Exploring the relationship between epigenetic DNA methylation and cardiac fibrosis through Raman microspectroscopy.

Cardiomyopathies are associated with fibrotic remodeling of the heart, which is characterized by the excessive accumulation of collagen type I (COL I) due to chronic inflammation and suspected epigenetic influences. Despite the severity and high mortality rate of cardiac fibrosis, current treatment options are often inadequate, underscoring the importance of gaining a deeper understanding of the disease's underlying molecular and cellular mechanisms. In this study, the extracellular matrix (ECM) and nuclei in fibrotic areas of different cardiomyopathies were molecularly characterized by Raman microspectroscopy and imaging and compared with the control myocardium. Patient samples were obtained from heart tissue affected by ischemia, hypertrophy, and dilated cardiomyopathy and analyzed for fibrosis through conventional histology and marker-independent Raman microspectroscopy (RMS). Prominent differences between control myocardium and cardiomyopathies were revealed by spectral deconvolution of COL I Raman spectra. Statistically significant differences were identified in the amide I region of spectral subpeak at 1,608 cm-1, which is a representative endogenous marker for alterations in the structural conformation of COL I fibers. Moreover, epigenetic 5mC DNA modification was identified within cell nuclei by multivariate analysis. A statistically significant increase in signal intensities of spectral features indicative of DNA methylation was detected in cardiomyopathies in accordance with immunofluorescence 5mC staining. Overall, RMS is a versatile technology in the discrimination of cardiomyopathies based on molecular evaluation of COL I and nuclei while providing insights into the pathogenesis of the diseases.NEW & NOTEWORTHY Cardiomyopathies are associated with severe fibrotic remodeling of the heart, which is characterized by the excessive accumulation of collagen type I (COL I). In this study, we used marker-independent Raman microspectroscopy (RMS) to gain a deeper understanding of the disease's underlying molecular and cellular mechanisms.

Patient-derived xenograft models of ALK+ ALCL reveal preclinical promise for therapy with brigatinib.

Anaplastic large-cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by the oncogenic anaplastic lymphoma kinase (ALK), accounting for approximately 15% of all paediatric non-Hodgkin lymphoma. Patients with central nervous system (CNS) relapse are particularly difficult to treat with a 3-year overall survival of 49% and a median survival of 23.5 months. The second-generation ALK inhibitor brigatinib shows superior penetration of the blood-brain barrier unlike the first-generation drug crizotinib and has shown promising results in ALK+ non-small-cell lung cancer. However, the benefits of brigatinib in treating aggressive paediatric ALK+ ALCL are largely unknown. We established a patient-derived xenograft (PDX) resource from ALK+ ALCL patients at or before CNS relapse serving as models to facilitate the development of future therapies. We show in vivo that brigatinib is effective in inducing the remission of PDX models of crizotinib-resistant (ALK C1156Y, TP53 loss) ALCL and furthermore that it is superior to crizotinib as a second-line approach to the treatment of a standard chemotherapy relapsed/refractory ALCL PDX pointing to brigatinib as a future therapeutic option.

Raman microspectroscopy identifies fibrotic tissues in collagen-related disorders via deconvoluted collagen type I spectra.

Fibrosis is a consequence of the pathological remodeling of extracellular matrix (ECM) structures in the connective tissue of an organ. It is often caused by chronic inflammation, which over time, progressively leads to an excess deposition of collagen type I (COL I) that replaces healthy tissue structures, in many cases leaving a stiff scar. Increasing fibrosis can lead to organ failure and death; therefore, developing methods that potentially allow real-time monitoring of early onset or progression of fibrosis are highly valuable. In this study, the ECM structures of diseased and healthy human tissue from multiple organs were investigated for the presence of fibrosis using routine histology and marker-independent Raman microspectroscopy and Raman imaging. Spectral deconvolution of COL I Raman spectra allowed the discrimination of fibrotic and non-fibrotic COL I fibers. Statistically significant differences were identified in the amide I region of the spectral subpeak at 1608 cm-1, which was deemed to be representative for structural changes in COL I fibers in all examined fibrotic tissues. Raman spectroscopy-based methods in combination with this newly discovered spectroscopic biomarker potentially offer a diagnostic approach to non-invasively track and monitor the progression of fibrosis. STATEMENT OF SIGNIFICANCE: Current diagnosis of fibrosis still relies on histopathological examination with invasive biopsy procedures. Although, several non-invasive imaging techniques such as positron emission tomography, single-photon emission computed tomography and second harmonic generation are gradually employed in preclinical or clinical studies, these techniques are limited in spatial resolution and the morphological interpretation highly relies on individual experience and knowledge. In this study, we propose a non-destructive technique, Raman microspectroscopy, to discriminate fibrotic changes of collagen type I based on a molecular biomarker. The changes of the secondary structure of collagen type I can be identified by spectral deconvolution, which potentially can provide an automatic diagnosis for fibrotic tissues in the clinical applicaion.

