Spatially resolved whole-transcriptomic and proteomic profiling of lung cancer and its immune-microenvironment according to PD-L1 expression.

The expression of PD-L1 on tumor cells (TCs) is used as an immunotherapy biomarker in lung cancer, but heterogeneous intratumoral expression is often observed. Using a Digital Spatial Profiling, we performed proteomic and whole-transcriptomic analyses of TCs and immune cells (ICs) in spatially matched areas based on tumor PD-L1 expression and the status of the immune microenvironment. Our findings were validated using immunohistochemistry, The Cancer Genome Atlas, and immunotherapy cohorts. ICs in areas with high PD-L1 expression on TCs showed more features indicative of immunosuppression and exhaustion than ICs in areas with low PD-L1 expression on TCs. TCs highly expressing PD-L1 within immune-inflamed (IF) areas show up-regulation of pro-inflammatory processes, whereas TCs highly expressing PD-L1 within immune-deficient (ID) areas show up-regulation of various metabolic processes. Using differentially expressed genes of TCs between the IF and ID areas, we identified a novel prognostic gene signature for lung cancer. In addition, a high ratio of CD8+ cells to M2 macrophages was found to predict favorable outcomes in patients with PD-L1-expressing lung cancer after immune checkpoint inhibitor therapy. This study demonstrates that TCs and ICs have distinct spatial features within the tumor microenvironment that are related to tumor PD-L1 expression and IC infiltration.

Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor DLBCL.

The pathogenesis of MYC and BCL2 double expressor diffuse large B-cell lymphoma (DE-DLBCL) remains unclear. To investigate how MYC and BCL2 contribute to tumor aggressiveness, we analyzed tumors from 14 patients each with DE- and non-DE-DLBCL patients by whole transcriptome sequencing. Validation was performed using publicly available datasets, tumor tissues from 126 patients, DLBCL cell lines, and a syngeneic mouse lymphoma model. Our transcriptome analysis revealed significantly elevated mRNA levels of C-C motif chemokine ligand 2 (CCL2) and C-C chemokine receptor type 2 (CCR2) in DE-DLBCLs compared to non-DE-DLBCLs (Padj < 0.05). Transcriptomic analysis with public datasets and immunohistochemistry corroborated these findings, indicating heightened M2 macrophage presence but diminished T-cell infiltration in DE-DLBCLs compared to non-DE-DLBCLs (all, P < 0.05). CCR2 expression was observed mainly in tumor-infiltrating macrophages rather than DLBCL cells. Increased CCL2 and CCR2 expression were significantly associated with the poor prognosis of patients with DLBCL. In vitro analyses, MYChigh/BCL2high DLBCL cells showed higher CCL2 expression and secretion than MYClow/BCL2low cells. MYC and BCL2 increased CCL2 expression and secretion by upregulation of nuclear factor-κB p65 in DLBCL cells and the CCL2 promoted M2 polarization of macrophages. In a mouse lymphoma model, CCL2 contributed to the immunosuppressive microenvironment and tumor growth of MYChigh/BCL2high tumor. We demonstrated that the increased CCL2/CCR2 axis confers aggressiveness to DE-DLBCL by increasing M2 polarization and can be a potential therapeutic target.

A humanized ACE2 mouse model recapitulating age- and sex-dependent immunopathogenesis of COVID-19.

In the ongoing battle against coronavirus disease 2019 (COVID-19), understanding its pathogenesis and developing effective treatments remain critical challenges. The creation of animal models that closely replicate human infection stands as a critical step forward in this research. Here, we present a genetically engineered mouse model with specifically-humanized knock-in ACE2 (hiACE2) receptors. This model, featuring nine specific amino acid substitutions for enhanced interaction with the viral spike protein, enables efficient severe acute respiratory syndrome coronavirus 2 replication in respiratory organs without detectable infection in the central nervous system. Moreover, it mirrors the age- and sex-specific patterns of morbidity and mortality, as well as the immunopathological features observed in human COVID-19 cases. Our findings further demonstrate that the depletion of eosinophils significantly reduces morbidity and mortality, depending on the infecting viral dose and the sex of the host. This reduction is potentially achieved by decreasing the pathogenic contribution of eosinophil-mediated inflammation, which is strongly correlated with neutrophil activity in human patients. This underscores the model's utility in studying the immunopathological aspects of COVID-19 and represents a significant advancement in COVID-19 modeling. It offers a valuable tool for testing vaccines and therapeutics, enhancing our understanding of the disease mechanisms and potentially guiding more targeted and effective treatments.

