Profile of the multicenter cohort of the German Cancer Consortium's Clinical Communication Platform.

Treatment concepts in oncology are becoming increasingly personalized and diverse. Successively, changes in standards of care mandate continuous monitoring of patient pathways and clinical outcomes based on large, representative real-world data. The German Cancer Consortium's (DKTK) Clinical Communication Platform (CCP) provides such opportunity. Connecting fourteen university hospital-based cancer centers, the CCP relies on a federated IT-infrastructure sourcing data from facility-based cancer registry units and biobanks. Federated analyses resulted in a cohort of 600,915 patients, out of which 232,991 were incident since 2013 and for which a comprehensive documentation is available. Next to demographic data (i.e., age at diagnosis: 2.0% 0-20 years, 8.3% 21-40 years, 30.9% 41-60 years, 50.1% 61-80 years, 8.8% 81+ years; and gender: 45.2% female, 54.7% male, 0.1% other) and diagnoses (five most frequent tumor origins: 22,523 prostate, 18,409 breast, 15,575 lung, 13,964 skin/malignant melanoma, 9005 brain), the cohort dataset contains information about therapeutic interventions and response assessments and is connected to 287,883 liquid and tissue biosamples. Focusing on diagnoses and therapy-sequences, showcase analyses of diagnosis-specific sub-cohorts (pancreas, larynx, kidney, thyroid gland) demonstrate the analytical opportunities offered by the cohort's data. Due to its data granularity and size, the cohort is a potential catalyst for translational cancer research. It provides rapid access to comprehensive patient groups and may improve the understanding of the clinical course of various (even rare) malignancies. Therefore, the cohort may serve as a decisions-making tool for clinical trial design and contributes to the evaluation of scientific findings under real-world conditions.

Pathogen Detection by Metagenomic Next-Generation Sequencing During Neutropenic Fever in Patients With Hematological Malignancies.

Background: Febrile neutropenia (FN) after chemotherapy is a major cause of morbidity during cancer treatment. The performance of metagenomic next-generation sequencing (mNGS) of circulating cell-free deoxyribonucleic acid from plasma may be superior to blood culture (BC) diagnostics for identification of causative pathogens. The aim of this study was to validate mNGS (DISQVER test) for the detection of pathogens in hematologic patients with FN. Methods: We collected paired whole blood specimens from central venous catheter and peripheral vein during FN for BC and mNGS testing. We repeated paired sampling at the earliest after 3 days of fever, which was defined as 1 FN episode. All clinical data were retrospectively reviewed by an infectious disease expert panel. We calculated percent positive agreement (PPA), percent negative agreement (PNA), percent overall agreement (POA), and sensitivity and specificity. Results: We analyzed a total of 98 unselected FN episodes in 61 patients who developed predominantly FN after conditioning therapy for allogeneic (n = 22) or autologous (n = 21) hematopoietic stem cell transplantation. Success rate of mNGS was 99% (97 of 98). Positivity rate of mNGS was 43% (42 of 97) overall and 32% (31 of 97) excluding viruses compared to 14% (14 of 98) in BC. The PPA, PNA, and POA between mNGS and BC were 84.6% (95% confidence interval [CI], 54.6% to 98.1%), 63.1% (95% CI, 51.9% to 73.4%), and 66% (95% CI, 55.7% to 75.3%), respectively. Sensitivity for bacteria or fungi was 40% (95% CI, 28.0% to 52.9%) and 18.5% (95% CI, 9.9% to 30.0%), respectively. Conclusions: Pathogen detection by mNGS (DISQVER) during unselected FN episodes shows 2-fold higher sensitivity and a broader pathogen spectrum than BC.

Distinct Chemokine Receptor Expression Profiles in De Novo DLBCL, Transformed Follicular Lymphoma, Richter's Trans-Formed DLBCL and Germinal Center B-Cells.

