Molecular profiling of patients with cytogenetically normal acute myeloid leukemia and hyperleukocytosis.

BACKGROUND: Acute myeloid leukemia (AML) with initial hyperleukocytosis is associated with high early mortality and a poor prognosis. The aims of this study were to delineate the underlying molecular landscape in the largest cytogenetic risk group, cytogenetically normal acute myeloid leukemia (CN-AML), and to assess the prognostic relevance of recurrent mutations in the context of hyperleukocytosis and clinical risk factors. METHODS: The authors performed a targeted sequencing of 49 recurrently mutated genes in 56 patients with newly diagnosed CN-AML and initial hyperleukocytosis of ≥100 G/L treated in the AMLCG99 study. The median number of mutated genes per patient was 5. The most common mutations occurred in FLT3 (73%), NPM1 (75%), and TET2 (45%). RESULTS: The predominant pathways affected by mutations were signaling (84% of patients), epigenetic modifiers (75% of patients), and nuclear transport (NPM1; 75%) of patients. AML with hyperleukocytosis was enriched for molecular subtypes that negatively affected the prognosis, including a high percentage of patients presenting with co-occurring mutations in signaling and epigenetic modifiers such as FLT3 internal tandem duplications and TET2 mutations. CONCLUSIONS: Despite these unique molecular features, clinical risk factors, including high white blood count, hemoglobin level, and lactate dehydrogenase level at baseline, remained the predictors for overall survival and relapse-free survival in hyperleukocytotic CN-AML.

Post-transplant Malignancies Show Reduced T-cell Abundance and Tertiary Lymphoid Structures as Correlates of Impaired Cancer Immunosurveillance.

PURPOSE: An increased risk to develop cancer is one of the most challenging negative side effects of long-term immunosuppression in organ transplant recipients and impaired cancer immunosurveillance is assumed as underlying mechanism. This study aims to elucidate transplant-related changes in the tumor immune microenvironment (TME) of cancer. EXPERIMENTAL DESIGN: Data from 123 organ transplant recipients (kidney, heart, lung, and liver) were compared with historic data from non-immunosuppressed patients. Digital image analysis of whole-section slides was used to assess abundance and spatial distribution of T cells and tertiary lymphoid structures (TLS) in the TME of 117 tumor samples. Expression of programmed cell death 1 ligand 1 (PD-L1) and human-leucocyte-antigen class I (HLA-I) was assessed on tissue microarrays. RESULTS: We found a remarkably reduced immune infiltrate in the center tumor (CT) regions as well as the invasive margins (IM) of post-transplant cancers. These differences were more pronounced in the IM than in the CT and larger for CD8+ T cells than for CD3+ T cells. The Immune-score integrating results from CT and IM was also lower in transplant recipients. Density of TLS was lower in cancer samples of transplant recipients. The fraction of samples with PD-L1 expression was higher in controls whereas decreased expression of HLA-I was more common in transplant recipients. CONCLUSIONS: Our study demonstrates the impact of immunosuppression on the TME and supports impaired cancer immunosurveillance as important cause of post-transplant cancer. Modern immunosuppressive protocols and cancer therapies should consider the distinct immune microenvironment of post-transplant malignancies.

T-cells of invasive candidiasis patients show patterns of T-cell-exhaustion suggesting checkpoint blockade as treatment option.

OBJECTIVE: Recent data imply that strengthening host immunity by checkpoint inhibition improves outcome in invasive fungal infections (IFI), particularly in candidiasis. METHODS: To assess T-cell exhaustion in this context, we compared peripheral blood mononuclear cells (PBMCs) and serum samples of patients with invasive Candida albicans infection (IC, n = 21) to PBMCs or tumor-infiltrating lymphocytes (TILs) from cancer patients (n = 14) and PBMCs of healthy controls (n = 20). Type and differentiation of lymphocytes and expression of 29 immune-regulatory molecules were analyzed by flow cytometry. C. albicans specific responses were assessed by FluoroSpot (n = 8) and antibody measurement (n = 14). RESULTS: Fractions and phenotypes of lymphocyte subsets in PBMCs of IC patients were similar compared to PBMCs of controls, while they were different in TILs. PBMCs of patients with IC showed increased expression of immune-checkpoint molecules. The pattern of upregulated molecules was similar to TILs, but not present in PBMCs of control cancer patients. Fractions of T-cells expressing PD-1 and TIGIT were higher in IC patients that died. FluoroSpot analysis showed a Candida-specific IFN-y or IL-2 response in 5/8 patients, enhanced by addition of nivolumab in vitro. CONCLUSIONS: Together with preclinical data and preliminary evidence of clinical efficacy in mucormycosis, our results support clinical evaluation of immune-checkpoint inhibition in IFI treatment. TRIAL REGISTRATION: NCT04533087; retrospectively registered on August 31, 2020.

