Search

Purpose In 2016, the University of Munich Molecular Tumor Board (MTB) was implemented to initiate a precision oncology program. This review of cases was conducted to assess clinical implications and functionality of the program, to identify current limitations and to inform future directions of these efforts. Methods Charts, molecular profiles, and tumor board decisions of the first 1000 consecutive cases (01/2016–03/2020) were reviewed. Descriptive statistics were applied to describe relevant findings. Results Of the first 1000 patients presented to the MTB; 914 patients received comprehensive genomic profiling. Median age of patients was 56 years and 58% were female. The most prevalent diagnoses were breast (16%) and colorectal cancer (10%). Different types of targeted or genome-wide sequencing assays were used; most of them offered by the local department of pathology. Testing was technically successful in 88%. In 41% of cases, a genomic alteration triggered a therapeutic recommendation. The fraction of patients receiving a tumor board recommendation differed significantly between malignancies ranging from over 50% in breast or biliary tract to less than 30% in pancreatic cancers. Based on a retrospective chart review, 17% of patients with an MTB recommendation received appropriate treatment. Conclusion Based on these retrospective analyses, patients with certain malignancies (breast and biliary tract cancer) tend to be more likely to have actionable variants. The low rate of therapeutic implementation (17% of patients receiving a tumor board recommendation) underscores the importance of meticulous follow-up for these patients and ensuring broad access to innovative therapies for patients receiving molecular tumor profiling.

Clonal hematopoiesis (CH) is an age-related condition predisposing to blood cancer and cardiovascular disease (CVD). Murine models demonstrate CH-mediated altered immune function and proinflammation. Low-grade inflammation has been implicated in the pathogenesis of osteoarthritis (OA), the main indication for total hip arthroplasty (THA). THA-derived hip bones serve as a major source of healthy hematopoietic cells in experimental hematology. We prospectively investigated frequency and clinical associations of CH in 200 patients without known hematologic disease who were undergoing THA. Prevalence of CH was 50%, including 77 patients with CH of indeterminate potential (CHIP, defined as somatic variant allele frequencies [VAFs] ≥2%), and 23 patients harboring CH with lower mutation burden (VAF, 1% to 2%). Most commonly mutated genes were DNMT3A (29.5%), TET2 (15.0%), and ASXL1 (3.5%). CHIP is significantly associated with lower hemoglobin, higher mean corpuscular volume, previous or present malignant disease, and CVD. Strikingly, we observed a previously unreported association of CHIP with autoimmune diseases (AIDs; multivariable adjusted odds ratio, 6.6; 95% confidence interval, 1.7-30; P = .0081). These findings underscore the association between CH and inflammatory diseases. Our results have considerable relevance for managing patients with OA and AIDs or mild anemia and question the use of hip bone–derived cells as healthy experimental controls.

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR–based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR–based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

Background It was tested whether the difference in carcinogenesis between noxa and human papillomavirus (HPV)-driven head and neck squamous cell carcinoma (HNSCC) is associated with a variation in genomic instability. Methods Conventional and molecular cytogenetics in HPV-positive and HPV-negative HNSCC cell lines. Results Numerical aneuploidy determined by multicolor fluorescence in situ hybridization and DNA ploidy was very similar for both entities with most chromosomes being present either in quadruplicate or triplicate, and only few are still diploid with, however, a striking similarity in the overall pattern. A clear difference was seen concerning the translocations formed, with no difference in the total amount but with a significantly higher genomic instability of HPV-positive cell lines at chromosome 3 as compared to HPV-negative cells. Conclusion The different processes of carcinogenesis of HPV-positive and HPV-negative HNSCC appear to result in a similar pattern of numerical but a clear difference in structural chromosomal aberrations.

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR-based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR-based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

The PI3K/Akt/mTOR pathway is frequently altered in human papillomavirus (HPV)-positive and negative squamous cell carcinoma of the head and neck (HNSCC) and overstimulation is associated with poor prognosis. PI3K drives Akt activation and constitutive signaling acts pro-proliferative, supports cell survival, DNA repair, and contributes to radioresistance. Since the small molecule NVP-BEZ235 (BEZ235) is a potent dual inhibitor of this pathway, we were interested whether BEZ235 could be an efficient radiosensitizer. The 50 nM BEZ235 was found to abrogate endogenous and irradiation-induced phosphorylation of Akt (Ser473). The anti-proliferative capacity of the drug resulted in an increase in G1-phase cells. Repair of radiation-induced DNA double-strand breaks (DSBs) was strongly suppressed. Reduction in DSB repair was only apparent in G1- but not in G2-phase cells, suggesting that BEZ235 primarily affects non-homologous end joining. This finding was confirmed using a DSB repair reporter gene assay and could be attributed to an impaired phosphorylation of DNA-PKcs (S2056). Cellular radiosensitivity increased strongly after BEZ235 addition in all HNSCC cell lines used, especially when irradiated in the G0 or G1 phase. Our data indicate that targeting the PI3K/Akt/mTOR pathway by BEZ235 with concurrent radiotherapy may be considered an effective strategy for the treatment of HNSCC, regardless of the HPV and Akt status.