Chemokine receptors and their ligands have been identified as playing an important role in the development of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and Richter syndrome (RS). Our aim was to investigate the different expression profiles in de novo DLBCL, transformed follicular lymphoma (tFL), and RS. Here, we profiled the mRNA expression levels of 18 chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, CX3CR1 and XCR1) using RQ-PCR, as well as immunohistochemistry of seven chemokine receptors (CCR1, CCR4-CCR8 and CXCR2) in RS, de novo DLBCL, and tFL biopsy-derived tissues. Tonsil-derived germinal center B-cells (GC-B) served as non-neoplastic controls. The chemokine receptor expression profiles of de novo DLBCL and tFL substantially differed from those of GC-B, with at least 5-fold higher expression of 15 out of the 18 investigated chemokine receptors (CCR1-CCR9, CXCR1, CXCR2, CXCR6, CXCR7, CX3CR1 and XCR1) in these lymphoma subtypes. Interestingly, the de novo DLBCL and tFL exhibited at least 22-fold higher expression of CCR1, CCR5, CCR8, and CXCR6 compared with RS, whereas no significant difference in chemokine receptor expression profile was detected when comparing de novo DLBCL with tFL. Furthermore, in de novo DLBCL and tFLs, a high expression of CCR7 was associated with a poor overall survival in our study cohort, as well as in an independent patient cohort. Our data indicate that the chemokine receptor expression profile of RS differs substantially from that of de novo DLBCL and tFL. Thus, these multiple dysregulated chemokine receptors could represent novel clinical markers as diagnostic and prognostic tools. Moreover, this study highlights the relevance of chemokine signaling crosstalk in the tumor microenvironment of aggressive lymphomas.

Impact of the microenvironment on the pathogenesis of mucosa-associated lymphoid tissue lymphomas.

Approximately 8% of all non-Hodgkin lymphomas are extranodal marginal zone B cell lymphomas of mucosa-associated lymphoid tissue (MALT), also known as MALT lymphomas. These arise at a wide range of different extranodal sites, with most cases affecting the stomach, the lung, the ocular adnexa and the thyroid. The small intestine is involved in a lower percentage of cases. Lymphoma growth in the early stages is associated with long-lasting chronic inflammation provoked by bacterial infections (e.g., Helicobacter pylori or Chlamydia psittaci infections) or autoimmune conditions (e.g., Sjögren's syndrome or Hashimoto thyroiditis). While these inflammatory processes trigger lymphoma cell proliferation and/or survival, they also shape the microenvironment. Thus, activated immune cells are actively recruited to the lymphoma, resulting in either direct lymphoma cell stimulation via surface receptor interactions and/or indirect lymphoma cell stimulation via secretion of soluble factors like cytokines. In addition, chronic inflammatory conditions cause the acquisition of genetic alterations resulting in autonomous lymphoma cell growth. Recently, novel agents targeting the microenvironment have been developed and clinically tested in MALT lymphomas as well as other lymphoid malignancies. In this review, we aim to describe the composition of the microenvironment of MALT lymphoma, the interaction of activated immune cells with lymphoma cells and novel therapeutic approaches in MALT lymphomas using immunomodulatory and/or microenvironment-targeting agents.

Immune Regulatory Processes of the Tumor Microenvironment under Malignant Conditions.

The tumor microenvironment (TME) is a critical regulator of tumor growth, progression, and metastasis. Since immune cells represent a large fraction of the TME, they play a key role in mediating pro- and anti-tumor immune responses. Immune escape, which suppresses anti-tumor immunity, enables tumor cells to maintain their proliferation and growth. Numerous mechanisms, which have been intensively studied in recent years, are involved in this process and based on these findings, novel immunotherapies have been successfully developed. Here, we review the composition of the TME and the mechanisms by which immune evasive processes are regulated. In detail, we describe membrane-bound and soluble factors, their regulation, and their impact on immune cell activation in the TME. Furthermore, we give an overview of the tumor/antigen presentation and how it is influenced under malignant conditions. Finally, we summarize novel TME-targeting agents, which are already in clinical trials for different tumor entities.

CD19+IgD+CD27- Naïve B Cells as Predictors of Humoral Response to COVID 19 mRNA Vaccination in Immunocompromised Patients.

Immunocompromised patients are considered high-risk and prioritized for vaccination against COVID-19. We aimed to analyze B-cell subsets in these patients to identify potential predictors of humoral vaccination response. Patients (n=120) suffering from hematologic malignancies or other causes of immunodeficiency and healthy controls (n=79) received a full vaccination series with an mRNA vaccine. B-cell subsets were analyzed prior to vaccination. Two independent anti-SARS-CoV-2 immunoassays targeting the receptor-binding domain (RBD) or trimeric S protein (TSP) were performed three to four weeks after the second vaccination. Seroconversion occurred in 100% of healthy controls, in contrast to 67% (RBD) and 82% (TSP) of immunocompromised patients, while only 32% (RBD) and 22% (TSP) achieved antibody levels comparable to those of healthy controls. The number of circulating CD19+IgD+CD27- naïve B cells was strongly associated with antibody levels (ρ=0.761, P<0.001) and the only independent predictor for achieving antibody levels comparable to healthy controls (OR 1.07 per 10-µL increase, 95%CI 1.02-1.12, P=0.009). Receiver operating characteristic analysis identified a cut-off at ≥61 naïve B cells per µl to discriminate between patients with and without an optimal antibody response. Consequently, measuring of naïve B cells in immunocompromised hematologic patients could be useful in predicting their humoral vaccination response.

Vinorelbine as substitute for vincristine in patients with diffuse large B cell lymphoma and vincristine-induced neuropathy.

BACKGROUND: A combination of rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is the standard first-line therapy for diffuse large B cell lymphoma (DLBCL), the most common aggressive lymphoma in adults. One of the major adverse effects of this regimen is vincristine-induced polyneuropathy which leads to discontinuation of vincristine in up to 30% of DLBCL-patients. Dose reduction of vincristine might worsen treatment outcomes of DLBCL but identification of treatment alternatives for patients exhibiting peripheral neuropathy during R-CHOP is an unmet need in hematology. METHODS: In this retrospective cohort study, comprising 987 patients with de novo DLBCL, we delineated the role of vinorelbine as a substitute for vincristine in R-CHOP by measuring improvements in neuropathy and outcome variables. RESULTS: Five-year overall survival (OS) and progression-free survival (PFS) were 72.6% and 63.1% in patients who received regular doses of vincristine, as compared to 60.6% and 51.7% in patients who received reduced doses of vincristine (p = 0.022 and p = 0.003, respectively). Of 199 patients who switched to vinorelbine, the majority experienced an improvement of neuropathy Furthermore, vinorelbine-switched patients showed favorable oncologic outcomes. CONCLUSION: Replacement of vincristine by vinorelbine due to neuropathy is effective and safe, and results in a significant improvement in neuropathy as compared to treatment with R-CHOP.

Micro-RNA-125a mediates the effects of hypomethylating agents in chronic myelomonocytic leukemia.

BACKGROUND: Chronic myelomonocytic leukemia (CMML) is an aggressive hematopoietic malignancy that arises from hematopoietic stem and progenitor cells (HSPCs). Patients with CMML are frequently treated with epigenetic therapeutic approaches, in particular the hypomethylating agents (HMAs), azacitidine (Aza) and decitabine (Dec). Although HMAs are believed to mediate their efficacy via re-expression of hypermethylated tumor suppressors, knowledge about relevant HMA targets is scarce. As silencing of tumor-suppressive micro-RNAs (miRs) by promoter hypermethylation is a crucial step in malignant transformation, we asked for a role of miRs in HMA efficacy in CMML. RESULTS: Initially, we performed genome-wide miR-expression profiling in a KrasG12D-induced CMML mouse model. Selected candidates with prominently decreased expression were validated by qPCR in CMML mice and human CMML patients. These experiments revealed the consistent decrease in miR-125a, a miR with previously described tumor-suppressive function in myeloid neoplasias. Furthermore, we show that miR-125a downregulation is caused by hypermethylation of its upstream region and can be reversed by HMA treatment. By employing both lentiviral and CRISPR/Cas9-based miR-125a modification, we demonstrate that HMA-induced miR-125a upregulation indeed contributes to mediating the anti-leukemic effects of these drugs. These data were validated in a clinical context, as miR-125a expression increased after HMA treatment in CMML patients, a phenomenon that was particularly pronounced in cases showing clinical response to these drugs. CONCLUSIONS: Taken together, we report decreased expression of miR-125a in CMML and delineate its relevance as mediator of HMA efficacy within this neoplasia.

