biological information of relevance for different treatment options. To enable continuous monitoring of patients, non-invasive companion diagnostic tools are needed which can further reduce cost and patient distress and enable efficient measurements of biological markers. METHODS: We have assessed if serum-based protein profiling can identify immune related proteins that stratify relapsed MCL patients based on risk. Overall, 371 scFv targeting 158 proteins were assessed using an antibody microarray platform. We profiled patients (n = 44) who had been treated within the MCL6-Philemon trial combining targeted and immune-modulatory treatment. RESULTS: The downstream processing led to the identification of the relapsed immune signature (RIS) consisting of 11 proteins with potential to stratify patients with long and short overall survival (OS). Moreover, in this population, MIPI alone failed to separate high, intermediate and low risk patients, but a combined index based on MIPI together with RIS, MIPIris, showed improved performance and significantly stratified all three risk groups based on OS. CONCLUSIONS: Our results show that addition of biological parameters to previous prognostic indices improves patient stratification among patients treated with BTK inhibitor triplet combination, particularly, in the identification of an extreme high risk group.

Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness.

TP53 mutations identify younger mantle cell lymphoma patients who do not benefit from intensive chemoimmunotherapy.

Despite recent advances in lymphoma treatment, mantle cell lymphoma (MCL) remains incurable, and we are still unable to identify patients who will not benefit from the current standard of care. Here, we explore the prognostic value of recurrent genetic aberrations in diagnostic bone marrow (BM) specimens from 183 younger patients with MCL from the Nordic MCL2 and MCL3 trials, which represent current standard-of-care regimens. In the univariate model, mutations of TP53 (11%) and NOTCH1 (4%), and deletions of TP53 (16%) and CDKN2A (20%), were significantly associated with inferior outcomes (together with MIPI, MIPI-c, blastoid morphology, and Ki67 > 30%); however, in multivariate analyses, only TP53 mutations (HR, 6.2; P < .0001) retained prognostic impact for overall survival (OS), whereas TP53 mutations (HR, 6.9; P < .0001) and MIPI-c high-risk (HR, 2.6; P = .003) had independent prognostic impact on time to relapse. TP53-mutated cases had a dismal outcome, with a median OS of 1.8 years, and 50% relapsed at 1.0 years, compared to a median OS of 12.7 years for TP53-unmutated cases (P < .0001). TP53 mutations were significantly associated with Ki67 > 30%, blastoid morphology, MIPI high-risk, and inferior responses to both induction- and high-dose chemotherapy. In conclusion, we show that TP53 mutations identify a phenotypically distinct and highly aggressive form of MCL with poor or no response to regimens including cytarabine, rituximab, and autologous stem-cell transplant (ASCT). We suggest patients with MCL should be stratified according to TP53 status, and that patients with TP53 mutations should be considered for experimental frontline trials exploring novel agents.

Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity.

BACKGROUND: The addition of high-dose cytarabine to the treatment of mantle cell lymphoma (MCL) has significantly prolonged survival of patients, but relapses are common and are normally associated with increased resistance. To elucidate the mechanisms responsible for cytarabine resistance, and to create a tool for drug discovery investigations, we established a unique and molecularly reproducible cytarabine resistant model from the Z138 MCL cell line. METHODS: Effects of different substances on cytarabine-sensitive and resistant cells were evaluated by assessment of cell proliferation using [methyl-14C]-thymidine incorporation and molecular changes were investigated by protein and gene expression analyses. RESULTS: Gene expression profiling revealed that major transcriptional changes occur during the initial phase of adaptation to cellular growth in cytarabine containing media, and only few key genes, including SPIB, are deregulated upon the later development of resistance. Resistance was shown to be mediated by down-regulation of the deoxycytidine kinase (dCK) protein, responsible for activation of nucleoside analogue prodrugs. This key event, emphasized by cross-resistance to other nucleoside analogues, did not only effect resistance but also levels of SPIB and NF-κB, as assessed through forced overexpression in resistant cells. Thus, for the first time we show that regulation of drug resistance through prevention of conversion of pro-drug into active drug are closely linked to increased proliferation and resistance to apoptosis in MCL. Using drug libraries, we identify several substances with growth reducing effect on cytarabine resistant cells. We further hypothesized that co-treatment with bortezomib could prevent resistance development. This was confirmed and show that the dCK levels are retained upon co-treatment, indicating a clinical use for bortezomib treatment in combination with cytarabine to avoid development of resistance. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that a majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial clinical response to cytarabine treatment. CONCLUSION: We show that cytarabine resistance potentially can be avoided or at least delayed through co-treatment with bortezomib, and that down-regulation of dCK and up-regulation of SPIB and NF-κB are the main molecular events driving cytarabine resistance development.

