From antibodies to diagnosis: FOXP3 and PD-1 markers of human lymphoid malignancies

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 17-12-201

Artificial Intelligence Predicted Overall Survival and Classified Mature B-Cell Neoplasms Based on Immuno-Oncology and Immune Checkpoint Panels

Artificial intelligence (AI) can identify actionable oncology biomarkers. This research integrates our previous analyses of non-Hodgkin lymphoma. We used gene expression and immunohistochemical data, focusing on the immune checkpoint, and added a new analysis of macrophages, including 3D rendering. The AI comprised machine learning (C5, Bayesian network, C&R, CHAID, discriminant analysis, KNN, logistic regression, LSVM, Quest, random forest, random trees, SVM, tree-AS, and XGBoost linear and tree) and artificial neural networks (multilayer perceptron and radial basis function). The series included chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, Burkitt, diffuse large B-cell lymphoma, marginal zone lymphoma, and multiple myeloma, as well as acute myeloid leukemia and pan-cancer series. AI classified lymphoma subtypes and predicted overall survival accurately. Oncogenes and tumor suppressor genes were highlighted (MYC, BCL2, and TP53), along with immune microenvironment markers of tumor-associated macrophages (M2-like TAMs), T-cells and regulatory T lymphocytes (Tregs) (CD68, CD163, MARCO, CSF1R, CSF1, PD-L1/CD274, SIRPA, CD85A/LILRB3, CD47, IL10, TNFRSF14/HVEM, TNFAIP8, IKAROS, STAT3, NFKB, MAPK, PD-1/PDCD1, BTLA, and FOXP3), apoptosis (BCL2, CASP3, CASP8, PARP, and pathway-related MDM2, E2F1, CDK6, MYB, and LMO2), and metabolism (ENO3, GGA3). In conclusion, AI with immuno-oncology markers is a powerful predictive tool. Additionally, a review of recent literature was made

TOX Expression in Mycosis Fungoides and Sezary Syndrome.

Mycosis fungoides (MF) and Sezary syndrome (SS) are the two most common type of cutaneous T-cell lymphoma (CTCL). Currently, no markers can be clearly related to prognosis or to differential diagnosis between early stages and inflammatory benign diseases (IBD). The thymocyte selection-associated high mobility group box factor (TOX), has been proposed as a possible marker in differential diagnosis between early CTCL stages and IBD. Recently TOX has been related to prognosis. We aimed to investigate whether TOX may be a diagnostic or prognostic marker. MF and SS biopsies between 2010 and 2020 were retrieved. New tissues slides were stained with an anti-TOX antibody, (Clone NAN448B). On each slide, 5 fields were examined at high magnification (400×), to evaluate the percentage of marker-positivity in a quantitative way. Thirty-six patients (12 females and 24 males) and 48 biopsies were collected. Nine patients had multiple biopsies. TOX expression in MF/SS cases showed an increase from early to advanced phases. TOX was not regarded as a prognostic marker due to the absence of significant changes by comparing early MF cases with reactive conditions. TOX statistical significance increased in patients alive with disease and in those dead of disease (p = 0.013 and = 0.0005, respectively) as compared with patients in complete remission. Our results show that TOX should be regarded more as a prognostic than a diagnostic marker.This research received no external fundingS

Expression of clec9a in the oral cancer microenvironment. A preliminary immunohistochemical pilot study

The search for treatments to improve cancer survival has led to the emergence of immunotherapy and the study of the tumour microenvironment existing in neoplasms. This preliminary study aims to understand the clinical and pathological relationship of clec9a expression in oral cancer and to explore survival models for future studies. Material and methods: Immunohistochemical study that included 26 patients with a diagnosis of oral squamous cell carcinoma (OSCC) in mobile tongue and floor of the mouth. Clinical and histopathological variables were recorded, and the biomarkers clec9a for dendritic cells and CD8 and CD4 for lymphocytes were used. Clec9a was expressed in 58% of the sample. It was more common in cases with low lymphoplasmacytic infiltration and in type 2 invasion patterns. It was significantly related to CD8 expression (p=0.055 and p=0.007). No prognostic risks were evident in the survival models studied (overall survival, disease-specific survival, disease-free survival). CLEC9A expression is present in the OSCC microenvironment and is mainly related to the presence of CD8 lymphocytes. The relationship of its expression with survival prognosis in OSCC could not be confirmed; however, this needs to be confirmed through future studies with larger sample size

Copy Number Alteration and Mutational Profile of High-Grade B-Cell Lymphoma with MYC and BCL2 and/or BCL6 Rearrangements, Diffuse Large B-Cell Lymphoma with MYC-Rearrangement, and Diffuse Large B-Cell Lymphoma with MYC-Cluster Amplification.