Antibiotic use and ileocolonic immune cells in patients receiving fecal microbiota transplantation for refractory intestinal GvHD: a prospective cohort study.

INTRODUCTION: Treatment-refractory, acute graft-versus-host disease (GvHD) of the lower gastrointestinal tract (GI) after allogeneic hematopoietic stem cell transplantation is life threatening and lacks effective treatment options. While fecal microbiota transplantation (FMT) was shown to ameliorate GI-GvHD, its mechanisms of action and the factors influencing the treatment response in humans remain unclear.The objective of this study is to assess response to FMT treatment, factors influencing response, and to study the mucosal immune cell composition in treatment-refractory GI-GvHD. METHODS: Consecutive patients with treatment-refractory GI-GvHD were treated with up to six endoscopically applied FMTs. RESULTS: We observed the response to FMT in four out of nine patients with severe, treatment refractory GI-GvHD, associated with a significant survival benefit (p = 0.017). The concomitant use of broad-spectrum antibiotics was the main factor associated with FMT failure (p = 0.048). In addition, antibiotic administration hindered the establishment of donor microbiota after FMT. Unlike in non-responders, the microbiota characteristics (e.g. α- and ß-diversity, abundance of anaerobe butyrate-producers) in responders were more significantly similar to those of FMT donors. During active refractory GI-GvHD, an increased infiltrate of T cells, mainly Th17 and CD8+ T cells, was observed in the ileocolonic mucosa of patients, while the number of immunomodulatory cells such as regulatory T-cells and type 3 innate lymphoid cells decreased. After FMT, a change in immune cell patterns was induced, depending on the clinical response. CONCLUSION: This study increases the knowledge about the crucial effects of antibiotics in patients given FMT for treatment refractory GI-GvHD and defines the characteristic alterations of ileocolonic mucosal immune cells in this setting.

Increased Expression of Micro-RNA-23a Mediates Chemoresistance to Cytarabine in Acute Myeloid Leukemia.

Resistance to chemotherapy is one of the primary obstacles in acute myeloid leukemia (AML) therapy. Micro-RNA-23a (miR-23a) is frequently deregulated in AML and has been linked to chemoresistance in solid cancers. We, therefore, studied its role in chemoresistance to cytarabine (AraC), which forms the backbone of all cytostatic AML treatments. Initially, we assessed AraC sensitivity in three AML cell lines following miR-23a overexpression/knockdown using MTT-cell viability and soft-agar colony-formation assays. Overexpression of miR-23a decreased the sensitivity to AraC, whereas its knockdown had the opposite effect. Analysis of clinical data revealed that high miR-23a expression correlated with relapsed/refractory (R/R) AML disease stages, the leukemic stem cell compartment, as well as with inferior overall survival (OS) and event-free survival (EFS) in AraC-treated patients. Mechanistically, we demonstrate that miR-23a targets and downregulates topoisomerase-2-beta (TOP2B), and that TOP2B knockdown mediates AraC chemoresistance as well. Likewise, low TOP2B expression also correlated with R/R-AML disease stages and inferior EFS/OS. In conclusion, we show that increased expression of miR-23a mediates chemoresistance to AraC in AML and that it correlates with an inferior outcome in AraC-treated AML patients. We further demonstrate that miR-23a causes the downregulation of TOP2B, which is likely to mediate its effects on AraC sensitivity.

The CXCR4-CXCL12-Axis Is of Prognostic Relevance in DLBCL and Its Antagonists Exert Pro-Apoptotic Effects In Vitro.