B cells in esophago-gastric adenocarcinoma are highly differentiated, organize in tertiary lymphoid structures and produce tumor-specific antibodies.

Tumor-infiltrating lymphocytes (TILs) are correlated to prognosis of several kinds of cancer. Most studies focused on T cells, while the role of tumor-associated B cells (TABs) has only recently gained more attention. TABs contain subpopulations with distinct functions, potentially promoting or inhibiting immune responses. This study provides a detailed analysis of TABs in gastro-esophageal adenocarcinoma (EAC). Flow cytometric analyses of single cell suspensions of tumor samples, mucosa, lymph nodes and peripheral blood mononuclear cells (PBMC) of EAC patients and healthy controls revealed a distinct B cell compartment in cancer patients. B cells were increased in tumor samples and subset-analyses of TILs showed increased proportions of differentiated and activated B cells and an enrichment for follicular T helper cells. Confocal microscopy demonstrated that TABs were mainly organized in tertiary lymphoid structures (TLS), which resemble lymphoid follicles in secondary lymphoid organs. A panel of 34 tumor-associated antigens (TAAs) expressed in EAC was identified based on public databases and TCGA data to analyze tumor-specific B cell responses using a LUMINEXTM bead assay and flow cytometry. Structural analyses of TLS and the detection of tumor-specific antibodies against one or more TAAs in 48.1% of analyzed serum samples underline presence of anti-tumor B cell responses in EAC. Interestingly, B cells were decreased in tumors with expression of Programmed Death Ligand 1 or impaired HLA-I expression. These data demonstrate that anti-tumor B cell responses are an additional and underestimated aspect of EAC. Our results are of immediate translational relevance to emerging immunotherapies.

Cytokine release syndrome.

During the last decade the field of cancer immunotherapy has witnessed impressive progress. Highly effective immunotherapies such as immune checkpoint inhibition, and T-cell engaging therapies like bispecific T-cell engaging (BiTE) single-chain antibody constructs and chimeric antigen receptor (CAR) T cells have shown remarkable efficacy in clinical trials and some of these agents have already received regulatory approval. However, along with growing experience in the clinical application of these potent immunotherapeutic agents comes the increasing awareness of their inherent and potentially fatal adverse effects, most notably the cytokine release syndrome (CRS). This review provides a comprehensive overview of the mechanisms underlying CRS pathophysiology, risk factors, clinical presentation, differential diagnoses, and prognostic factors. In addition, based on the current evidence we give practical guidance to the management of the cytokine release syndrome.

Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor originating from the epithelial lining of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. The natural killer group 2D (NKG2D) receptors on natural killer (NK) cells and several T cell subsets play an important role for immunosurveillance of HNSCC and are thus targeted by tumor immune evasion strategies in particular by shedding of various NKG2D ligands (NKG2DLs). Based on plasma and tumor samples of 44 HNSCC patients, we found that despite compositional heterogeneity the total plasma level of NKG2DLs correlates with NK cell inhibition and disease progression. Strikingly, based on tumor spheroids and primary tumors of HNSCC patients, we found that NK cells failed to infiltrate HNSCC tumors in the presence of high levels of NKG2DLs, demonstrating a novel mechanism of NKG2DL-dependent tumor immune escape. Therefore, the diagnostic acquisition of the plasma level of all NKG2DLs might be instrumental for prognosis and to decipher a patient cohort, which could benefit from restoration of NKG2D-dependent tumor immunosurveillance. Along these lines, we could show that removal of shed NKG2DLs (sNKG2DLs) from HNSCC patients' plasma restored NK cell function in vitro and in individual patients following surgical removal of the primary tumor. In order to translate these findings into a therapeutic setting, we performed a proof-of-concept study to test the efficacy of adsorption apheresis of sNKG2DLs from plasma after infusion of human MICA in rhesus monkeys. Complete removal of MICA was achieved after three plasma volume exchanges. Therefore, we propose adsorption apheresis of sNKG2DLs as a future preconditioning strategy to improve the efficacy of autologous and adoptively transferred immune cells in cellular cancer immunotherapy.