Cell-cell communication is essential in healthy bone marrow (BM) and seems to be disturbed in acute myeloid leukemia (AML). To understand whether the communication patterns are different in healthy and malignant BM, we need to perform a comparative analysis--a task which most algorithms published to date are not designed to do. We solved this issue with our new computational tool which analyses differential communication in two cohorts of multiple samples and allows us to do systematic studies of communication changes in large cohorts of individuals. Our algorithm works on single-cell RNA-seq or bulk RNA-seq data of individual FACS-sorted cell types. Here we use two published datasets of human bone marrow single-cell RNA-seq containing samples from AML patients at diagnosis, as well as healthy donors. We selected 9 AML samples from van Galen, 2019 by excluding samples collected later than day 0 after diagnosis, having less than 50% blasts at diagnosis, or having less than 5 cell types captured. As a control group, we used 29 samples from healthy donors (4 from van Galen, 2019 and 25 from Oetjen, 2018). To unify the cell type annotation, we merged the original cell type annotation into bigger classes: hematopoietic stem and progenitor cells (HSPCs), monocytes, erythrocytes, B cells, T cells, natural killer cells (NK), and dendritic cells (DC). Due to low capturing rate, we excluded NK from the subsequent analysis. After filtering out lowly expressed genes and cells with low number of reads, we normalised the data using scran package (Lun, 2016). We next proceeded to calculate the communication between the 6 cell types for each sample. A communication edge represents a signal which is sent by a "sending" cell type via its ligand and is received by a "receiving" cell type via its receptor. Each edge has a weight that indicates the intensity of communication and varies between 0 (no communication) and 1 (communication of maximum intensity). We calculate edge weights using a formula which addresses the proportion of the cells within each cell type that expressed the ligand/receptor, the relative level of expression of ligand/receptor in sending/receiving cell type compared to other cell types and the discrepancy of expression between the ligand in the sending cell type and the receptor in the receiving cell type. We used iTALK (Wang, 2019) database of ligand-receptor pair interactions as a reference. After calculating communication edges in all samples, we filter low-weight edges and proceed to differential communication analysis. Here we perform Wilcoxon-Mann-Whitney test with Bonferroni correction in the two groups of samples (AML vs healthy). We identified over 4.500 communication edges in the dataset, of which 58 were significantly differentially used (adjusted p-value < 0.1). Markedly, the majority of the communication edges, as well as all significantly differential edges were down-regulated in the AML samples, indicating overall decrease in communicative activity among the investigated cell types in AML patients at diagnosis. In the significantly differential edges -- which were actively used in the healthy bone marrow, but lost in the AML -- the two most active "sending" cell types were the erythrocytes (in 19 edges) and monocytes (in 17 edges), followed by the DC (in 9 edges), B cells (in 7 edges), T cells (in 4 edges), and HSCP (in 2 edges). The most active receiving cell types were the DC (in 24 edges) and the erythrocyte (in 17 edges), followed by the monocytes (in 8 edges), B cells (in 6 edges), HSCP (in 2 edges) and T cells (in 1 edge). The top 20 significantly differential edges showed communication from erythrocytes, monocytes, B cells and T cells to DC using CALM1/3 : SELL, as well as ADAM10 : AXL interaction, indicating reduced cell-cell adhesive contact among the immune cell types in the AML. In our study, we developed a computational tool which allows us to characterize differential cell-cell communication in two cohorts of patients using scRNA-seq data. We applied our tool to the published scRNA-seq data from the bone marrow of 8 AML patients and 29 healthy donors. We identified disease-driven loss in communication patterns in the AML such as disrupted adhesion among the immune cells, which might indicate immunoevasion. This analysis is crucial to generate new data driven hypotheses for a deeper understanding of haematopoietic neoplasms. Disclosures No relevant conflicts of interest to declare.

Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p < 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p < 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p < 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p < 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p < 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p < 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p < 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p < 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p < 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC > 1 or < -1; p < 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj < 0.0001) and protein secretion (NES=-1.49; p adj < 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.

At present, advanced stage human Papillomavirus (HPV) negative and positive head and neck squamous cell carcinoma (HNSCC) are treated by intense multimodal therapy that includes radiochemotherapy, which are associated with relevant side effects. Patients with HPV positive tumors possess a far better prognosis than those with HPV negative cancers. Therefore, new therapeutic strategies are needed to improve the outcome especially of the latter one as well as quality of life for all HNSCC patients. Here we tested whether roscovitine, an inhibitor of cyclin-dependent kinases (CDKs), which hereby also blocks homologous recombination (HR), can be used to enhance the radiation sensitivity of HNSCC cell lines. In all five HPV negative and HPV positive cell lines tested, roscovitine caused inhibition of CDK1 and 2. Surprisingly, all HPV positive cell lines were found to be defective in HR. In contrast, HPV negative strains demonstrated efficient HR, which was completely suppressed by roscovitine. In line with this, for HPV negative but not for HPV positive cell lines, treatment with roscovitine resulted in a pronounced enhancement of the radiation-induced G2 arrest as well as a significant increase in radiosensitivity. Due to a defect in HR, all HPV positive cell lines were efficiently radiosensitized by the PARP-1 inhibitor olaparib. In contrast, in HPV negative cell lines a significant radiosensitization by olaparib was only achieved when combined with roscovitine.

Der Fortbildungsschwerpunkt dieser Ausgabe ist Zufallsbefunden gewidmet, die u. a. auf onkologische Erkrankungen hindeuten konnen. Wie immer haben Experten die Thematik so aufbereitet, dass sie fur den Arzt in der niedergelassenen Praxis verstandlich und praxisrelevant ist.

Squamous cell carcinoma of the head and neck region (HNSCC), which is related to an infection with human papilloma virus (HPV), responds better to simultaneous radio-chemotherapy with Cisplatin based regimens than HPV-negative tumors. The underlying molecular mechanisms for this clinical observation are not fully understood. Therefore, the response of four HPV-positive (HPV+) (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (HPV-) (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) HNSCC cell lines to x-irradiation ± Cisplatin incubation in terms of clonogenic survival, cell cycle progression, protein expression (cyclin A2, cyclin E2, E6, E7, p53) and induction of apoptosis, was investigated. HPV+ cells were more radio- and chemosensitive and were more effectively sensitized to x-irradiation by simultaneous Cisplatin incubation than HPV- cell lines. HPV+ cell lines revealed an increased and prolonged G2/M arrest after irradiation, whereas Cisplatin induced a blockage of cells in S phase. In comparison to irradiation only, addition of Cisplatin significantly enhanced apoptosis especially in HPV+ cell lines. While irradiation alone increased the amount of HPV E6 and E7 proteins, both were down-regulated by Cisplatin incubation either alone or in combination with x-rays, which however did not increase the expression of endogenous p53. Our results demonstrate that cell cycle deregulation together with downregulation of HPV E6 and E7 proteins facilitating apoptosis after Cisplatin incubation promote the enhanced sensitivity of HPV+ HNSCC cells to simultaneous radio-chemotherapy. Combined effects of irradiation and Cisplatin appear to be relevant in mediating the enhanced therapeutic response of HPV-related HNSCC and are indicative of the benefit of combined modality approaches in future treatment optimization strategies.

Human Papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) respond favourably to radiotherapy as compared to HPV-unrelated HNSCC. We investigated DNA damage response in HPV-positive and HPV-negative HNSCC cell lines aiming to identify mechanisms, which illustrate reasons for the increased sensitivity of HPV-positive cancers of the oropharynx.Radiation response including clonogenic survival, apoptosis, DNA double-strand break (DSB) repair, and cell cycle redistribution in four HPV-positive (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) cell lines was evaluated.HPV-positive cells were more radiosensitive (mean SF2: 0.198 range: 0.22-0.18) than HPV-negative cells (mean SF2: 0.34, range: 0.45-0.27; p = 0.010). Irradiated HPV-positive cell lines progressed faster through S-phase showing a more distinct accumulation in G2/M. The abnormal cell cycle checkpoint activation was accompanied by a more pronounced increase of cell death after x-irradiation and a higher number of residual and unreleased DSBs.The enhanced responsiveness of HPV-related HNSCC to radiotherapy might be caused by a higher cellular radiosensitivity due to cell cycle dysregulation and impaired DNA DSB repair.