miR-18b overexpression identifies mantle cell lymphoma patients with poor outcome and improves the MIPI-B prognosticator.

Recent studies show that mantle cell lymphoma (MCL) express aberrant microRNA (miRNA) profiles; however, the clinical effect of miRNA expression has not previously been examined and validated in large prospective homogenously treated cohorts. We performed genome-wide miRNA microarray profiling of 74 diagnostic MCL samples from the Nordic MCL2 trial (screening cohort). Prognostic miRNAs were validated in diagnostic MCL samples from 94 patients of the independent Nordic MCL3 trial (validation cohort). Three miRNAs (miR-18b, miR-92a, and miR-378d) were significantly differentially expressed in patients who died of MCL in both cohorts. MiR-18b was superior to miR-92a and miR-378d in predicting high risk. Thus, we generated a new biological MCL International Prognostic Index (MIPI-B)-miR prognosticator, combining expression levels of miR-18b with MIPI-B data. Compared to the MIPI-B, this prognosticator improved identification of high-risk patients with regard to cause-specific, overall, and progression-free survival. Transfection of 2 MCL cell lines with miR-18b decreased their proliferation rate without inducing apoptosis, suggesting that miR-18b may render MCL cells resistant to chemotherapy by decelerating cell proliferation. We conclude that overexpression of miR-18b identifies patients with poor prognosis in 2 large prospective MCL cohorts and adds prognostic information to the MIPI-B. MiR-18b may reduce the proliferation rate of MCL cells as a mechanism of chemoresistance.

Identification of V-ATPase as a molecular sensor of SOX11-levels and potential therapeutic target for mantle cell lymphoma.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive disease with short median survival. Molecularly, MCL is defined by the t(11;14) translocation leading to overexpression of the CCND1 gene. However, recent data show that the neural transcription factor SOX11 is a disease defining antigen and several involved signaling pathways have been pin-pointed, among others the Wnt/ß-catenin pathway that is of importance for proliferation in MCL. Therefore, we evaluated a compound library focused on the Wnt pathway with the aim of identifying Wnt-related targets that regulate growth and survival in MCL, with particular focus on SOX11-dependent growth regulation. METHODS: An inducible SOX11 knock-down system was used to functionally screen a library of compounds (n = 75) targeting the Wnt signaling pathway. A functionally interesting target, vacuolar-type H(+)-ATPase (V-ATPase), was further evaluated by western blot, siRNA-mediated gene silencing, immunofluorescence, and flow cytometry. RESULTS: We show that 15 out of 75 compounds targeting the Wnt pathway reduce proliferation in all three MCL cell lines tested. Furthermore, three substances targeting two different targets (V-ATPase and Dkk1) showed SOX11-dependent activity. Further validation analyses were focused on V-ATPase and showed that two independent V-ATPase inhibitors (bafilomycin A1 and concanamycin A) are sensitive to SOX11 levels, causing reduced anti-proliferative response in SOX11 low cells. We further show, using fluorescence imaging and flow cytometry, that V-ATPase is mainly localized to the plasma membrane in primary and MCL cell lines. CONCLUSIONS: We show that SOX11 status affect V-ATPase dependent pathways, and thus may be involved in regulating pH in intracellular and extracellular compartments. The plasma membrane localization of V-ATPase indicates that pH regulation of the immediate extracellular compartment may be of importance for receptor functionality and potentially invasiveness in vivo.

DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells.

BACKGROUND: The neural transcription factor SOX11 is present at specific stages during embryo development with a very restricted expression in adult tissue, indicating precise regulation of transcription. SOX11 is strongly up-regulated in some malignancies and have a functional role in tumorgenesis. With the aim to explore differences in epigenetic regulation of SOX11 expression in normal versus neoplastic cells, we investigated methylation and histone modifications related to the SOX11 promoter and the possibility to induce re-expression using histone deacetylase (HDAC) or EZH2 inhibitors. METHODS: The epigenetic regulation of SOX11 was investigated in distinct non-malignant cell populations (n = 7) and neoplastic cell-lines (n = 42) of different cellular origins. DNA methylation was assessed using bisulfite sequencing, methylation-specific melting curve analysis, MethyLight and pyrosequencing. The presence of H3K27me3 was assessed using ChIP-qPCR. The HDAC inhibitors Vorinostat and trichostatin A were used to induce SOX11 in cell lines with no endogenous expression. RESULTS: The SOX11 promoter shows a low degree of methylation and strong enrichment of H3K27me3 in non-malignant differentiated cells, independent of cellular origin. Cancers of the B-cell lineage are strongly marked by de novo methylation at the SOX11 promoter in SOX11 non-expressing cells, while solid cancer entities display a more varying degree of SOX11 promoter methylation. The silencing mark H3K27me3 was generally present at the SOX11 promoter in non-expressing cells, and an increased enrichment was observed in cancer cells with a low degree of SOX11 methylation compared to cells with dense methylation. Finally, we demonstrate that the HDAC inhibitors (vorinostat and trichostatin A) induce SOX11 expression in cancer cells with low levels of SOX11 methylation. CONCLUSIONS: We show that SOX11 is strongly marked by repressive histone marks in non-malignant cells. In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications. The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation. In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.

SOX11 and TP53 add prognostic information to MIPI in a homogenously treated cohort of mantle cell lymphoma--a Nordic Lymphoma Group study.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma, where survival has been remarkably improved by use of protocols including high dose cytarabine, rituximab and autologous stem cell transplantation, such as the Nordic MCL2/3 protocols. In 2008, a MCL international prognostic index (MIPI) was created to enable stratification of the clinical diverse MCL patients into three risk groups. So far, use of the MIPI in clinical routine has been limited, as it has been shown that it inadequately separates low and intermediate risk group patients. To improve outcome and minimize treatment-related morbidity, additional parameters need to be evaluated to enable risk-adapted treatment selection. We have investigated the individual prognostic role of the MIPI and molecular markers including SOX11, TP53 (p53), MKI67 (Ki-67) and CCND1 (cyclin D1). Furthermore, we explored the possibility of creating an improved prognostic tool by combining the MIPI with information on molecular markers. SOX11 was shown to significantly add prognostic information to the MIPI, but in multivariate analysis TP53 was the only significant independent molecular marker. Based on these findings, we propose that TP53 and SOX11 should routinely be assessed and that a combined TP53/MIPI score may be used to guide treatment decisions.

Molecular serum portraits in patients with primary breast cancer predict the development of distant metastases.

The risk of distant recurrence in breast cancer patients is difficult to assess with current clinical and histopathological parameters, and no validated serum biomarkers currently exist. Using a recently developed recombinant antibody microarray platform containing 135 antibodies against 65 mainly immunoregulatory proteins, we screened 240 sera from 64 patients with primary breast cancer. This unique longitudinal sample material was collected from each patient between 0 and 36 mo after the primary operation. The velocity for each serum protein was determined by comparing the samples collected at the primary operation and then 3-6 mo later. A 21-protein signature was identified, using leave-one-out cross-validation together with a backward elimination strategy in a training cohort. This signature was tested and evaluated subsequently in an independent test cohort (prevalidation). The risk of developing distant recurrence after primary operation could be assessed for each patient, using her molecular portraits. The results from this prevalidation study showed that patients could be classified into high- versus low-risk groups for developing metastatic breast cancer with a receiver operating characteristic area under the curve of 0.85. This risk assessment was not dependent on the type of adjuvant therapy received by the patients. Even more importantly, we demonstrated that this protein signature provided an added value compared with conventional clinical parameters. Consequently, we present here a candidate serum biomarker signature able to classify patients with primary breast cancer according to their risk of developing distant recurrence, with an accuracy outperforming current procedures.