In tumor cells of more than 20 different cancer types, the CXCR4-CXCL12-axis is involved in multiple key processes including proliferation, survival, migration, invasion, and metastasis. Since data on this axis in diffuse large B cell lymphoma (DLBCL) are inconsistent and limited, we comprehensively studied the CXCR4-CXCL12-axis in our DLBCL cohort as well as the effects of CXCR4 antagonists on lymphoma cell lines in vitro. In DLBCL, we observed a 140-fold higher CXCR4 expression compared to non-neoplastic controls, which was associated with poor clinical outcome. In corresponding bone marrow biopsies, we observed a correlation of CXCL12 expression and lymphoma infiltration rate as well as a reduction of CXCR4 expression in remission of bone marrow involvement after treatment. Additionally, we investigated the effects of three CXCR4 antagonists in vitro. Therefore, we used AMD3100 (Plerixafor), AMD070 (Mavorixafor), and WKI, the niacin derivative of AMD070, which we synthesized. WK1 demonstrated stronger pro-apoptotic effects than AMD070 in vitro and induced expression of pro-apoptotic genes of the BCL2-family in CXCR4-positive lymphoma cell lines. Finally, WK1 treatment resulted in the reduced expression of JNK-, ERK1/2- and NF-κB/BCR-target genes. These data indicate that the CXCR4-CXCL12-axis impacts the pathogenesis of DLBCL and represents a potential therapeutic target in aggressive lymphomas.

Significance of PITX2 Promoter Methylation in Colorectal Carcinoma Prognosis.

BACKGROUND: New treatment modalities and a growing understanding of the complex genetic tumor landscape have improved the outcome of colorectal cancer (CRC) patients. Nonetheless, more individualized treatment regimens, taking individual tumor characteristics into account, have been recently postulated and prognostic biomarkers are needed. We therefore evaluated the prognostic potential of paired-like homeodomain transcription factor 2 (PITX2) promoter methylation in CRC patients. MATERIALS AND METHODS: Data of 2 independent cohorts were investigated. Tissue specimens of cohort A (n = 179) were analyzed for their methylation in the PITX2 promoter region using quantitative methylation-specific polymerase chain reaction and compared with publicly available data (PITX2 promoter methylation and PITX2 mRNA expression levels) from "The Cancer Genome Atlas Research Network" (cohort B, n = 443). Data were correlated with clinicopathological parameters and outcome. RESULTS: Tumor samples of both cohorts showed a decreased PITX2 promoter methylation level (both P < .001) compared with nonmalignant tissue. Additionally, PITX2 promoter hypomethylation was prognostic in univariate and multivariate analysis (hazard ratio [HR], 1.97 [95% confidence interval (CI), 1.12-3.47], P = .018 and HR, 1.89 [95% CI, 1.09-3.29], P = .023), and Kaplan-Meier analysis (median overall survival, 53.2 vs. 70.4 months, P = .004). Subanalysis of high-risk vs. low-risk stage II CRC patients also showed a PITX2 hypomethylation of the promoter region in the high-risk group (P = .006). CONCLUSION: Our results suggest a prognostic role of PITX2 promoter methylation in CRC as biomarker for risk stratification in stage II CRC patients although the results need to be independently validated.

PITX2 DNA Methylation as Biomarker for Individualized Risk Assessment of Prostate Cancer in Core Biopsies.

Hypermethylation of the paired-like homeodomain transcription factor 2 (PITX2) gene is a strong predictor of the risk of biochemical recurrence in patients with prostate cancer (PCa) after radical prostatectomy. We investigate whether PITX2 methylation is feasible for individualized risk assessment in prostate core biopsies before surgery. A quantitative, methylation-specific real-time PCR was used to measure PITX2 in three cohorts: i) matched samples of neoplastic and nonneoplastic tissue from 24 patients with PCa, ii) a well-characterized cohort of 300 patients with PCa after radical prostatectomy, and iii) core biopsy specimens from 32 patients with PCa and 31 patients with benign prostatic disease. PITX2 methylation discriminated between neoplastic and nonneoplastic tissue in patients with PCa (P < 0.001). In the second cohort, PITX2 methylation significantly correlated with clinicopathologic parameters, and PITX2 hypermethylation predicted an increased risk of biochemical recurrence in univariate Cox proportional hazards regression analysis (hazard ratio, 1.77; P = 0.046) and Kaplan-Meier analysis (P = 0.043). In 753 prostate biopsies, 720 (95.6%) were applicable for analysis, rendering the assay feasible for diagnostic biopsies. PITX2 methylation was furthermore significantly increased in tumor-positive biopsies and strongly correlated with International Society of Urological Pathology (ISUP) grade groups. This study indicates that the PITX2 methylation assay is feasible in prostate biopsies and might add valuable prognostic information for risk assessment in a presurgical diagnostic setting.

