T-bet suppresses proliferation of malignant B cells in chronic lymphocytic leukemia.

ABSTRACT: The T-box transcription factor T-bet is known as a master regulator of the T-cell response but its role in malignant B cells has not been sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with a genetic knockout of Tbx21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity, induced by inflammatory signals provided by the microenvironment, triggered T-bet expression, which affected promoter-proximal and distal chromatin coaccessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling and negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of patients with CLL. Our study uncovered a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling, which has implications for the stratification and therapy of patients with CLL. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in the inflammatory signaling pathways in CLL.

[Primary small cell neuroendocrine carcinoma of the larynx: a review of literature and case series]. / Kleinzelliges neuroendokrines Karzinom des Kopf-Hals-Bereichs: eine Übersichtsarbeit und Fallserie.

INTRODUCTION: Small cell neuroendocrine carcinoma (SCNC) of the larynx is a rare tumor entity with a 5-year overall survival (OS) of only 5 % after treatment with chemoradiotherapy. METHODS: A systematic review of the literature was performed for "SCNC" and "SCNC in head and neck". Our hospital's own electronic patient file database was investigated for patients diagnosed with a SCNC over the last 12 years. RESULTS: The effectiveness of chemoradiotherapy in SCNC is still unclear since randomized clinical trials are missing for the evaluation of standard of care treatment. Common therapy approaches are based on experiences with small cell lung cancer. 0.5 % of all SCNC occur in the head and neck region. In the last 12 years, we diagnosed 9 patients with SCNC, two of which were located in the larynx. Exemplarily, we report the case of a 29-year-old male with the initial diagnosis of a SCNC of the larynx with concurrent lymph node metastasis. This case is particularly interesting due to the young age at disease onset and the lack of major risk factors. Treatment was modified to nivolumab due to progressive disease after treatment with chemoradiotherapy. After an OS of 22 months, the patient deceased due to a tumor-associated major bleeding with airway obstruction. CONCLUSION: So far there are no clinical reports evaluating the use of nivolumab in third-line-therapy of SCNC. NTRK fusion (neurotrophic tyrosine receptor kinase gene fusion) or the folate receptor expression analysis should be considered to evaluate the potential use of a tropomyosin receptor kinase inhibitor or a folate receptor targeting therapy.

INHBA is Enriched in HPV-negative Oropharyngeal Squamous Cell Carcinoma and Promotes Cancer Progression.

Patients with oropharyngeal squamous cell carcinoma (OPSCC) caused by human papilloma virus (HPV) exhibit a better prognosis than those with HPV-negative OPSCC. This study investigated the distinct molecular pathways that delineate HPV-negative from HPV-positive OPSCC to identify biologically relevant therapeutic targets. Bulk mRNA from 23 HPV-negative and 39 HPV-positive OPSCC tumors (n = 62) was sequenced to uncover the transcriptomic profiles. Differential expression followed by gene set enrichment analysis was performed to outline the top enriched biological process in the HPV-negative compared with HPV-positive entity. INHBA, the highest overexpressed gene in the HPV-negative tumor, was knocked down. Functional assays (migration, proliferation, cell death, stemness) were conducted to confirm the target's oncogenic role. Correlation analyses to reveal its impact on the tumor microenvironment were performed. We revealed that epithelial-to-mesenchymal transition (EMT) is the most enriched process in HPV-negative compared with HPV-positive OPSCC, with INHBA (inhibin beta A subunit) being the top upregulated gene. INHBA knockdown downregulated the expression of EMT transcription factors and attenuated migration, proliferation, stemness, and cell death resistance of OPSCC cells. We uncovered that INHBA associates with a pro-tumor microenvironment by negatively correlating with antitumor CD8+ T and B cells while positively correlating with pro-tumor M1 macrophages. We identified three miRNAs that are putatively involved in repressing INHBA expression. Our results indicate that the upregulation of INHBA is tumor-promoting. We propose INHBA as an attractive therapeutic target for the treatment of INHBA-enriched tumors in patients with HPV-negative OPSCC to ameliorate prognosis. SIGNIFICANCE: Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC.

Multi-omics analysis of overexpressed tumor-associated proteins: gene expression, immunopeptide presentation, and antibody response in oropharyngeal squamous cell carcinoma, with a focus on cancer-testis antigens.

