Faculty Bibliography

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.

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.

Ultraviolet (UV)-induced DNA mutations generate genetic drivers of cutaneous melanoma and numerous neoantigens that can trigger anti-tumor immunity. Melanoma cells must therefore rapidly evade immune detection by modulating cell-autonomous epigenetic mechanisms and tumor-microenvironment interactions. Although angiogenesis typically facilitates immune infiltration, solid tumors increase vascularization while limiting immune cell entry. By comparing transcription factor (TF) expression across early-stage melanoma, naevi, and other cancers, we found that the homeodomain TF HOXD13 drives a melanoblast-like program upregulated in melanoma and strongly correlated with angiogenesis and immune cell exclusion. Using transcriptomics, 3D chromatin profiling, and in vivo models, we show that HOXD13 promotes tumor growth by enhancing angiogenesis and suppressing T-cell infiltration. HOXD13 orchestrates 3D enhancer-promoter contacts activating VEGFA, SEMA3A, and CD73, which remodel vasculature and elevate immunosuppressive adenosine. Consistently, HOXD13-induced tumor growth is reversed by combined VEGFR and adenosine receptor (AdR) inhibition, revealing a dual pro-angiogenic and immunosuppressive HOXD13 axis with therapeutic relevance.

Lymphatic vessels activate anti-tumor immune surveillance and support metastasis. Whether there are distinct lymphatic phenotypes that govern immunity and metastasis remains unclear. Here we reveal that cytotoxic immunity normalizes lymphatic function and uncouples immune and metastatic potential. We demonstrate that intratumoral lymphatic vessel density negatively correlates with cytotoxic immunity and that IFNγ reprograms the intratumoral lymphatic state. Lymphatic deletion of Ifngr1 expanded the intratumoral lymphatic network and drove the emergence of a tip-like state that promotes lymph node metastasis but not dendritic cell migration or response to immune checkpoint blockade (ICB). Mechanistically, IFNγ restrains proliferation and cell state programs through inhibition of mitochondrial respiration. Lymphatic-specific inhibition of mitochondrial complex III restrained the intratumoral tip-like state, blocked metastasis, and enhanced the response to ICB. Our data reveal that IFNγ induces a metabolic and phenotypic switch in tumor-associated lymphatic vessels that blocks regional metastasis and reinforces immune surveillance.

PURPOSE/OBJECTIVE:Patients with stage II and resected stage III melanomas have variable clinical outcomes, evidence of underlying biological differences in tumors and/or the patients themselves, beyond stage. The approval of adjuvant immunotherapy for stage IIB/C and resected stage III/IV disease (and adjuvant targeted therapy for resected stage III disease), has created a pressing need to develop biomarkers to accurately distinguish patients at low- versus high-risk for recurrence and death from melanoma. MicroRNAs (miRNAs) are promising biomarkers because of their stability in tissues/fluids and their demonstrated functional and prognostic roles in melanoma. We hypothesized that miRNA expression could be integrated into prognostic models that would more accurately classify 5-year survival outcomes than clinical factors alone. PATIENTS AND METHODS/METHODS:Using a Nanostring miRNA Expression Assay, we analyzed 715 primary melanomas from patients with stage II or stage III disease within the InterMEL consortium, and examined associations between miRNA expression and melanoma-specific death. RESULTS:When integrated into clinical prognostic models for 5-year melanoma-specific survival, miRNA signatures improved the area under the receiver operating characteristic curve (AUC) for stage II patients from 0.71 for a 'clinical factors-only' model to 0.81 for a 'clinical plus miRNA' model in an independent test set, an improvement of 0.10 with 95% CI (0.03, 0.19). The improvement was more modest for stage III patients that were included in the analysis. CONCLUSIONS:Incorporating miRNA expression in primary melanomas may enhance the accuracy of clinical prognostic models, and potentially aid the selection of melanoma patients for adjuvant treatment and clinical trials.

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.

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.

BACKGROUND:The introduction of Trastuzumab deruxtecan (T-Dxd) has exposed clinically significant limitations in accurately detecting HER2-low expression testing when using immunohistochemistry (IHC) assays originally developed to detect HER2 over-expression. While HER2 testing is widely used to determine T-Dxd eligibility, no HER2-low assay was ever validated against HER2 protein expression. METHODS:To address this pressing need, the Consortium for Analytic Standardization in Immunohistochemistry (CASI) conducted the CASI-01 study, involving 54 IHC laboratories across Europe and the U.S. The study aimed to identify optimal assay conditions for accurate HER2 testing, differentiating between HER2 overexpression (3+) for Trastuzumab eligibility and HER2-low expression (1+ or ultra-low) for T-Dxd eligibility. The conventional FDA-cleared HER2 assay ("predicate") was compared with higher-sensitivity assays using pathologist versus image analysis readouts. HER2 overexpression was validated against HER2 gene amplification via in situ hybridisation (ISH), while HER2-low accuracy was evaluated using newly introduced HER2 reference standards and a novel IHC parameter-dynamic range. FINDINGS/RESULTS:CASI-01 revealed variability in predicate HER2 assays, with detection thresholds ranging from 30,000 to 60,000 among laboratories. Despite this variability, these assays demonstrated high accuracy for identifying HER2 overexpression (3+), with 85.7% (18/21) sensitivity (95% confidence limits 63.66-96.95%) and 100% (49/49) specificity (95% confidence limits 92.75-100%), though sensitivity may have been limited by the use of older tissue specimens, with loss or reduced expression levels of the HER2 protein. However, these same assays exhibited poor dynamic range for detecting HER2-low scores. Enhanced analytic sensitivity of IHC assays combined with image analysis overcame this limitation with HER2-low scores, achieving a six-fold improvement (p = 0.0017). INTERPRETATION/CONCLUSIONS:IHC assays with detection thresholds in the range of 30,000-60,000 HER2 molecules per cell yield accurate results for determination of Trastuzumab eligibility (HER2 3+) but fail to demonstrate the dynamic range for accurate HER2-low scores. Enhanced analytic sensitivity of HER2 assays combined with image analysis addresses this critical gap in HER2-low testing. More generally, CASI-01 introduces pivotal advancements in precision medicine: (a) the importance of reporting IHC analytic sensitivity and ability to demonstrate an assay dynamic range, and (b) image analysis can surpass pathologist readout accuracy in specific clinical contexts. FUNDING/BACKGROUND:This work was supported by the National Cancer Institute of the National Institutes of Health under Award Number R44CA268484.

Targeted therapies and immunotherapy have improved treatment outcomes for many melanoma patients. However, patients whose melanomas harbor driver mutations in the neurofibromin 1 (NF1) tumor suppressor gene often lack effective targeted treatment options when their tumors do not respond to immunotherapy. In this study, we utilized patient-derived short-term cultures (STCs) and multiomics approaches to identify molecular features that could inform the development of therapies for patients with NF1 mutant melanoma. Differential gene expression analysis revealed that the epidermal growth factor receptor (EGFR) is highly expressed and active in NF1 mutant melanoma cells, where it hyper-activates ERK and AKT, leading to increased tumor cell proliferation, survival, and growth. In contrast, genetic or pharmacological inhibition of EGFR hindered cell proliferation and survival and suppressed tumor growth in patient-derived NF1 mutant melanoma models but not in NF1 wild-type models. These results reveal a connection between NF1 loss and increased EGFR expression that is critical for the survival and growth of NF1 mutant melanoma cells in patient-derived culture and xenograft models, irrespective of their BRAF and NRAS mutation status.