Critical care of patients with cancer.

Answer questions and earn CME/CNE The increasing prevalence of patients living with cancer in conjunction with the rapid progress in cancer therapy will lead to a growing number of patients with cancer who will require intensive care treatment. Fortunately, the development of more effective oncologic therapies, advances in critical care, and improvements in patient selection have led to an increased survival of critically ill patients with cancer. As a consequence, critical care has become an important cornerstone in the continuum of modern cancer care. Although, in many aspects, critical care for patients with cancer does not differ from intensive care for other seriously ill patients, there are several challenging issues that are unique to this patient population and require special knowledge and skills. The optimal management of critically ill patients with cancer necessitates expertise in oncology, critical care, and palliative medicine. Cancer specialists therefore have to be familiar with key principles of intensive care for critically ill patients with cancer. This review provides an overview of the state-of-the-art in the individualized management of critically ill patients with cancer. CA Cancer J Clin 2016;66:496-517. © 2016 American Cancer Society.

Processing and MHC class II presentation of exogenous soluble antigen involving a proteasome-dependent cytosolic pathway in CD40-activated B cells.

Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4 + T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4 + T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway.

Phase I clinical study of the toll-like receptor 9 agonist MGN1703 in patients with metastatic solid tumours.

PURPOSE: This study was initiated to evaluate safety, toxicity, pharmacokinetics, and pharmacodynamics of treatment with MGN1703, a novel synthetic DNA-based toll-like receptor 9 (TLR9)-immunomodulator. METHODS: The study consisted of an escalating single dose regimen followed by a multiple dose part. Dose levels of 0.25, 2, 10, 30, and 60 mg of MGN1703 were administered subcutaneously over 6 weeks twice weekly. Patients with at least stable disease (SD) could participate in the extension phase of the study for six further weeks. Effects on the immune status were monitored. RESULTS: 28 patients with metastatic solid tumours were included. Fatigue and activated partial thromboplastin time (aPTT) prolongation were the only two cases of drug-related grade 3 Common Terminology Criteria adverse events (CTCAE). The most frequently reported drug-related adverse events were of CTC Grade ⩽2. There was no relationship between toxicity and dose and no patient was withdrawn from the study due to drug-related AE. No drug-related serious AE (SAE) were reported. Six out of 24 patients had SD after 6 weeks of treatment and three of those remained in SD after a total of 12 weeks. Four patients were further treated in a compassionate use programme showing long-term disease stabilisation for up to 18 months. Immune assessment of cell compartments showed a non-significant increase of TLR9 expressing naïve B cells during therapy. CONCLUSION: Twice weekly subcutaneous applications of MGN1703 in a dose of up to 60 mg are safe and well tolerated without dose-limiting toxicities. MGN1703 shows immune activation and anti-tumour efficacy in heavily pretreated patients. The recommended dose of 60 mg twice weekly is currently used in a phase II trial in small cell lung cancer and a phase III trial in colorectal cancer patients.

Inhibition of protein geranylgeranylation specifically interferes with CD40-dependent B cell activation, resulting in a reduced capacity to induce T cell immunity.

Ab-independent effector functions of B cells, such as Ag presentation and cytokine production, have been shown to play an important role in a variety of immune-mediated conditions such as autoimmune diseases, transplant rejection, and graft-versus-host disease. Most current immunosuppressive treatments target T cells, are relatively unspecific, and result in profound immunosuppression that places patients at an increased risk of developing severe infections and cancer. Therapeutic strategies, which interfere with B cell activation, could therefore be a useful addition to the current immunosuppressive armamentarium. Using a transcriptomic approach, we identified upregulation of genes that belong to the mevalonate pathway as a key molecular event following CD40-mediated activation of B cells. Inhibition of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme of the mevalonate pathway, by lipophilic statins such as simvastatin and atorvastatin resulted in a specific inhibition of B cell activation via CD40 and impaired their ability to act as stimulatory APCs for allospecific T cells. Mechanistically, the inhibitory effect resulted from the inhibition of protein geranylgeranylation subsequent to the depletion of mevalonate, the metabolic precursor for geranylgeranyl. Thus, inhibition of geranylgeranylation either directly through geranylgeranyl transferase inhibitors or indirectly through statins represents a promising therapeutic approach for the treatment of diseases in which Ag presentation by B cells plays a role.

Characterization of tumor-associated B-cell subsets in patients with colorectal cancer.

PURPOSE: A precise understanding of the mechanisms by which human immune cell subsets affect tumor biology will be critical for successful treatment of cancer using immunotherapeutic approaches. Recent evidence suggests that B cells can both promote and inhibit the development and progression of tumors. The aim of this study was to characterize the composition of the B-cell infiltrates in colorectal cancers (CRC) in order to gain further insight into the role of B cells in CRC. EXPERIMENTAL DESIGN: In this study we characterized B-cell subsets in primary tumors (n=38), metastases (n=6) and blood (n=46) of 51 patients with a diagnosis of CRC and blood of 10 healthy controls. B-cell subsets were analyzed by flow cytometry or immunohistochemistry. RESULTS: Peripheral blood of CRC patients contained a higher percentage of memory B cells than that of age-matched healthy controls. Furthermore, the percentage of B cells within tumors was higher than that in the peripheral blood of CRC patients while metastases were typically devoid of tumor-infiltrating B cells. Tumor-associated B cells were enriched for activated and terminally differentiated B cells. Relevant proportions of regulatory B cells could only be detected in advanced cancer and metastases. CONCLUSION: B cells constitute a significant proportion of the immune infiltrate in CRC. The B-cell infiltrate of primary CRC is characterized by an accumulation of terminally differentiated memory B cells or plasma cells suggestive of a specific immune response against the tumor. However advanced tumors and metastases are also infiltrated by a considerable number of regulatory B cells.

Pretransplant comorbidities maintain their impact on allogeneic stem cell transplantation outcome 5 years posttransplant: a retrospective study in a single german institution.

The introduction of reduced-intensity conditioning regimens has allowed elderly patients with preexisting comorbidities access to the potentially curative allogeneic stem cell transplantation. Patient's comorbidities at the time of treatment consideration play a significant role in transplant outcome in terms of both overall survival (OS) and nonrelapse mortality (NRM). The hematopoietic stem cell transplantation comorbidity index (HCT-CI) quantifies these patient specific risks and has established itself as a major tool in the pretransplant assessment of patients. Many single center and multicenter studies have assessed the HCT-CI score and reported conflicting outcomes. The present study aimed to evaluate the HCT-CI in a single large European transplant centre. 245 patients were retrospectively analyzed and the predictive value of the score was assessed with respect to OS and NRM. We confirm that the HCT-CI predicts outcome for both OS and NRM. Moreover, we identified age of the patient as an independent prognostic parameter for OS. Incorporation of age in the HCT-CI would improve its ability to prognosticate and allow the transplant physician to assess the patient specific risks appropriately at the time of counseling for transplant.

The ratio between dendritic cells and T cells determines whether prostaglandin E2 has a stimulatory or inhibitory effect.

Prostaglandin E2 has been shown to enhance the maturation, migration, and antigen-presenting capacity of DCs. It is therefore included in many maturation cocktails for the generation of monocyte-derived DCs. Paradoxically, PGE2 is also an important tumor-derived immunosuppressive factor and has inhibitory effects on DC differentiation and function. To further investigate these seemingly contradictory results we studied whether the DC:T cell ratio has an impact on the outcome of the interaction between PGE2-treated DCs and T cells. Surprisingly, at high DC:T cell ratios T cell proliferation was inhibited while at low ratios PGE2-treated DCs displayed enhanced T cell-stimulatory properties. The inhibitory function of PGE2-treated DCs depended primarily on the PGE2-induced induction of indoleamine 2,3-dioxygenase competence. In summary, we show that PGE2-treated DCs can have either an immunogenic or tolerogenic function depending on the DC:T cell ratio. This finding could explain the conflicting results regarding the influence of PGE2 on DC function.

The immunosuppressive factors IL-10, TGF-ß, and VEGF do not affect the antigen-presenting function of CD40-activated B cells.

BACKGROUND: Progress in recent years strengthened the concept of cellular tumor vaccinations. However, a crucial barrier to successful cancer immunotherapy is tumor-mediated immunosuppression. Tumor-derived soluble factors such as IL-10, TGF-ß, and VEGF suppress effector cells either directly or indirectly by disruption of dendritic cell (DC) differentiation, migration and antigen presentation. Human B cells acquire potent immunostimulatory properties when activated via CD40 and have been shown to be an alternative source of antigen-presenting cells (APCs) for cellular cancer vaccines. Nevertheless, in contrast to DCs little knowledge exists about their susceptibility to tumor derived immunosuppressive factors. Thus, we assessed whether IL-10, TGF-ß, or VEGF do affect key aspects of the immunostimulatory function of human CD40-activated B cells. METHODS: Cell surface expression of adhesion and costimulatory molecules and the proliferation capacity of CD40-activated B cells were compared to untreated controls by flow cytometry. Migration towards important chemokines of secondary lymph organs was measured with or without exposure to the immunosuppressive cytokines. Finally, an influence on T cell stimulation was investigated by allogeneic mixed lymphocyte reactions. For statistical analysis Student's t test or two-way analysis of variance followed by Bonferroni's post-hoc test was used to compare groups. P values of <0.05 were considered statistically significant. RESULTS: Neither cell adhesion nor the expression of MHC class II and costimulatory molecules CD80 and CD86 was inhibited by addition of IL-10, TGF-ß, or VEGF. Likewise, the proliferation of CD40-activated B cells was not impaired. Despite being exposed to IL-10, TGF-ß, or VEGF the B cells migrated equally well as untreated controls to the chemokines SLC and SDF-1α. Most importantly, the capacity of CD40-activated B cells to stimulate CD4+ and CD8+ T cells remained unaffected. CONCLUSION: Our findings suggest that key immunostimulatory functions of CD40-activated B cells are resistant to inhibition by the immunosuppressive factors IL-10, TGF-ß, and VEGF. This supports considerations to use ex vivo generated CD40-activated B cells as a promising alternative or additional APC for cellular immunotherapy, especially in settings where these immunosuppressive cytokines are present in tumor environment.

Targeting malignant B cells as antigen-presenting cells: TLR-9 agonist induces systemic regression of lymphoma.

Evaluation of: Brody JD, Ai WZ, Czerwinski DK et al. In situ vaccination with a TLR9 agonist induces systemic lymphoma regression: a Phase I/II study. J. Clin. Oncol. 28(28), 4324-4332 (2010). Despite high response rates of the follicular B-cell lymphoma to monoclonal antibodies, the clinical course of lymphoma is still characterized by a continuous pattern of relapse. Brody and colleagues treated 15 patients with relapsed, low-grade lymphoma using low-dose radiotherapy applied to one of the tumor sites with combined injection of a TLR-9 agonist at the same site. This strategy induced specific immunity and tumor regression in several patients with an objective response rate of 27%. The results indicate an involvement of the tumor TLR-9-expressing B cells acting as antigen-presenting cells. TLR-9 in situ vaccination combined with local radiotherapy clearly warrants further in-depth analysis and investigation in a Phase III randomized trial, and may provide a new opportunity for the treatment of B-cell malignancies.

The cyclins: a family of widely expressed tumor antigens?

Continuous cell division is a hallmark of cancer and cell-cycle regulators therefore represent relevant target molecules for tumor therapy. Among these targets the cyclins are of particular interest as they are overexpressed in various tumor entities with little expression in normal tissue. Here we review evidence that these molecules are recognized by the immune system, summarize why cyclins A, B and D in particular appear to be interesting targets for active and passive immunotherapy, and discuss whether the entire family could be an interesting novel class of tumor antigens for cancer treatment and prevention.

Toll-like receptor 9 agonists as cancer therapeutics.

INTRODUCTION: Toll-like receptor 9 (TLR9) agonists, commonly referred to as CpG oligodeoxynucleotides (ODN), have been added to the arsenal of anti-cancer drugs as monotherapy or in combination with chemotherapy, radiotherapy and other immunotherapeutic approaches as they increase antigen presentation and boost anti-tumor T- and B-cell responses. Several synthetic TLR9 agonists have been developed for clinical grade use and displayed substantial efficacy in the preclinical and clinical models. AREAS COVERED: This review summarizes TLR9 signaling and the impact of TLR9 agonists on the immune response. The most recent experimental and clinical data are analyzed as well as the development of new TLR9 agonists in current clinical trials. EXPERT OPINION: Application of TLR9 agonists, in particular, combination strategies with chemo- or radiotherapy seem a promising and efficient immunotherapeutic approach in cancer patients even with refractory disease. Simultaneous application of TLR9 agonists aims at supporting the patient's immune response and overcoming specific immunosuppressant strategies developed by tumors. Combinatory approaches of the future might also seek for synergism of TLR9 agonists with other immunomodulatory strategies such as B-cell activation using the CD40-CD40L system.