Comparison of Tumor Microenvironments between Primary Tumors and Lymph Node Metastases in Head and Neck Squamous Cell Carcinoma and Their Predictive Role in Immune Checkpoint Inhibitor Treatment.

The relationship between tumor microenvironments (TMEs) of regional lymph node metastases (LNMs) and primary tumors in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study compared tumor-infiltrating lymphocytes (TILs) and the immune phenotype (IP), characterized by spatial TIL distribution, between primary tumors and LNMs. Twenty-one HNSCC patients with regional LNM who received immune checkpoint inhibitors (ICIs) were included. A paired comparative analysis of TIL densities and IP between primary tumors and LNMs revealed no significant difference or correlation between TIL densities in primary tumors and LNMs. Their IPs were discordant in 12 patients (57.1%). Patients with high intratumoral TIL exhibited longer progression-free survival (PFS) than those with low intratumoral TIL in both primary tumors (median, 5.2 vs. 1.3 months, p = 0.003) and LNMs (median, 30.2 vs. 1.3 months, p = 0.012). Patients with inflamed IP exhibited longer PFS than those with non-inflamed IP in both primary tumors (median, 4.5 vs. 1.3 months, p = 0.043) and LNMs (median, 4.1 vs. 1.3 months, p = 0.037). Given the lack of correlation in TIL densities, the discrepancies in IP, and the predictive value of both TMEs, evaluating the TMEs of both primary tumors and LNMs may be beneficial for the precise use of ICIs in HNSCC. There was a significant discordance between the TME of primary tumors and LNMs, with implications in survival outcomes. Therefore, evaluating the TME of both the primary tumor and LNM could be beneficial for the precise use of ICIs in HNSCC.

Innate immune signatures in the nasopharynx after SARS-CoV-2 infection and links with the clinical outcome of COVID-19 in Omicron-dominant period.

While severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is characterized by impaired induction of interferons (IFNs) and IFN-stimulated genes (ISGs), the IFNs and ISGs in upper airway is essential to restrict the spread of respiratory virus. Here, we identified the prominent IFN and ISG upregulation in the nasopharynx (NP) of mild and even severe coronavirus disease 2019 (COVID-19) patients (CoV2+) in Omicron era and to compare their clinical outcome depending on the level of IFNs and ISGs. Whereas the induction of IFNB was minimal, transcription of IFNA, IFNG, and IFNLs was significantly increased in the NP of CoV2 + patients. IFNs and ISGs may be more upregulated in the NP of CoV2 + patients at early phases of infection according to viral RNA levels and this is observed even in severe cases. IFN-related innate immune response might be characteristic in macrophages and monocytes at the NP and the CoV2 + patients with higher transcription of IFNs and ISGs in the NP showed a correlation with good prognosis of COVID-19. This study presents that IFNs and ISGs may be upregulated in the NP, even in severe CoV2 + patients depending on viral replication during Omicron-dominant period and the unique IFN-responsiveness in the NP links with COVID-19 clinical outcomes.

Transcriptomic heterogeneity of EGFR-mutant non-small cell lung cancer evolution towards small cell lung cancer.

PURPOSE: Histological transformation from epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is a key mechanism of resistance to EGFR tyrosine kinase inhibitors (TKIs). However, transcriptomic changes between NSCLC and transformed SCLC (t-SCLC) remain unexplored. EXPERIMENTAL DESIGN: We conducted whole transcriptome analysis of 59 regions of interest (ROIs) through the spatial profiling of FFPE tissues obtained from ten patients (lung adenocarcinoma, 22; combined SCLC/NSCLC, 7; t-SCLC, 30 ROIs). Transcriptomic profiles and differentially expressed genes (DEGs) were compared between pre- and post-transformed tumors. RESULTS: Following EGFR-TKI treatment, 93.7% (15/16) of transformed-SCLC (t-SCLC) components evolved into neuroendocrine-high subtypes (SCLC-A or SCLC-N). The transition to t-SCLC occurred regardless of EGFR-TKI treatment and EGFR mutational status, with a notable decrease in EGFR expression (P < 0.001) at both mRNA and protein levels. Pathway analysis revealed that gene overexpression was related to epigenetic alterations in t-SCLC. Interestingly, Histone deacetylase (HDAC) inhibitors restored EGFR expression in SNU-2962A cells and their organoid model. The synergistic effects of third-generation EGFR-TKI osimertinib and the HDAC inhibitor fimepinostat were validated in both in vitro and in vivo models. CONCLUSIONS: Our study demonstrated most t-SCLC showed neuronal subtypes with low EGFR expression. DEGs analysis and t-SCLC preclinical models identified an epigenetic modifier as a promising treatment strategy for t-SCLC.