Introduction: The human leukocyte antigen complex (HLA) is essential for inducing specific immune responses to cancer by presenting tumor-associated peptides (TAP) to T cells. Overexpressed tumor associated antigens, mainly cancer-testis antigens (CTA), are outlined as essential targets for immunotherapy in oropharyngeal squamous cell carcinoma (OPSCC). This study assessed the degree to which presentation, gene expression, and antibody response (AR) of TAP, mainly CTA, are correlated in OPSCC patients to evaluate their potential as immunotherapy targets. Materials and methods: Snap-frozen tumor (NLigand/RNA=40), healthy mucosa (NRNA=6), and healthy tonsils (NLigand=5) samples were obtained. RNA-Seq was performed using Illumina HiSeq 2500/NovaSeq 6000 and whole exome sequencing (WES) utilizing NextSeq500. HLA ligands were isolated from tumor tissue using immunoaffinity purification, UHPLC, and analyzed by tandem MS. Antibodies were measured in serum (NAb=27) utilizing the KREX™ CT262 protein array. Data analysis focused on 312 proteins (KREX™ CT262 panel + overexpressed self-proteins). Results: 183 and 94 of HLA class I and II TAP were identified by comparative profiling with healthy tonsils. Genes from 26 TAP were overexpressed in tumors compared to healthy mucosa (LFC>1; FDR<0.05). Low concordance (r=0.25; p<0.0001) was found between upregulated mRNA and class I TAP. The specific mode of correlation of TAP was found to be dependent on clinical parameters. A lack of correlation was observed both between mRNA and class II TAP, as well as between class II tumor-unique TAP (TAP-U) presentation and antibody response (AR) levels. Discussion: This study demonstrates that focusing exclusively on gene transcript levels fails to capture the full extent of TAP presentation in OPSCC. Furthermore, our findings reveal that although CTA are presented at relatively low levels, a few CTA TAP-U show potential as targets for immunotherapy.

Heterogeneous expression of predictive biomarkers PD-L1 and TIGIT in non-mucinous lung adenocarcinoma and corresponding lymph node metastasis: A challenge for clinical biomarker testing.

The use of immune checkpoint inhibitors (ICI) targeting the PD-L1:PD1 interaction revolutionized tumor treatment by re-activating the anti-tumoral capacity of the immune system. Assessment of tumor mutational burden, microsatellite instability, or expression of the surface marker PD-L1 have been used to predict individual response to ICI therapy. However, the predicted response does not always correspond to the actual therapy outcome. We hypothesize that tumor heterogeneity might be a major cause of this inconsistency. In this respect we recently demonstrated that PD-L1 shows heterogenous expression in the different growth patterns of non-small cell lung cancer (NSCLC) - lepidic, acinar, papillary, micropapillary and solid. Furthermore, additional inhibitory receptors, like T cell immunoglobulin and ITIM domain (TIGIT), appear to be heterogeneously expressed and affect the outcome of anti-PD-L1 treatment. Given this heterogeneity in the primary tumor, we set out to analyze the situation in corresponding lymph node metastases, since these are often used to obtain biopsy material for tumor diagnosis, staging and molecular analysis. Again, we observed heterogeneous expression of PD-1, PD-L1, TIGIT, Nectin-2 and PVR in relation to different regions and growth pattern distribution that varied between the primary tumor and their metastases. Together, our study underscores the complex situation regarding the heterogeneity of NSCLC samples and suggest that the analysis of a small biopsy from lymph node metastases may not be sufficient to ensure a reliable prediction of ICI therapy success.

The Small GTPase RHOA Links SLP65 Activation to PTEN Function in Pre B Cells and Is Essential for the Generation and Survival of Normal and Malignant B Cells.

The generation, differentiation, survival and activation of B cells are coordinated by signals emerging from the B cell antigen receptor (BCR) or its precursor, the pre-BCR. The adaptor protein SLP65 (also known as BLNK) is an important signaling factor that controls pre-B cell differentiation by down-regulation of PI3K signaling. Here, we investigated the mechanism by which SLP65 interferes with PI3K signaling. We found that SLP65 induces the activity of the small GTPase RHOA, which activates PTEN, a negative regulator of PI3K signaling, by enabling its translocation to the plasma membrane. The essential role of RHOA is confirmed by the complete block in early B cell development in conditional RhoA-deficient mice. The RhoA-deficient progenitor B cells showed defects in activation of immunoglobulin gene rearrangement and fail to survive both in vitro and in vivo. Reconstituting the RhoA-deficient cells with RhoA or Foxo1, a transcription factor repressed by PI3K signaling and activated by PTEN, completely restores the survival defect. However, the defect in differentiation can only be restored by RhoA suggesting a unique role for RHOA in B cell generation and selection. In full agreement, conditional RhoA-deficient mice develop increased amounts of autoreactive antibodies with age. RHOA function is also required at later stage, as inactivation of RhoA in peripheral B cells or in a transformed mature B cell line resulted in cell loss. Together, these data show that RHOA is the key signaling factor for B cell development and function by providing a crucial SLP65-activated link between BCR signaling and activation of PTEN. Moreover, the identified essential role of RHOA for the survival of transformed B cells offers the opportunity for targeting B cell malignancies by blocking RHOA function.

Usefulness of BATF3 Immunohistochemistry in Diagnosing Classical Hodgkin Lymphoma.