Multiparametric Classification of Non-Muscle Invasive Papillary Urothelial Neoplasms: Combining Morphological, Phenotypical, and Molecular Features for Improved Risk Stratification.

Diagnosis and grading of non-invasive papillary urothelial tumors according to the current WHO classification poses some challenges for pathologists. The diagnostic reproducibility of separating low-grade and high-grade lesions is low, which impacts their clinical management. Whereas papillary urothelial neoplasms with low malignant potential (PUN-LMP) and low-grade papillary non-invasive carcinoma (LG-PUC) are comparable and show frequent local recurrence but rarely metastasize, high-grade papillary non-invasive carcinoma (HG-PUC) has a poor prognosis. The main objective of this work is to develop a multiparametric classification to unambiguously distinguish low-grade and high-grade tumors, considering immunohistochemical stains for p53, FGFR3, CK20, MIB-1, p16, p21 and p-HH3, and pathogenic mutations in TP53, FGFR3, TP53, ERCC2, PIK3CA, PTEN and STAG2. We reviewed and analyzed the clinical and histological data of 45 patients with a consensus diagnosis of PUN-LMP (n = 8), non-invasive LG-PUC (n = 23), and HG-PUC (n = 14). The proliferation index and mitotic count assessed with MIB-1 and P-HH3 staining, respectively correlated with grading and clinical behavior. Targeted sequencing confirmed frequent FGFR3 mutations in non-invasive papillary tumors and identified mutations in TP53 as high-risk. Cluster analysis of the different immunohistochemical and molecular parameters allowed a clear separation in two different clusters: cluster 1 corresponding to PUN-LMP and LG-PUC (low MIB-1 and mitotic count/FGFR3 and STAG2 mutations) and cluster 2, HG-PUC (high MIB-1 and mitosis count/CK20 +++ expression, FGFR3 WT and TP53 mutation). Further analysis is required to validate and analyze the reproducibility of these clusters and their biological and clinical implication.

Elevated Expression of the Immune Checkpoint Ligand CD276 (B7-H3) in Urothelial Carcinoma Cell Lines Correlates Negatively with the Cell Proliferation.

The cell surface molecule CD276 (B7-H3) is an immune checkpoint antigen. The elevated expression of CD276 on tumors contributes to the suppression of anti-tumor T-cell responses and correlates with poor prognosis. METHODS: The expression of CD276 was explored in vitro on eight urothelial carcinoma cell lines (UM-UC) in comparison to eight normal urothelial cells (NUCs) by RT-qPCR, Western blotting, and flow cytometry. Cell proliferation was enumerated over consecutive passages. The expression of cancer stem cell markers CD24 and CD44, cytokeratins, and vimentin was investigated by immunofluorescence. The expression of CD276 in bladder tumor samples and metastases was explored by immunohistochemistry. RESULTS: Expression of CD276 on cell surfaces was elevated on UM-UCs when compared to NUCs. In UM-UCs, CD276 transcripts correlated moderately positive with CD276 protein expression (ρ = 0.660) and strongly positive with CD276 surface-expression (ρ = 0.810). CD276 mRNA expression (ρ = -0.475) and CD276 protein expression (ρ = -0.417) had a significant negative correlation with proliferation, while a significant correlation between proliferation and cell surface expression was not observed in UM-UCs. CONCLUSION: The expression of CD276 on UM-UC bladder tumor cell surfaces is elevated. Slow proliferating UM-UC cells express more CD276 mRNA and protein than fast proliferating cells. In patients, slow proliferating CD276high tumor (stem) cells may evade immune surveillance. However, cancer therapy targeting CD276 may be effective in the treatment of slow proliferating tumor cells.