Harnessing Institutionally Developed Clinical Targeted Sequencing to Improve Patient Survival in Breast Cancer: A Seven-Year Experience.

Purpose: Considering the high disease burden and unique features of Asian patients with breast cancer (BC), it is essential to have a comprehensive view of genetic characteristics in this population. An institutional targeted sequencing platform was developed through the Korea Research-Driven Hospitals project and was incorporated into clinical practice. This study explores the use of targeted next-generation sequencing (NGS) and its outcomes in patients with advanced/metastatic BC in the real world. Materials and Methods: We reviewed the results of NGS tests administered to BC patients using a customized sequencing platform - FiRST Cancer Panel (FCP) - over seven years. We systematically described clinical translation of FCP for precise diagnostics, personalized therapeutic strategies, and unraveling disease pathogenesis. Results: NGS tests were conducted on 548 samples from 522 patients with BC. 97.6% of tested samples harbored at least one pathogenic alteration. The common alterations included mutations in TP53(56.2%), PIK3CA(31.2%), GATA3(13.8%), BRCA2(10.2%), and amplifications of CCND1(10.8%), FGF19(10.0%), and ERBB2(9.5%). NGS analysis of ERBB2 amplification correlated well with HER2 immunohistochemistry and in situ hybridization. RNA panel analyses found potentially actionable and prognostic fusion genes. FCP effectively screened for potentially germline pathogenic/likely pathogenic mutation. 10.3% of BC patients received matched therapy guided by NGS, resulting in a significant overall survival advantage (p=0.022), especially for metastatic BCs. . Conclusion: Clinical NGS provided multifaceted benefits, deepening our understanding of the disease, improving diagnostic precision, and paving the way for targeted therapies. The concrete advantages of FCP highlight the importance of multi-gene testing for BC, especially for metastatic conditions.

Resistance mechanisms of EGFR tyrosine kinase inhibitors, in EGFR exon 20 insertion-mutant lung cancer.

BACKGROUND: Mobocertinib, an EGFR exon 20 insertion (Ex20ins)-specific tyrosine kinase inhibitor has been used for treatment of advanced/metastatic EGFR Ex20ins-mutant non-small cell lung cancer (NSCLC). However, resistance mechanisms to EGFR Ex20ins-specific inhibitors and the efficacy of subsequent amivantamab treatment is unknown. METHODS: To investigate resistance mechanisms, tissue and cfDNA samples were collected before treatment initiation and upon development of resistance from NSCLC patients with EGFR Ex20ins mutations received mobocertinib, poziotinib, and amivantamab treatments. Genetic alterations were analyzed using whole-genome and targeted sequencing, and in vitro resistant cell lines were generated for validation. RESULTS: EGFR amplification (n = 6, including 2 broad copy number gain) and EGFR secondary mutation (n = 3) were observed at the resistance of mobocertinib. One patient had both EGFR secondary mutation and high EGFR focal amplification. In vitro models harboring EGFR alterations were constructed to validate resistance mechanisms and identify overcoming strategies to resistance. Acquired EGFR-dependent alterations were found to mediate resistance to mobocertinib in patients and in vitro models. Furthermore, two of six patients who received sequential amivantamab followed by an EGFR tyrosine kinase inhibitor had MET amplification and showed partial response. CONCLUSIONS: Our study revealed EGFR-dependent and -independent mechanisms of mobocertinib resistance in patients with advanced EGFR Ex20ins-mutant NSCLC.

Transformation of t(14;18)-negative follicular lymphoma to plasmablastic lymphoma: a case report with analysis of genetic evolution.

BACKGROUND: Follicular lymphoma (FL) is characterized by t(14;18)(q32;q21) involving the IGH and BCL2 genes. However, 10-15% of FLs lack the BCL2 rearrangement. These BCL2-rearrangement-negative FLs are clinically, pathologically, and genetically heterogeneous. The biological behavior and histological transformation of such FLs are not adequately characterized. Here, we report the first case of t(14;18)-negative FL that rapidly progressed to plasmablastic lymphoma (PBL). CASE PRESENTATION: A previously healthy 51-year-old man presented with leg swelling. Computed tomography (CT) showed enlarged lymph nodes (LNs) throughout the body, including both inguinal areas. Needle biopsy of an inguinal LN suggested low-grade B-cell non-Hodgkin lymphoma. Excisional biopsy of a neck LN showed proliferation of centrocytic and centroblastic cells with follicular and diffuse growth patterns. Immunohistochemical analysis showed that the cells were positive for CD20, BCL6, CD10, and CD23. BCL2 staining was negative in the follicles and weak to moderately positive in the interfollicular areas. BCL2 fluorescence in situ hybridization result was negative. Targeted next-generation sequencing (NGS) revealed mutations in the TNFRSF14, CREBBP, STAT6, BCL6, CD79B, CD79A, and KLHL6 genes, without evidence of BCL2 or BCL6 rearrangement. The pathologic and genetic features were consistent with t(14;18)-negative FL. Two months after one cycle of bendamustine and rituximab chemotherapy, the patient developed left flank pain. Positron emission tomography/CT showed new development of a large hypermetabolic mass in the retroperitoneum. Needle biopsy of the retroperitoneal mass demonstrated diffuse proliferation of large plasmablastic cells, which were negative for the B-cell markers, BCL2, BCL6, and CD10; they were positive for MUM-1, CD138, CD38, and C-MYC. The pathologic findings were consistent with PBL. The clonal relationship between the initial FL and subsequent PBL was analyzed via targeted NGS. The tumors shared the same CREBBP, STAT6, BCL6, and CD79B mutations, strongly suggesting that the PBL had transformed from a FL clone. The PBL also harbored BRAF V600E mutation and IGH::MYC fusion in addition to IGH::IRF4 fusion. CONCLUSIONS: We propose that transformation or divergent clonal evolution of FL into PBL can occur when relevant genetic mutations are present. This study broadens the spectrum of histological transformation of t(14;18)-negative FL and emphasizes its biological and clinical heterogeneity.

Epithelial-mesenchymal transition induced by tumor cell-intrinsic PD-L1 signaling predicts a poor response to immune checkpoint inhibitors in PD-L1-high lung cancer.

BACKGROUND: We investigated the role of tumor cell-intrinsic PD-L1 signaling in the epithelial-mesenchymal transition (EMT) in non-small-cell lung cancer (NSCLC) and the role of EMT as a predictive biomarker for immune checkpoint inhibitor (ICI) therapy. METHODS: PD-L1-overexpressing or PD-L1-knockdown NSCLC cells underwent RNA-seq and EMT phenotype assessment. Mouse lung cancer LLC cells were injected into nude mice. Two cohorts of patients with NSCLC undergoing ICI therapy were analyzed. RESULTS: RNA-seq showed that EMT pathways were enriched in PD-L1-high NSCLC cells. EMT was enhanced by PD-L1 in NSCLC cells, which was mediated by transforming growth factor-ß (TGFß). PD-L1 promoted the activation of p38-MAPK by binding to and inhibiting the protein phosphatase PPM1B, thereby increasing the TGFß production. Tumor growth and metastasis increased in nude mice injected with PD-L1-overexpressing LLC cells. In the ICI cohort, EMT signature was higher in patients with progressive disease than in those with responses, and EMT was significantly associated with poor survival in PD-L1-high NSCLC. In PD-L1-high NSCLC, EMT was associated with increased M2-macrophage and regulatory T-cell infiltrations and decreased cytotoxic T-cell infiltration. CONCLUSIONS: Tumor cell-intrinsic PD-L1 function contributes to NSCLC progression by promoting EMT. EMT may predict an unfavorable outcome after ICI therapy in PD-L1-high NSCLC.

CRIF1 deficiency induces FOXP3LOW inflammatory non-suppressive regulatory T cells, thereby promoting antitumor immunity.

Recently identified human FOXP3lowCD45RA- inflammatory non-suppressive (INS) cells produce proinflammatory cytokines, exhibit reduced suppressiveness, and promote antitumor immunity unlike conventional regulatory T cells (Tregs). In spite of their implication in tumors, the mechanism for generation of FOXP3lowCD45RA- INS cells in vivo is unclear. We showed that the FOXP3lowCD45RA- cells in human tumors demonstrate attenuated expression of CRIF1, a vital mitochondrial regulator. Mice with CRIF1 deficiency in Tregs bore Foxp3lowINS-Tregs with mitochondrial dysfunction and metabolic reprograming. The enhanced glutaminolysis activated α-ketoglutarate-mTORC1 axis, which promoted proinflammatory cytokine expression by inducing EOMES and SATB1 expression. Moreover, chromatin openness of the regulatory regions of the Ifng and Il4 genes was increased, which facilitated EOMES/SATB1 binding. The increased α-ketoglutarate-derived 2-hydroxyglutarate down-regulated Foxp3 expression by methylating the Foxp3 gene regulatory regions. Furthermore, CRIF1 deficiency-induced Foxp3lowINS-Tregs suppressed tumor growth in an IFN-γ-dependent manner. Thus, CRIF1 deficiency-mediated mitochondrial dysfunction results in the induction of Foxp3lowINS-Tregs including FOXP3lowCD45RA- cells that promote antitumor immunity.

Fetal Lung Interstitial Tumor: A Comprehensive Case Study with an Emphasis on Next-Generation Sequencing.

Fetal lung interstitial tumor (FLIT), which is characterized by immature interstitial cells resembling the fetal lung parenchyma of 20 to 24 weeks of gestation, is a rare respiratory neoplasm. This study presents the first reported FLIT in Korea. It also aims to refine the diagnostic method of FLIT and increase the accuracy of prognostic assessment by using next-generation sequencing to check for anaplastic lymphoma receptor tyrosine kinase (anaplastic lymphoma kinase) gene rearrangement. Although the initial prognosis for FLIT has been promising since its first report in 2010, certain pathological features are associated with poorer outcomes. Therefore, achieving an accurate diagnosis of FLIT is crucial for avoiding unnecessary treatments beyond surgical resection.

Clinicopathological implications of immunohistochemical expression of TBX21, CXCR3, GATA3, CCR4, and TCF1 in nodal follicular helper T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified.

BACKGROUND: The classification of nodal peripheral T-cell lymphoma (PTCL) has evolved according to histology, cell-of-origin, and genetic alterations. However, the comprehensive expression pattern of follicular helper T-cell (Tfh) markers, T-cell factor-1 (TCF1), and Th1- and Th2-like molecules in nodal PTCL is unclear. METHODS: Eighty-two cases of nodal PTCL were classified into 53 angioimmunoblastic T-cell lymphomas (AITLs)/nodal T-follicular helper cell lymphoma (nTFHL)-AI, 18 PTCLs-Tfh/nTFHL-not otherwise specified (NOS), and 11 PTCLs-NOS according to the revised 4th/5th World Health Organization classifications. Immunohistochemistry for TCF1, TBX21, CXCR3, GATA3, and CCR4 was performed. RESULTS: TCF1 was highly expressed in up to 68% of patients with nTFHL but also in 44% of patients with PTCL-NOS (p > .05). CXCR3 expression was higher in AITLs than in non-AITLs (p = .035), whereas GATA3 expression was higher in non-AITL than in AITL (p = .007) and in PTCL-Tfh compared to AITL (p = .010). Of the cases, 70% of AITL, 44% of PTCLTfh/ nTFHL-NOS, and 36% of PTCL-NOS were subclassified as the TBX21 subtype; and 15% of AITL, 38% of PTCL-Tfh/nTFHL-NOS, and 36% of PTCL-NOS were subclassified as the GATA3 subtype. The others were an unclassified subtype. CCR4 expression was associated with poor progression-free survival (PFS) in patients with PTCL-Tfh (p < .001) and nTFHL (p = .023). The GATA3 subtype showed poor overall survival in PTCL-NOS compared to TBX21 (p = .046) and tended to be associated with poor PFS in patients with non-AITL (p = .054). CONCLUSIONS: The TBX21 subtype was more prevalent than the GATA3 subtype in AITL. The GATA3 subtype was associated with poor prognosis in patients with non-AITL and PTCL-NOS.

De novo fatty-acid synthesis protects invariant NKT cells from cell death, thereby promoting their homeostasis and pathogenic roles in airway hyperresponsiveness.

Invariant natural-killer T (iNKT) cells play pathogenic roles in allergic asthma in murine models and possibly also humans. While many studies show that the development and functions of innate and adaptive immune cells depend on their metabolic state, the evidence for this in iNKT cells is very limited. It is also not clear whether such metabolic regulation of iNKT cells could participate in their pathogenic activities in asthma. Here, we showed that acetyl-coA-carboxylase 1 (ACC1)-mediated de novo fatty-acid synthesis is required for the survival of iNKT cells and their deleterious functions in allergic asthma. ACC1, which is a key fatty-acid synthesis enzyme, was highly expressed by lung iNKT cells from WT mice that were developing asthma. Cd4-Cre::Acc1fl/fl mice failed to develop OVA-induced and HDM-induced asthma. Moreover, iNKT cell-deficient mice that were reconstituted with ACC1-deficient iNKT cells failed to develop asthma, unlike when WT iNKT cells were transferred. ACC1 deficiency in iNKT cells associated with reduced expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity that promoted iNKT-cell death. Furthermore, circulating iNKT cells from allergic-asthma patients expressed higher ACC1 and PPARG levels than the corresponding cells from non-allergic-asthma patients and healthy individuals. Thus, de novo fatty-acid synthesis prevents iNKT-cell death via an ACC1-FABP-PPARγ axis, which contributes to their homeostasis and their pathogenic roles in allergic asthma.

Targeting CD73 to Overcomes Resistance to First-Generation EGFR Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

PURPOSE: In patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC), EGFR tyrosine kinase inhibitors (TKIs) improve response rate and survival. However, most patients eventually develop resistance. This study aimed to identify the role of CD73 in EGFR-mutant NSCLC and explore whether CD73 inhibition may serve as a therapeutic strategy in NSCLC patients with acquired resistance to EGFR-TKIs. MATERIALS AND METHODS: We evaluated the prognostic role of CD73 expression in EGFR-mutant NSCLC using tumor samples from a single institution. We silenced CD73 in EGFR-TKI-resistant cell lines using short hairpin RNA (shRNA) targeting CD73 and also transfected a vector alone as a negative control. Using these cell lines, cell proliferation and viability assays, immunoblot assays, cell cycle analysis, colony-forming assays, flow cytometry, and apoptosis analysis were performed. RESULTS: High expression of CD73 was associated with shorter survival in patients with metastatic EGFR-mutant NSCLC treated with first-generation EGFR-TKI. CD73 inhibition synergistically inhibited cell viability with first-generation EGFR-TKI treatment compared with the negative control. When CD73 inhibition and EGFR-TKI treatment were combined, G0/G1 cell cycle arrest was induced through the regulation of p21 and cyclin D1. In addition, the apoptosis rate was increased in CD73 shRNA-transfected cells treated with EGFR-TKI. CONCLUSION: High expression of CD73 adversely affects the survival of patients with EGFR-mutant NSCLC. The study demonstrated that inhibiting CD73 in EGFR-TKI-resistant cell lines resulted in increased apoptosis and cell cycle arrest, which overcame the acquired resistance to first-generation EGFR-TKIs. Further research is needed to determine whether blocking CD73 plays a therapeutic role in EGFR-TKI-resistant patients with EGFR-mutant NSCLC.

Prognostic Impact of the Immunoscore Based on Whole-Slide Image Analysis of CD3+ Tumor-Infiltrating Lymphocytes in Diffuse Large B-Cell Lymphoma.

An Immunoscore based on tumor-infiltrating T-cell density was validated as a prognostic factor in patients with solid tumors. However, the potential utility of the Immunoscore in predicting the prognosis of patients with diffuse large B-cell lymphoma (DLBCL) is unclear. Here, the prognostic value of an Immunoscore based on tumor-infiltrating CD3+ T-cell density was evaluated in 104 patients with DLBCL who underwent R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) therapy. Digitally scanned whole-slide images were analyzed using Aperio ImageScope software. CD3+ cell densities in the whole tumor area were quantitated using 3 different methods, including number of CD3+ cells/area (mm2), ratio of CD3+ cells to total cells, and ratio of CD3+ cells to CD20+ cells. There was a high concordance among the 3 methods. Patients with low CD3+ cell density had an elevated serum lactate dehydrogenase level and a high Ki-67 proliferation index (all, P < .05). Patients with low CD3+ cell density, according to all 3 methods, had worse overall survival (OS) and worse progression-free survival (P < .05, all). They also had poor OS, independent of MYC/BCL2 double expression (DE) status, Eastern Cooperative Oncology Group performance status, or Ann Arbor stage (all, P < .05). These results were validated using 2 publicly available data sets. In both validation cohorts, patients with low CD3E mRNA expression had an elevated serum lactate dehydrogenase level, extranodal site involvement, and DE status (P < .05). They also had worse progression-free survival (P = .067 and P = .002, respectively) and OS (both P < .05). A low CD3E mRNA level was predictive of poor OS, independent of DE status. An Immunoscore based on whole-slide image analysis of CD3+ T-cell infiltration was sufficient to predict survival in patients with DLBCL. Low CD3+ cell density was a poor prognostic factor, independent of other prognostic parameters and DE status.