Empowering macrophages: the cancer fighters within the tumour microenvironment in mantle cell lymphoma.

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.

Predictive and Prognostic Biomarkers in Patients With Mycosis Fungoides and Sézary Syndrome (BIO-MUSE): Protocol for a Translational Study.

BACKGROUND: Cutaneous T-cell lymphoma (CTCL) is a rare group of lymphomas that primarily affects the skin. Mycosis fungoides (MF) is the most common form of CTCL and Sézary syndrome (SS) is more infrequent. Early stages (IA-IIA) have a favorable prognosis, while advanced stages (IIB-IVB) have a worse prognosis. Around 25% of patients with early stages of the disease will progress to advanced stages. Malignant skin-infiltrating T-cells in CTCL are accompanied by infiltrates of nonmalignant T-cells and other immune cells that produce cytokines that modulate the inflammation. Skin infection, often with Staphylococcus aureus, is frequent in advanced stages and can lead to sepsis and death. S. aureus has also been reported to contribute to the progression of the disease. Previous reports indicate a shift from Th1 to Th2 cytokine production and dysfunction of the skin barrier in CTCL. Treatment response is highly variable and often unpredictable, and there is a need for new predictive and prognostic biomarkers. OBJECTIVE: This prospective translational study aims to identify prognostic biomarkers in the blood and skin of patients with MF and SS. METHODS: The Predictive and Prognostic Biomarkers in Patients With MF and SS (BIO-MUSE) study aims to recruit 120 adult patients with MF or SS and a control group of 20 healthy volunteers. The treatments will be given according to clinical routine. The sampling of each patient will be performed every 3 months for 3 years. The blood samples will be analyzed for lactate dehydrogenase, immunoglobulin E, interleukins, thymus and activation-regulated chemokine, and lymphocyte subpopulations. The lymphoma microenvironment will be investigated through digital spatial profiling and single-cell RNA sequencing. Microbiological sampling and analysis of skin barrier function will be performed. The life quality parameters will be evaluated. The results will be evaluated by the stage of the disease. RESULTS: Patient inclusion started in 2021 and is still ongoing in 2023, with 18 patients and 20 healthy controls enrolled. The publication of selected translational findings before the publication of the main results of the trial is accepted. CONCLUSIONS: This study aims to investigate blood and skin with a focus on immune cells and the microbiological environment to identify potential new prognostic biomarkers in MF and SS. TRIAL REGISTRATION: ClinicalTrials.gov NCT04904146; https://www.clinicaltrials.gov/study/NCT04904146. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/55723.

Spatial tumor immune microenvironment phenotypes in ovarian cancer.

Immunotherapy has largely failed in ovarian carcinoma (OC), likely due to that the vast tumor heterogeneity and variation in immune response have hampered clinical trial outcomes. Tumor-immune microenvironment (TIME) profiling may aid in stratification of OC tumors for guiding treatment selection. Here, we used Digital Spatial Profiling combined with image analysis to characterize regions of spatially distinct TIME phenotypes in OC to assess whether immune infiltration pattern can predict presence of immuno-oncology targets. Tumors with diffuse immune infiltration and increased tumor-immune spatial interactions had higher presence of IDO1, PD-L1, PD-1 and Tim-3, while focal immune niches had more CD163 macrophages and a preliminary worse outcome. Immune exclusion was associated with presence of Tregs and Fibronectin. High-grade serous OC showed an overall stronger immune response and presence of multiple targetable checkpoints. Low-grade serous OC was associated with diffuse infiltration and a high expression of STING, while endometrioid OC had higher presence of CTLA-4. Mucinous and clear cell OC were dominated by focal immune clusters and immune-excluded regions, with mucinous tumors displaying T-cell rich immune niches.

Enhancing cell death in B-cell malignancies through targeted inhibition of Bcl-3.

The t(14;19)(q32;q13) is a rare recurring translocation found in B-cell lymphoproliferative malignancies, involving the Bcl-3 gene. This chromosomal translocation is often found in patients under the age of 50 and causes a more progressive disease. The Bcl-3 gene encodes a protein belonging to the IκB family of proteins, which tightly regulates NFκB signaling by acting as an activator or repressor of transcription. Previously, we developed a second-generation Bcl-3 inhibitor that could directly interfere with Bcl-3 signaling pathway, resulting in reduced melanoma cell proliferation, invasion, and migration. The present study aimed to investigate the effect of a Bcl-3 inhibitor on B-cell lymphoma and leukemia cells. It was found that treatment of cells with this inhibitor caused a decrease in cell proliferation and cell survival. Furthermore, Bcl-3 inhibition in B-cell malignant cells resulted in the loss of mitochondrial membrane potential and functionality, as well as the increased expression of cleaved caspase 3, indicating that cell death occurs through the intrinsic apoptotic pathway. This observation is further supported by reduced expression of cIAP1 protein 1 (cIAP1) upon treatment of cancer cells. Given the current lack of clinical advancements targeting Bcl-3 in oncology, this opens a novel avenue for the development and investigation of highly specific therapeutic interventions against B-cell malignancies.

CD163+ macrophages in mantle cell lymphoma induce activation of prosurvival pathways and immune suppression.

ABSTRACT: 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.

Quantification and Profiling of Early and Late Differentiation Stage T Cells in Mantle Cell Lymphoma Reveals Immunotherapeutic Targets in Subsets of Patients.

With the aim to advance the understanding of immune regulation in MCL and to identify targetable T-cell subsets, we set out to combine image analysis and spatial omic technology focused on both early and late differentiation stages of T cells. MCL patient tissue (n = 102) was explored using image analysis and GeoMx spatial omics profiling of 69 proteins and 1812 mRNAs. Tumor cells, T helper (TH) cells and cytotoxic (TC) cells of early (CD57-) and late (CD57+) differentiation stage were analyzed. An image analysis workflow was developed based on fine-tuned Cellpose models for cell segmentation and classification. TC and CD57+ subsets of T cells were enriched in tumor-rich compared to tumor-sparse regions. Tumor-sparse regions had a higher expression of several key immune suppressive proteins, tentatively controlling T-cell expansion in regions close to the tumor. We revealed that T cells in late differentiation stages (CD57+) are enriched among MCL infiltrating T cells and are predictive of an increased expression of immune suppressive markers. CD47, IDO1 and CTLA-4 were identified as potential targets for patients with T-cell-rich MCL TIME, while GITR might be a feasible target for MCL patients with sparse T-cell infiltration. In subgroups of patients with a high degree of CD57+ TC-cell infiltration, several immune checkpoint inhibitors, including TIGIT, PD-L1 and LAG3 were increased, emphasizing the immune-suppressive features of this highly differentiated T-cell subset not previously described in MCL.

Protein expression profiling of formalin-fixed paraffin-embedded tissue using recombinant antibody microarrays.

Proteomics, the large-scale analysis of proteins, is a rapidly evolving field with an increasing number of key clinical applications, such as diagnosis, prognosis, and classification. In order to generate complete protein expression profiles, or protein atlases, any crude sample format must be addressable in a rapid, multiplex, and sensitive manner. A common and clinically central sample format, formalin-fixed, paraffin-embedded (FFPE) tissue material, holds great potential as a source for disease-associated biomarker signatures. However, despite major efforts, extraction and subsequent profiling of proteins from FFPE tissue has proven to be challenging. In this proof-of-concept study, we have demonstrated for the first time that proteins could be extracted, labeled, and subsequently profiled in a multiplex, sensitive, and reproducible manner using recombinant scFv antibody microarrays. Thus, we have added FFPE samples to the list of sample formats available for high-throughput analysis by affinity proteomics, paving the way for the next generation of biomarker-driven discovery projects.