CD24: A Marker for an Extended Expansion Potential of Urothelial Cancer Cell Organoids In Vitro?

BACKGROUND: Bladder cancer is the most cost-intensive cancer due to high recurrence rates and long follow-up times. Bladder cancer organoids were considered interesting tools for investigating better methods for the detection and treatment of this cancer. METHODS: Organoids were generated from urothelial carcinoma tissue samples, then expanded and characterized; the expression of immune modulatory antigens and tumor stem cells markers CD24 and CD44 was explored in early (P ≤ 3) and later (P ≥ 5) passages (P) by immunofluorescence and by quantitative PCR of cDNA. The expression of these factors was investigated in the corresponding cancer tissue samples by immunohistochemistry. RESULTS: The expression of the PD-L1 was detected on some but not all organoids. CD276 and CD47 were observed on organoids in all passages investigated. Organoids growing beyond passage 8 expressed both CD24 and CD44 at elevated levels in early and late cultures. Organoids proliferating to the eighth passage initially expressed both CD24 and CD44, but lost CD24 expression over time, while CD44 remained. Organoids growing only up to the 6th passage failed to express CD24 but expressed CD44. CONCLUSIONS: The data indicate that the expression of CD24 in urothelial cancer cell organoids may serve as an indicator for the prolonged proliferation potential of the cells.

Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL.

EBV and the Pathogenesis of NK/T Cell Lymphoma.

Epstein-Barr virus (EBV) is a ubiquitous gamma herpes virus with tropism for B cells. EBV is linked to the pathogenesis of B cell, T cell and NK cell lymphoproliferations, with extranodal NK/T cell lymphoma, nasal type (ENKTCL) being the prototype of an EBV-driven lymphoma. ENKTCL is an aggressive neoplasm, particularly widespread in East Asia and the native population of Latin America, which suggests a strong genetic predisposition. The link between ENKTCL and different populations has been partially explored. EBV genome sequencing analysis recognized two types of strains and identified variants of the latent membrane protein 1 (LMP1), which revealed different oncogenic potential. In general, most ENKTCL patients carry EBV type A with LMP1 wild type, although the LMP1 variant with a 30 base pair deletion is also common, especially in the EBV type B, where it is necessary for oncogenic transformation. Contemporary high-throughput mutational analyses have discovered recurrent gene mutations leading to activation of the JAK-STAT pathway, and mutations in other genes such as BCOR, DDX3X and TP53. The genomic landscape in ENKTCL highlights mechanisms of lymphomagenesis, such as immune response evasion, secondary to alterations in signaling pathways or epigenetics that directly or indirectly interfere with oncogenes or tumor suppressor genes. This overview discusses the most important findings of EBV pathogenesis and genetics in ENKTCL.

Genetic evolution of in situ follicular neoplasia to aggressive B-cell lymphoma of germinal center subtype.

In situ follicular neoplasia (ISFN) is the earliest morphologically identifiable precursor of follicular lymphoma (FL). Although it is genetically less complex than FL and has low risk for progression, ISFN already harbors secondary genetic alterations, in addition to the defining t(14;18)(q32;q21) translocation. FL, in turn, frequently progresses to diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (HGBL). By BCL2 staining of available reactive lymphoid tissue obtained at any time point in patients with aggressive B-cell lymphoma (BCL), we identified ten paired cases of ISFN and DLBCL/HGBL, including six de novo tumors and four tumors transformed from FL as an intermediate step, and investigated their clonal evolution using microdissection and next-generation sequencing. A clonal relationship between ISFN and aggressive BCL was established by immunoglobulin and/or BCL2 rearrangements and/or the demonstration of shared somatic mutations for all ten cases. Targeted sequencing revealed CREBBP, KMT2D, EZH2, TNFRSF14 and BCL2 as the genes most frequently mutated already in ISFN. Based on the distribution of private and shared mutations, two patterns of clonal evolution were evident. In most cases, the aggressive lymphoma, ISFN and, when present, FL revealed divergent evolution from a common progenitor, whereas linear evolution with sequential accumulation of mutations was less frequent. In conclusion, we demonstrate for the first time that t(14;18)+ aggressive BCL can arise from ISFN without clinically evident FL as an intermediate step and that during this progression, branched evolution is common.