The authors thank the technicians and scientists of the Genomics Core Facility of the IDIBAPS for their assistance in performing the NGS analysis.Diffuse large B-cell lymphoma (DLBCL) with MYC alteration is classified as high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (double/triple-hit lymphoma; DHL/THL), DLBCL with MYC rearrangement (single-hit lymphoma; SHL) and DLBCL with MYC-cluster amplification (MCAD). To elucidate the genetic features of DHL/THL, SHL, and MCAD, 23 lymphoma cases from Tokai University Hospital were analyzed. The series included 10 cases of DHL/THL, 10 cases of SHL and 3 cases of MCAD. The analysis used whole-genome copy number microarray analysis (OncoScan) and a custom-made next-generation sequencing (NGS) panel of 115 genes associated with aggressive B-cell lymphomas. The copy number alteration (CNA) profiles were similar between DHL/THL and SHL. MCAD had fewer CNAs than those of DHL/THL and SHL, except for +8q24. The NGS profile characterized DHL/THL with a higher "mutation burden" than SHL (17 vs. 10, p = 0.010), and the most relevant genes for DHL/THL were BCL2 and SOCS1, and for SHL was DTX1. MCAD was characterized by mutations of DDX3X, TCF3, HLA-A, and TP53, whereas MYC was unmutated. In conclusion, DHL/THL, SHL, and MCAD have different profiles.S

Overlap at the molecular and immunohistochemical levels between angioimmunoblastic T-cell lymphoma and a subgroup of peripheral T-cell lymphomas without specific morphological features

The overlap of morphology and immunophenotype between angioimmunoblastic T-cell lymphoma (AITL) and other nodal peripheral T-cell lymphomas (n-PTCLs) is a matter of current interest whose clinical relevance and pathogenic background have not been fully established. We studied a series of 98 n-PTCL samples (comprising 57 AITL and 41 PTCL-NOS) with five TFH antibodies (CD10, BCL-6, PD-1, CXCL13, ICOS), looked for mutations in five of the genes most frequently mutated in AITL (TET2, DNMT3A, IDH2, RHOA and PLCG1) using the Next-Generation-Sequencing Ion Torrent platform, and measured the correlations of these characteristics with morphology and clinical features. The percentage of mutations in the RHOA and TET2 genes was similar (23.5% of cases). PLCG1 was mutated in 14.3%, IDH2 in 11.2% and DNMT3A in 7.1% of cases, respectively. In the complete series, mutations in RHOA gene were associated with the presence of mutations in IDH2, TET2 and DNMT3A (p < 0.001, p = 0.043, and p = 0.029, respectively). Fourteen cases featured RHOA mutations without TET2 mutations. A close relationship was found between the presence of these mutations and a TFH-phenotype in AITL and PTCL-NOS patients. Interestingly, BCL-6 expression was the only TFH marker differentially expressed between AITL and PTCL-NOS cases. There were many fewer mutated cases than there were cases with a TFH phenotype. Overall, these data suggest alternative ways by which neoplastic T-cells overexpress these proteins. On the other hand, no clinical or survival differences were found between any of the recognized subgroups of patients with respect to their immunohistochemistry or mutational profile.This work was supported by grants from the Instituto de Salud Carlos III, from the Ministerio de Economía, Industria y Competitividad (RTICC RD06/0020/0107, RD12/0036/0060, PI 12/1682, PT13/0010/0007, PI16/ 01294, SAF2013-47416-R, CIBERONC-ISCIII, PIE15/ 0081, ISCIII-MINECO AES-FEDER (Plan Estatal I+D+I 2013–2016): PI14/00221, PIE14/0064, PIE15/0081 and PIE16/01294)) and the Asociación Española Contra el Cáncer, Spain. JG-R is a recipient of an iPFIS predoctoral fellowship (IFI14/00003) from ISCIII-MINECO-AESFEDER (Plan Estatal I+D+I 2013–2016). MSB was supported by a Miguel Servet contract (CP11/00018) from the ISCIII-MINECO-AES-FEDER (Plan Nacional I+D+I 2008–2011), and currently holds a Miguel Servet II contract (CPII16/00024), supported by ISCIII-MINECOAES- FEDER (Plan Estatal I+D+I 2013–2016) and the Fundación de Investigación Biomédica Puerta de Hierro.S

Genetic deletion of HVEM in a leukemia B cell line promotes a preferential increase of PD-1- stem cell-like T cells over PD-1+ T cells curbing tumor progression