Computed Tomography-based Prognostication in Lung Adenocarcinomas through Histopathological Feature Learning: A Retrospective Multicenter Study.

Rationale: Modeling imaging surrogates for well-validated histopathological risk factors would enable prognostication in early-stage lung adenocarcinomas. Objectives: We aimed to develop and validate computed tomography (CT)-based deep learning (DL) models for the prognostication of early-stage lung adenocarcinomas through learning histopathological features and to investigate the models' reproducibility using retrospective, multicenter datasets. Methods: Two DL models were trained to predict visceral pleural invasion and lymphovascular invasion, respectively, using preoperative chest CT scans from 1,426 patients with stage I-IV lung adenocarcinomas. The averaged model output was defined as the composite score and evaluated for the prognostic discrimination and its added value to clinicopathological factors in temporal (n = 610) and external test sets (n = 681) of stage I lung adenocarcinomas. The study outcomes were freedom from recurrence (FFR) and overall survival (OS). Interscan and interreader reproducibility were analyzed in 31 patients with lung cancer who underwent same-day repeated CT scans. Results: For the temporal test set, the time-dependent area under the receiver operating characteristic curve was 0.76 (95% confidence interval [CI], 0.71-0.81) for 5-year FFR and 0.67 (95% CI, 0.59-0.75) for 5-year OS. For the external test set, the area under the curve was 0.69 (95% CI, 0.63-0.75) for 5-year OS. The discrimination performance remained stable in 10-year follow-up for both outcomes. The prognostic value of the composite score was independent of and complementary to the clinical factors (adjusted per-percent hazard ratio for FFR [temporal test], 1.04 [95% CI, 1.03-1.05; P < 0.001]; OS [temporal test], 1.03 [95% CI, 1.02-1.04; P < 0.001]; OS [external test], 1.03 [95% CI, 1.02-1.04; P < 0.001]). The likelihood ratio tests indicated added value of the composite score (all P < 0.05). The interscan and interreader reproducibility were excellent (Pearson's correlation coefficient, 0.98 for both). Conclusions: The CT-based composite score obtained from DL of histopathological features predicted survival in early-stage lung adenocarcinomas with high reproducibility.

The transcription factor Mef2d regulates B:T synapse-dependent GC-TFH differentiation and IL-21-mediated humoral immunity.

Communication between CD4 T cells and cognate B cells is key for the former to fully mature into germinal center-T follicular helper (GC-TFH) cells and for the latter to mount a CD4 T cell-dependent humoral immune response. Although this interaction occurs in a B:T synapse-dependent manner, how CD4 T cells transcriptionally regulate B:T synapse formation remains largely unknown. Here, we report that Mef2d, an isoform of the myocyte enhancer factor 2 (Mef2) transcription factor family, is a critical regulator of this process. In CD4 T cells, Mef2d negatively regulates expression of Sh2d1a, which encodes SLAM-associated protein (SAP), a critical regulator of B:T synapses. We found that Mef2d regulates Sh2d1a expression via DNA binding-dependent transcriptional repression, inhibiting SAP-dependent B:T synapse formation and preventing antigen-specific CD4 T cells from differentiating into GC-TFH cells. Mef2d also impeded IL-21 production by CD4 T cells, an important B cell help signaling molecule, via direct repression of the Il21 gene. In contrast, CD4 T cell-specific disruption of Mef2d led to a substantial increase in GC-TFH differentiation in response to protein immunization, concurrent with enhanced SAP expression. MEF2D mRNA expression inversely correlates with human systemic lupus erythematosus (SLE) patient autoimmune parameters, including circulating TFH-like cell frequencies, autoantibodies, and SLEDAI scores. These findings highlight Mef2d as a pivotal rheostat in CD4 T cells for controlling GC formation and antibody production by B cells.