IL10RA modulates crizotinib sensitivity in NPM1-ALK+ anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive anaplastic lymphoma kinase (ALK) fusion protein. ALK inhibitors, such as crizotinib, provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by nucleophosmin 1 (NPM1)-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54% to 90% in clinical trials; however, a subset of patients progressed within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) activation and knockout screens in ALCL cell lines, combined with RNA sequencing data derived from ALK inhibitor-relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of interleukin 10 receptor subunit alpha (IL10RA). Elevated IL10RA expression rewires the STAT3 signaling pathway, bypassing otherwise critical phosphorylation by NPM1-ALK. IL-10RA expression does not correlate with response to standard chemotherapy in pediatric patients, suggesting that a combination of crizotinib and chemotherapy could prevent ALK inhibitor resistance-specific relapse.

Epstein - Barr virus positive T and NK-cell lymphoproliferations: Morphological features and differential diagnosis.

The spectrum of Epstein-Barr virus (EBV)-positive T and NK-cell lymphoproliferations is broad and ranges from reactive self-limited disorders to neoplastic processes with a fulminant clinical course. EBV plays an important role promoting lymphomagenesis, although the precise mechanisms remain elusive. EBV-positive lymphoproliferative disorders (LPD) are more common in East Asia (China, Japan, Korea and Taiwan), and Latin America suggesting a strong genetic predisposition. The revised 2016 World Health Organization (WHO) lymphoma classification recognizes the following malignant NK- and T-cell lymphomas; extranodal NK/T-cell lymphoma, nasal type (ENKTCL), aggressive NK-cell leukemia (ANKL), and the provisional entity within the group of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) "primary EBV-positive nodal T or NK cell lymphoma". Disorders presenting mainly in children and young adults include chronic active EBV infection (CAEBV) - systemic and cutaneous forms - which are not considered malignant disorders but were included in the WHO classification for the first time because of the differential diagnosis with other T- or NK-cell lymphomas. CAEBV, cutaneous form, includes hydroa vacciniforme-like LPD (HV-LPD) and severe mosquito bite allergy (SMBA). Finally, systemic EBV-positive T-cell lymphoma of childhood was recognized as lymphoma because of its fulminant clinical course. Given the shared pathogenesis of these disorders, overlapping features are common demanding a close clinical, morphological and molecular correlation for an accurate diagnosis. This review summarizes the clinical, histopathological and molecular features of EBV-associated T and NK-cell LPD, highlighting the main features that might aid in the differential diagnosis.

Mutational profile and EBV strains of extranodal NK/T-cell lymphoma, nasal type in Latin America.

Extranodal NK/T-cell lymphoma (ENKTL) is an Epstein-Barr virus (EBV) associated lymphoma, prevalent in Asia and Latin America. Studies in Asian cohorts have identified some recurrent gene mutations in ENKTL; however, the mutational landscape of ENKTL in Latin America is unknown. In this study, we investigated the mutational profile and EBV strains of 71 ENKTL cases from Latin America (42 from Mexico, 17 from Peru, and 12 from Argentina) and compared it with Asian cohorts. The mutational analysis was performed by next generation sequencing (NGS) using an Ion AmpliSeq™ custom panel covering for the most frequently mutated genes identified in ENKTL. STAT3 was the most frequent mutated gene (16 cases: 23%), followed by MSN (10 cases; 14%), BCOR (9 cases; 13%), DDX3X (6 cases; 8%), TP53 (6 cases; 8%), MGA (3 cases; 4%), JAK3 (2 cases; 3%), and STAT5B (1 case; 1%). Mutations in STAT3, BCOR, and DDX3X were nearly mutually exclusive, suggesting different molecular pathways involved in the pathogenesis of ENKTL; whereas mutations in MGA, MSN, and TP53 were concomitant with other mutations. Most cases (75%) carried Type A EBV without the 30-bp LMP1 gene deletion. The overall survival was significantly associated with serum LDH level, Eastern Cooperative Oncology Group (ECOG) performance status, International Prognostic Index (IPI) score, and therapy (p < 0.05), but not associated with any mutation, EBV strain or deletion in EBV LMP1 gene. In conclusion, mutational analysis of ENKTL from Latin America reveals frequent gene mutations leading to activation of the JAK-STAT pathway (25%), mostly STAT3. Compared to Asian cohorts, BCOR, DDX3X and TP53 mutations were also identified but with different frequencies. None of these mutations were associated with prognosis.