[EN] Introduction: A high frequency of mutations affecting the gene encoding Herpes Virus Entry Mediator (HVEM, TNFRSF14) is a common clinical finding in a wide variety of human tumors, including those of hematological origin. Methods: We have addressed how HVEM expression on A20 leukemia cells influences tumor survival and its involvement in the modulation of the antitumor immune responses in a parental into F1 mouse tumor model of hybrid resistance by knocking-out HVEM expression. HVEM WT or HVEM KO leukemia cells were then injected intravenously into semiallogeneic F1 recipients and the extent of tumor dissemination was evaluated. Results: The loss of HVEM expression on A20 leukemia cells led to a significant increase of lymphoid and myeloid tumor cell infiltration curbing tumor progression. NK cells and to a lesser extent NKT cells and monocytes were the predominant innate populations contributing to the global increase of immune infiltrates in HVEM KO tumors compared to that present in HVEM KO tumors. In the overall increase of the adaptive T cell immune infiltrates, the stem cell-like PD-1- T cells progenitors and the effector T cell populations derived from them were more prominently present than terminally differentiated PD-1+ T cells. Conclusions: These results suggest that the PD-1- T cell subpopulation is likely to be a more relevant contributor to tumor rejection than the PD-1+ T cell subpopulation. These findings highlight the role of co-inhibitory signals delivered by HVEM upon engagement of BTLA on T cells and NK cells, placing HVEM/BTLA interaction in the spotlight as a novel immune checkpoint for the reinforcement of the anti-tumor responses in malignancies of hematopoietic originSIDepartment of Education of Castilla and Leon Regional Government (Grant # LE-006P20)Gerencia Regional de Salud (GRS1142/A/2015)Gerencia Regional de Salud (GRS1505/A/2017

Prognosis Value of Immunoregulatory Molecules in Oral Cancer Microenvironment: An Immunohistochemical Study

Objectives: To evaluate the relationship of the immune-checkpoint PD-1/PD-L1 with the clinical evolution of OSCC; to assess survival in OSCC based on the characteristics of TME and histologic risk score; to evaluate the clinical and histopathological relationship of OSCC with immunological TME. Material and Methods: A retrospective study was carried out on 65 samples from patients with OSCC on the floor of the mouth or tongue. Clinicopathological variables and the expression of the biomarkers PD-1, PD-L1, FoxP3, CD4, CD8, CSF1R, and p16 were recorded. The relationship of the clinical and histological variables with the expression of the biomarkers and survival was studied. Results: The univariate and multivariate analysis indicated that positive PD-1 expression was an independent protective factor for survival (overall, disease-free, disease-specific survival) and that high PD-L1 also improved survival. Poorly differentiated histological grades and metastasis were associated with a worse prognosis. Conclusions: PD-1 is a protective survival factor that is maintained independently of PD-L1 expression. High values of PD-L1 expression also improve survival. Higher expression of PD-1 is observed in smaller tumors, and higher expression of PD-L1 is more likely in women. No relationship between the tumor microenvironment and histologic risk score was found to influence the survival patterns studied in the OSCC. There is no evidence of a relationship between the histopathological features and the studied markers, although the positive PD-1 and PD-L1 cases have a lower risk of a high WPOI score, and positive PD-1 expression was associated with a lower DOIThis research was funded by the Fundación para la investigación Biomédica Hospital Universitario La Paz with the project number: EC_5474. The research has been partially funded through a predoctoral research grant awarded by the Official College of Dentists of Madrid (Madrid, Spain) with not project number associate

FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL

High Expression of Caspase-8 Associated with Improved Survival in Diffuse Large B-Cell Lymphoma: Machine Learning and Artificial Neural Networks Analyses

High expression of the anti-apoptotic TNFAIP8 is associated with poor survival of the patients with diffuse large B-cell lymphoma (DLBCL), and one of the functions of TNFAIP8 is to inhibit the pro-apoptosis Caspase-8. We aimed to analyze the immunohistochemical expression of Caspase-8 (active subunit p18; CASP8) in a series of 97 cases of DLBCL from Tokai University Hospital, and to correlate with other Caspase-8 pathway-related markers, including cleaved Caspase-3, cleaved PARP, BCL2, TP53, MDM2, MYC, Ki67, E2F1, CDK6, MYB and LMO2. After digital image quantification, the correlation with several clinicopathological characteristics of the patients showed that high protein expression of Caspase-8 was associated with a favorable overall and progression-free survival (Hazard Risks = 0.3; p = 0.005 and 0.03, respectively). Caspase-8 also positively correlated with cCASP3, MDM2, E2F1, TNFAIP8, BCL2 and Ki67. Next, the Caspase-8 protein expression was modeled using predictive analytics, and a high overall predictive accuracy (&gt;80%) was obtained with CHAID decision tree, Bayesian network, discriminant analysis, C5 tree, logistic regression, and Artificial Intelligence Neural Network methods (both Multilayer perceptron and Radial basis function); the most relevant markers were cCASP3, E2F1, TP53, cPARP, MDM2, BCL2 and TNFAIP8. Finally, the CASP8 gene expression was also successfully modeled in an independent DLBCL series of 414 cases from the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). In conclusion, high protein expression of Caspase-8 is associated with a favorable prognosis of DLBCL. Predictive modeling is a feasible analytic strategy that results in a solution that can be understood (i.e., explainable artificial intelligence, “white-box” algorithms)