DNA Methylation of PITX2 and PANCR Is Prognostic for Overall Survival in Patients with Resected Adenocarcinomas of the Biliary Tract.

Biliary tract cancers (BTC) are rare but highly aggressive malignant epithelial tumors. In order to improve the outcome in this lethal disease, novel biomarkers for diagnosis, prognosis, and therapy response prediction are urgently needed. DNA promoter methylation of PITX2 variants (PITX2ab, PITX2c) and intragenic methylation of the PITX2 adjacent non-coding RNA (PANCR) were investigated by methylations-specific qPCR assays in formalin-fixed paraffin-embedded tissue from 80 patients after resection for BTC. Results were correlated with clinicopathologic data and outcome. PITX2 variants and PANCR showed significant hypermethylation in tumor vs. normal adjacent tissue (p < 0.001 and p = 0.015), respectively. In survival analysis, dichotomized DNA methylation of variant PITX2c and PANCR were significantly associated with overall survival (OS). Patients with high tumor methylation levels of PITX2c had a shorter OS compared to patients with low methylation (12 vs. 40 months OS; HR 2.48 [1.38-4.48], p = 0.002). In contrast, PANCR hypermethylation was associated with prolonged survival (25 vs. 19 months OS; HR 0.54 [0.30-0.94], p = 0.015) and qualified as an independent prognostic factor on multivariate analysis. The biomarkers investigated in this study may help to identify BTC subpopulations at risk for worse survival. Further studies are needed to evaluate if PITX2 might be a clinically useful biomarker for an optimized and individualized treatment.

PITX3 promoter methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients after radical prostatectomy.

BACKGROUND: Molecular biomarkers that might help to distinguish between more aggressive and clinically insignificant prostate cancers (PCa) are still urgently needed. Aberrant DNA methylation as a common molecular alteration in PCa seems to be a promising source for such biomarkers. In this study, PITX3 DNA methylation (mPITX3) and its potential role as a prognostic biomarker were investigated. Furthermore, mPITX3 was analyzed in combination with the established PCa methylation biomarker PITX2 (mPITX2). METHODS: mPITX3 and mPITX2 were assessed by a quantitative real-time PCR and by means of the Infinium HumanMethylation450 BeadChip. BeadChip data were obtained from The Cancer Genome Atlas (TCGA) Research Network. DNA methylation differences between normal adjacent, benign hyperplastic, and carcinomatous prostate tissues were examined in the TCGA dataset as well as in prostatectomy specimens from the University Hospital Bonn. Retrospective analyses of biochemical recurrence (BCR) were conducted in a training cohort (n = 498) from the TCGA and an independent validation cohort (n = 300) from the University Hospital Bonn. All patients received radical prostatectomy. RESULTS: In PCa tissue, mPITX3 was increased significantly compared to normal and benign hyperplastic tissue. In univariate Cox proportional hazards analyses, mPITX3 showed a significant prognostic value for BCR (training cohort: hazard ratio (HR) = 1.83 (95 % CI 1.07-3.11), p = 0.027; validation cohort: HR = 2.56 (95 % CI 1.44-4.54), p = 0.001). A combined evaluation with PITX2 methylation further revealed that hypermethylation of a single PITX gene member (either PITX2 or PITX3) identifies an intermediate risk group. CONCLUSIONS: PITX3 DNA methylation alone and in combination with PITX2 is a promising biomarker for the risk stratification of PCa patients and adds relevant prognostic information to common clinically implemented parameters. Further studies are required to determine whether the results are transferable to a biopsy-based patient cohort. Trial registration: Patients for this unregistered study were enrolled retrospectively.