Epstein-Barr Virus-Associated T and NK-Cell Lymphoproliferative Diseases.

EBV-associated T and NK-cell lymphoproliferative diseases (EBV-T/NK LPDs) are characterized by the transformation and proliferation of EBV-infected T or NK cells. The 2016 revised World Health Organization classification recognizes the following EBV-positive lymphoproliferative disorders (LPD): chronic active EBV infection (CAEBV) of T- and NK-cell type (cutaneous and systemic forms), systemic EBV-positive T-cell lymphoma of childhood, aggressive NK-cell leukemia, extranodal NK/T-cell lymphoma, nasal type, and the new provisional entity primary EBV-positive nodal T/NK-cell lymphoma. EBV-associated hemophagocytic lymphohistiocytosis (HLH), although not included in the WHO classification because it is a reactive, inflammatory disease, is included in this review because it can be life-threatening and may have overlapping features with other EBV+ T/NK LPDs. EBV+ T/NK LPDs are rare diseases difficult to diagnose and manage properly, because some LPDs have unusual presentations, and discrepancies between clinical and histological findings might be encountered. Furthermore, EBV+ T/NK disorders share some clinico-pathological features, and may evolve into other categories during the clinical course, including malignant transformation of CAEBV. Here, we review the EBV+ T/NK LPDs in terms of their definitions, clinical features, histology, immunophenotype, molecular findings, and pathogenesis. This review aims to increase our understanding and awareness of the differential diagnosis among the different EBV+ T/NK LPDs. New insights into the genetic characteristics of these disorders will also be discussed.

The Pathological Spectrum of Systemic Anaplastic Large Cell Lymphoma (ALCL).

Anaplastic large cell lymphoma (ALCL) represents a group of malignant T-cell lymphoproliferations that share morphological and immunophenotypical features, namely strong CD30 expression and variable loss of T-cell markers, but differ in clinical presentation and prognosis. The recognition of anaplastic lymphoma kinase (ALK) fusion proteins as a result of chromosomal translocations or inversions was the starting point for the distinction of different subgroups of ALCL. According to their distinct clinical settings and molecular findings, the 2016 revised World Health Organization (WHO) classification recognizes four different entities: systemic ALK-positive ALCL (ALK+ ALCL), systemic ALK-negative ALCL (ALK− ALCL), primary cutaneous ALCL (pC-ALCL), and breast implant-associated ALCL (BI-ALCL), the latter included as a provisional entity. ALK is rearranged in approximately 80% of systemic ALCL cases with one of its partner genes, most commonly NPM1, and is associated with favorable prognosis, whereas systemic ALK− ALCL shows heterogeneous clinical, phenotypical, and genetic features, underlining the different oncogenesis between these two entities. Recognition of the pathological spectrum of ALCL is crucial to understand its pathogenesis and its boundaries with other entities. In this review, we will focus on the morphological, immunophenotypical, and molecular features of systemic ALK+ and ALK− ALCL. In addition, BI-ALCL will be discussed.

EMMPRIN (CD147) is induced by C/EBPß and is differentially expressed in ALK+ and ALK- anaplastic large-cell lymphoma.

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPß, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPß for the unusual phenotype of ALK+ ALCL cells, C/EBPß was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPß knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPß knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPß in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPß plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL.