Faculty Bibliography

Acute myeloid leukemia (AML) is a blood cancer with poor survival outcomes. Acute respiratory failure frequently occurs due to leukemia infiltration of the lungs. Underlying mechanisms remain unexplored and therapeutic interventions remain empiric. Here we map the AML lung microenvironment at spatial and single-cell resolution. We show that extensive remodeling is coupled with inflammation and impaired tissue integrity and function. Steroid treatment significantly reduces AML burden and lung infiltration, improving oxygenation and pulmonary function. As a mechanistic correlate, the S-type lectin Lgals9 is triggered by inflammation and mediates cell-cell interactions within infiltrated lungs. Also, the alarmin IL-33 and its receptor (IL-1RL1) are involved in cell-cell interactions within the leukemic lung microenvironment. Targeting either the Lgals9 or IL-33 axis significantly decreases overall AML burden and lung infiltration through effects on both the immune microenvironment and AML cells. Our studies delineate pulmonary infiltration phenotypes in acute leukemia, enabling new treatment strategies.

UNLABELLED:LKB1 mutations in lung cancer promote an immunosuppressive tumor microenvironment, but the underlying mechanisms remain unknown. Using genetically engineered mouse models and human tumor samples, we demonstrate that LKB1 loss leads to high expression of the cytokine leukemia-inhibitory factor (LIF), which through a cancer cell-autonomous autocrine loop, orchestrates the infiltration of immunosuppressive SiglecFHi neutrophils and Arg1+ interstitial macrophages. Genetic deletion of Lifr, the receptor for LIF, on Lkb1-mutant lung tumors revealed that autocrine LIF signaling induces tumor plasticity and the emergence of a Sox17+ dedifferentiated inflammatory cell state. Antibody-mediated LIF neutralization selectively eliminates the Sox17+ tumor cell state, reduces immunosuppressive myeloid cells, and enhances antitumor T-cell responses. Our study uncovers a novel LKB1-LIF axis driving immune evasion and identifies LIF as a potential therapeutic target in LKB1-mutant lung cancer. This work highlights the interplay between tumor genetics, cellular plasticity, and immune regulation in lung cancer progression. SIGNIFICANCE/UNASSIGNED:LKB1-mutant lung cancers express LIF, which induces an immunosuppressive Sox17+ tumor state. Anti-LIF therapy eliminates this state and restores antitumor immunity, revealing a novel vulnerability in this aggressive cancer subtype lacking effective targeted therapies.

Cancer cells activate the integrated stress response (ISR) to adapt to stress and resist therapy¹. ISR signals converge on activating transcription factor 4 (ATF4), which controls cell-intrinsic transcriptional programs that are involved in metabolic adaptation, survival and growth^2,3. However, whether the ISR-ATF4 axis influences anti-tumour immune responses remains mostly unknown. Here we show that loss of ATF4 decreases tumour progression considerably in immunocompetent mice, but not in immunocompromised ones, by enhancing T cell-dependent anti-cancer immune responses. An unbiased genetic screen of ATF4-regulated genes identifies lipocalin 2 (LCN2) as the principal ATF4-dependent effector that impairs anti-tumour immunity by favouring infiltration with immunosuppressive interstitial macrophages. Furthermore, we find that LCN2 promotes T cell exclusion and immune evasion in preclinical mouse models, and correlates with decreased T cell infiltration in patients with lung and pancreatic adenocarcinomas. Anti-LCN2 antibodies promote robust anti-tumour T cell responses in mouse models of aggressive solid tumours. Our study shows that the ATF4-LCN2 axis has a cell-extrinsic role in suppressing anti-cancer immunity, and could pave the way for an immunotherapy approach that targets LCN2.

Dendritic cells (DCs) facilitate the maintenance of immunological tolerance in the steady state. We report that transcription factor Etv3 is preferentially expressed in mature DCs, including tissue-derived migratory DCs (migDCs), and facilitates their homeostatic maturation and CCR7-dependent migration. Mice with global or DC-specific deletion of Etv3 manifested the expansion of CD25^low regulatory T (T_reg) cells, spontaneous activation of conventional T cells, and multiorgan T cell infiltration. Etv3 deficiency exacerbated TLR7-driven systemic lupus erythematosus (SLE)-like disease, supporting the reported genetic association of human ETV3 with SLE. Etv3-deficient migDCs up-regulated multiple costimulatory molecules, including OX40 ligand (OX40L/TNFSF4), whose blockade partially rescued the T_reg cell abnormalities. These results identify Etv3 as an essential regulator of the tolerogenic function of DCs and implicate it in the regulation of human autoimmunity.

OBJECTIVES/OBJECTIVE:With the development of genomic technologies, the isolation of genomic DNA (gDNA) from clinical samples has become more important for both clinical diagnostics and research studies. Blood samples collected in PAXgene Blood RNA Tubes, which are typically used for RNA extraction, can be used for gDNA extraction, particularly in clinical studies when only such blood samples are available. METHODS:We optimized the pre-treatment of blood samples collected in PAXgene Blood RNA Tubes. For the first time, three magnetic bead-based automated platforms (QIAsymphony SP, Maxwell RSC and KingFisher Apex) were compared for gDNA extraction from these blood samples. Additionally, the effects of storage at 4 °C or freeze-thaw cycles of the blood samples on gDNA yield were investigated. High-throughput extraction in 96-well format was evaluated. RESULTS:Systematic optimization of blood sample pre-treatment shows that prolonged incubation at room temperature and/or increased centrifugation speed and time improved gDNA yield from blood samples collected in PAXgene Blood RNA Tubes. QIAsymphony SP (4.27 ± 2.19 µg) and Maxwell RSC (4.82 ± 2.96 µg) produced significantly higher gDNA yields than KingFisher Apex (1.09 ± 0.61 µg). Higher gDNA yields were obtained with shorter storage time at 4 °C or fewer freeze-thaw cycles of the blood samples. In the 96-well format extraction, gDNA yields ranged from 0.24 to 13.46 µg. CONCLUSIONS:Not all magnetic bead-based automated platforms are suitable for gDNA extraction from blood samples collected in PAXgene Blood RNA Tubes. Systematic pre-treatments optimization provides guidance for routine and high-throughput workflows, and storage conditions and freeze-thaw cycles offer practical reference for biobanking.

Thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4d-UT) is an uncommon, aggressive lung neoplasm associated with smoking and characterized by loss of SMARCA4 (BRG-1) expression. Although originally considered to be a primary sarcoma, there is growing evidence that these lesions may represent transformation of conventional non-small cell carcinoma. In this study, we probe this relationship based on the clinical, histologic and molecular findings of 18 SMARCA4-deficient malignancies of the lung. Cases diagnosed as SMARCA4d-UT and SMARCA4-deficient carcinoma were retrospectively reviewed, including histologic and immunophenotypic features, and next generation sequencing studies. Of the 18 tumors, 5 were considered to represent undifferentiated SMARCA4d-UT, and 13 SMARCA4-deficient carcinomas, including 11 adenocarcinomas, 1 squamous cell carcinoma, and 1 poorly differentiated non-small cell carcinoma. All 13 carcinomas had a morphologically identifiable undifferentiated component. Survival outcomes were similar in both SMARCA4d-UT and carcinomas. Genetic alterations often seen in lung cancer were identified in 8 cases, including mutations in EGFR (in 2 SMARCA4-deficient adenocarcinomas), KRAS (1 SMARCA4d-UT and 1 SMARCA4-deficient adenocarcinoma), MAP2K1 (1 SMARCA4-deficient adenocarcinoma), and a gene fusion involving EML4::ALK (1 SMARCA4d-UT). The patient with EML4::ALK fusion was treated with alectinib with partial response. Fusions involving BRAF::CHCHD3 and FGFR1::FILIP1 were identified in 2 SMARCA4-deficient adenocarcinomas. High expression of PD-L1 (TPS >50 %) was seen in 12 cases (67 %). These finding further suggest that SMARCA4d-UT and carcinomas with SMARCA4 loss may be on the same spectrum of disease, and accurate histologic distinction between these lesions may be challenging. A unified terminology may be beneficial for appropriate diagnosis and treatment.

BACKGROUND AND OBJECTIVE/UNASSIGNED:Accurate diagnosis of mediastinal tumors is of critical importance to establish appropriate therapy. However, these lesions are relatively uncommon and may be challenging to evaluate, particularly in small biopsy specimens. Thymomas and thymic carcinomas are the most common primary malignant tumors of the mediastinum, but the site can be affected by many other neoplasms that can pose significant difficulty in diagnosis. The objective of this article is to bring awareness to these rarer tumors and offer a diagnostic approach using ancillary techniques guided by clinical and morphological features. METHODS/UNASSIGNED:We discuss and review six challenging cases of mediastinal tumors with overlapping morphologic features. We discuss their unique morphologic, immunophenotypic, and relevant molecular characteristics to support their definitive diagnosis, based on current literature. Sources were obtained via PubMed search and include original studies and review articles published in the English language between 1990 and 2025. Search terms include the diagnostic entities discussed in the article. KEY CONTENT AND FINDINGS/UNASSIGNED:Judicious use of immunohistochemistry and molecular studies is necessary to accurately diagnose mediastinal neoplasms with overlapping histologic features, such as those seen in the cases discussed. CONCLUSIONS/UNASSIGNED:Diagnosis of uncommon mediastinal lesions may be challenging, particularly in small biopsies, as morphological features may be shared among different entities. Awareness of these rare entities, their clinical characteristics and presentation, and differential diagnosis can guide in the selection of appropriate immunohistochemical panels, molecular markers, and molecular diagnostics when appropriate to support the diagnoses.

BACKGROUND:The diagnosis of cardiac sarcoidosis (CS) is often challenging, particularly in atypical cases. CASE SUMMARY/METHODS:This case involves a previously healthy 33-year-old woman who was found to have a biatrial mass and evidence of a diffuse inflammatory or neoplastic process on multimodality imaging. Percutaneous biopsy of the cardiac mass was performed, and histopathologic examination revealed granulomas consistent with CS. DISCUSSION/CONCLUSIONS:This case adds to the growing number of reports of CS manifesting as an intracardiac mass. TAKE-HOME MESSAGES/CONCLUSIONS:The clinical presentation of CS is highly variable, and it may rarely manifest as an intracardiac mass. The diagnosis of cardiac sarcoidosis is often challenging, particularly in patients with atypical presentations. Indeterminate cardiac masses often require direct tissue sampling because the changes in treatment and prognosis are substantial.

There is a body-wide network of interstitial spaces that includes three components: a large-scale fascial network made up of fluid-filled spaces containing collagens and other extracellular matrix components like hyaluronic acid (HA), the peri-vascular/capillary interstitium, and intercellular interstitial spaces. Staining for HA within the colon, skin, and liver has demonstrated spatial continuity of the fascial interstitium across tissue layers and between organs, while continuity of HA staining between perineurial and adventitial sheathes beyond organ boundaries confirmed that they also participate in this body-wide network. We asked whether the pulmonary interstitium comprises a continuous organ-wide network that also connects to the body-wide interstitium via routes along nerves and the vasculature. We studied archival lung lobectomy specimens containing normal tissues inclusive of all lung anatomical units from six females and three males (mean age 53+/- 16.5 years). For comparison, we also studied normal mouse lung. Multiplex immunohistochemical cocktails were used to identify: (1) HA, CD34, and vimentin - highlighting interstitium; (2) HA, CD34, and podoplanin (D2-40) - highlighting relationships between the interstitium, vasculature, and lymphatics. Sizes of extracellular APP were measured. Tissues from nine patients (six females, three males, mean age 53+/- 16.5 years) were studied. HA staining was continuous throughout the five major anatomic compartments of the lung: alveolar walls, subpleural connective tissue, centrilobular peribronchovascular compartment, interlobular septal compartment, and axial peribronchovascular of the hilum, with similar findings in murine lung tissue. Continuity with interstitial spaces of the perineurium and adventitia was confirmed. The distribution of APP corresponded to known routes of lymphatic drainage, superficial and deep. APP within perineurium and perivascular adventitia further demonstrated continuity between intra- and extrapulmonary interstitium. To conclude, all segments of the lung interstitium are connected and are linked along nerves and the vascular tree to a body-wide communication network. These findings have significant implications for understanding lung physiology and pathobiology, suggesting routes of passage for inflammatory cells and mediators, malignant cells, and infectious agents. Interstitial spaces may be important in microbiome signaling within and beyond the lung and may be a component of the lung-brain axis.

Subsolid nodules (SSNs) are increasingly encountered in chest computed tomography (CT) imaging and clinical practice, as awareness of their significance and CT utilization grows. Either part-solid or solely ground-glass in attenuation, SSNs are shown to correlate with lung adenocarcinomas and their precursors, although a differential diagnosis is to be considered that includes additional neoplastic and inflammatory etiologies. This review discusses the differential diagnosis for SSNs, imaging and clinical features, and pathology that are helpful when making management decisions that may include PET/CT, biopsy, or surgery. Potential pitfalls in nodule characterization and management will be highlighted, to aid in managing SSNs appropriately.