It is well recognized that the AP-1 transcription factor BATF3 is constitutively expressed in Hodgkin/Reed-Sternberg (HRS) cells, but its potential as a diagnostic marker for classical Hodgkin lymphoma (cHL) has not yet been addressed. In this study, we performed immunohistochemistry and analyzed the BATF3 expression in lymphoma cells on 218 lymphoma samples belonging to 14 different lymphoma entities. We observed varying degrees of BATF3 expression in nearly half of the cases (n = 100) with BATF3 expression being a constitutive feature of cHL (n = 53) and anaplastic large cell lymphoma (ALCL). By scoring BATF3 expression (BATF3-score) we observed constitutively high BATF3-scores in cHL and ALCL and low to moderate BATF3-scores in all other entities examined. Western blot analysis confirmed BATF3 protein expression in cell lysates from cHL cell lines (n = 7). Thus, BATF3 can be considered a useful IHC marker for the diagnosis of cHL as it is highly sensitive and sufficiently specific when analyzed by BATF3-scoring.

Age-related activity of Poly (ADP-Ribose) Polymerase (PARP) in men with localized prostate cancer.

Mutations in DNA repair genes have been connected with familial prostate cancer and sensitivity to targeted drugs like PARP-inhibitors. Clinical use of this information is limited by the small fraction of prostate cancer risk gene carriers, variants of unknown pathogenicity and the focus on monogenic disease mechanisms. Functional assays capturing mono- and polygenic defects were shown to detect breast and ovarian cancer risk in blood-derived cells. Here, we comparatively analyzed lymphocytes from prostate cancer patients and controls applying a sensitive DNA double-strand break (DSB) repair assay and a flow cytometrybased assay measuring the activity of Poly(ADP-Ribose)-Polymerase, a target in treatment of metastatic prostate cancer. Contrary to breast and ovarian cancer patients, error-prone DNA double-strand break repair was not activated in prostate cancer patients. Yet, the activity of PARP discriminated between prostate cancer cases and controls. PARylation also correlated with the age of male probands, suggesting male-specific links between mutation-based and aging-associated DNA damage accumulation and PARP. Our work identifies prostate cancer-specific DNA repair phenotypes characterized by increased PARP activities and carboplatin-sensitivities, detected by functional testing of lymphocytes. This provides new insights for further investigation of PARP and carboplatin sensitivity as biomarkers in peripheral cells of men and prostate cancer patients.

U-DCS: characterization of the first permanent human dendritic sarcoma cell line.

A dendritic cell sarcoma cell line, U-DCS, was established from a dendritic cell sarcoma in a 53-year-old Caucasian male patient. Since its establishment, U-DCS has maintained stable phenotypic characteristics in vitro and has a doubling time of approximately 2 days under standard culture conditions. U-DCS is growing with typical dendritic cell morphology in tissue and expresses the dendritic cell sarcoma immunophenotypic markers S100 protein, MHCI, MHCII, and vimentin. Expression analysis revealed transcripts for the toll-like receptors TLR3, -4, -9 and DDX58 (RIG-I), but not for TLR2. U-DCS shows functional features of dendritic cells with the ability of phagocytosis and antigen-specific T cell stimulation. Karyotype-, CGH-, and mFISH analysis point to a chromosomal instability and a hypotetraploid karyotype with approximately 130 chromosomes. U-DCS is the first immortalized human dendritic cell sarcoma cell line and has some morphological and functional features of dendritic cells without dependency on growth factors.

Amyloid precursor protein-fragments-containing inclusions in cardiomyocytes with basophilic degeneration and its association with cerebral amyloid angiopathy and myocardial fibrosis.

Cardiomyopathies with intracellular inclusions are a distinct subset of cardiomyopathies whereas basophilic degeneration (BD) of the heart describes inclusions in cardiomyocytes of the aging heart, which have not yet been related to a specific disease condition or to a distinct type of protein inclusion. To address the question whether BD represents a specific pathological feature and whether it is linked to a distinct disease condition we studied 62 autopsy cases. BD inclusions exhibited an immunohistochemical staining pattern related to glycosylated, δ- or η-secretase-derived N-terminal cleavage products of the amyloid precursor protein (sAPPδ/η) or shorter fragments of sAPPη. BD aggregates were found in the myocardium of both ventricles and atria with highest amounts in the atria and lowest in the interventricular septum. The frequency of BD-lesions correlated with age, degree of myocardial fibrosis in individuals with arterial hypertension, and the severity of cerebral amyloid angiopathy (CAA). The intracytoplasmic deposition of N-terminal sAPPδ/η fragments in BD indicates a specific inclusion body pathology related to APP metabolism. The correlation with the severity of CAA, which is related to the APP-derived amyloid ß-protein, supports this point of view and suggests a possible link between myocardial and cerebrovascular APP-related lesions.