Faculty Bibliography

Tumor mutations can influence the surrounding microenvironment leading to suppression of anti-tumor immune responses and thereby contributing to tumor progression and failure of cancer therapies. Here we use genetically engineered lung cancer mouse models and patient samples to dissect how LKB1 mutations accelerate tumor growth by reshaping the immune microenvironment. Comprehensive immune profiling of LKB1 -mutant vs wildtype tumors revealed dramatic changes in myeloid cells, specifically enrichment of Arg1 ⁺ interstitial macrophages and SiglecF ^Hi neutrophils. We discovered a novel mechanism whereby autocrine LIF signaling in Lkb1 -mutant tumors drives tumorigenesis by reprogramming myeloid cells in the immune microenvironment. Inhibiting LIF signaling in Lkb1 -mutant tumors, via gene targeting or with a neutralizing antibody, resulted in a striking reduction in Arg1 ⁺ interstitial macrophages and SiglecF ^Hi neutrophils, expansion of antigen specific T cells, and inhibition of tumor progression. Thus, targeting LIF signaling provides a new therapeutic approach to reverse the immunosuppressive microenvironment of LKB1 -mutant tumors.

Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.

Calcific aortic valve disease (CAVD) and stenosis have a complex pathogenesis, and no therapies are available that can halt or slow their progression. Several studies have shown the presence of apolipoprotein-related amyloid deposits in close proximity to calcified areas in diseased aortic valves. In this Perspective, we explore a possible relationship between amyloid deposits, calcification and the development of aortic valve stenosis. These amyloid deposits might contribute to the amplification of the inflammatory cycle in the aortic valve, including extracellular matrix remodelling and myofibroblast and osteoblast-like cell proliferation. Further investigation in this area is needed to characterize the amyloid deposits associated with CAVD, which could allow the use of antisense oligonucleotides and/or isotype gene therapies for the prevention and/or treatment of CAVD.

BACKGROUND:Mitral valve (MV) elongation is a primary hypertrophic cardiomyopathy (HCM) phenotype and contributes to obstruction. The residual MV leaflet that protrudes past the coaptation point is especially susceptible to flow-drag and systolic anterior motion. Histopathological features of MVs in obstructive hypertrophic cardiomyopathy (OHCM), and of residual leaflets specifically, are unknown. OBJECTIVES/OBJECTIVE:The purpose of this study was to characterize gross, structural, and cellular histopathologic features of MV residual leaflets in OHCM. On a cellular-level, we assessed for developmental dysregulation of epicardium-derived cell (EPDC) differentiation, adaptive endocardial-to-mesenchymal transition and valvular interstitial cell proliferation, and genetically-driven persistence of cardiomyocytes in the valve. METHODS:Structural and immunohistochemical staining were performed on 22 residual leaflets excised as ancillary procedures during myectomy, and compared with 11 control leaflets from deceased patients with normal hearts. Structural components were assessed with hematoxylin and eosin, trichrome, and elastic stains. We stained for EPDCs, EPDC paracrine signaling, valvular interstitial cells, endocardial-to-mesenchymal transition, and cardiomyocytes. RESULTS:= 0.08). No markers of primary cellular processes were identified. CONCLUSIONS:MV residual leaflets in HCM were characterized by histologic findings that were likely secondary to chronic hemodynamic stress and may further increase susceptibility to systolic anterior motion.

BACKGROUND:edition of UICC/AJCC TNM classification system the primary tumor pT stage is determined based on presence and size of the invasive components. The aim of this study was to identify histological features in tumors with lepidic growth pattern that may be used to establish criteria for distinguishing invasive from non-invasive areas. MATERIALS AND METHODS/METHODS:A Delphi approach was used with two rounds of blinded anonymized analysis of resected non-mucinous lung adenocarcinoma cases with presumed invasive and non-invasive components, followed by one round of reviewer de-anonymized and unblinded review of cases with known outcomes. A digital pathology platform was used for measuring total tumor size and invasive tumor size. RESULTS:The mean coefficient of variation for measuring total tumor size and tumor invasive size was 6.9% (range 1.7-22.3%) and 54% (range 14.7-155%), respectively, with substantial variations in interpretation of the size and location of invasion among pathologists. Following the presentation of the results and further discussion among members at large of the IASLC Pathology Committee, extensive epithelial proliferation (EEP) in areas of collapsed lepidic growth pattern is recognized as a feature likely to be associated with invasive growth. EEP is characterized by multilayered luminal epithelial cell growth, usually with high grade cytological features in several alveolar spaces. CONCLUSION/CONCLUSIONS:Collapsed alveoli and transition zones with EEP were identified by the Delphi process as morphologic features that were a source of interobserver variability. Definition criteria for collapse and EEP are proposed to improve reproducibility of invasion measurement.

Background: In mediastinal biopsies that show fibrosis, the differential diagnosis includes fibrosing mediastinitis, immunoglobulin G subclass 4-related disease, Hodgkin lymphoma, as well as reactive fibrotic and inflammatory changes adjacent to other processes including neoplasms. Cases Description: We report two cases of incidentally detected mediastinal seminoma that contained extensive areas of paucicellular fibrosis, which precluded accurate preoperative biopsy diagnosis. The fibrosis consisted of mildly inflamed, densely scarred tissue with thin dilated vessels, and was present to a significant extent that is suggestive of spontaneous regression. These features are not currently described in the World Health Organization Classification of Thoracic Tumors. In both patients, needle and open biopsies sampled only the fibrotic areas of the tumors, and the final diagnosis was not achieved until surgical excision was performed. After surgery, both patients received chemotherapy, and were alive without evidence of disease at 3.4 years and 1 year post-operatively, respectively. Tumor fibrosis composed approximately 95% and 50% of each patient"™s tumor, respectively. In one of the patients, correlation of the needle biopsy position with the positron emission tomography (PET) scan revealed that the biopsy needle had sampled a non-metabolically active portion of the tumor. Conclusions: While pathologic spontaneous regression is well-described in gonadal germ cell tumors, it is not well-reported in extragonadal locations. Prospective knowledge of this diagnostic pitfall and targeting PET-avid regions of the tumor may increase the diagnostic yield and help to avoid non-indicated surgical interventions.

Lung squamous cell carcinoma (LUSC) represents a major subtype of lung cancer with limited treatment options. KMT2D is one of the most frequently mutated genes in LUSC (>20%), and yet its role in LUSC oncogenesis remains unknown. Here, we identify KMT2D as a key regulator of LUSC tumorigenesis wherein Kmt2d deletion transforms lung basal cell organoids to LUSC. Kmt2d loss increases activation of receptor tyrosine kinases (RTKs), EGFR and ERBB2, partly through reprogramming the chromatin landscape to repress the expression of protein tyrosine phosphatases. These events provoke a robust elevation in the oncogenic RTK-RAS signaling. Combining SHP2 inhibitor SHP099 and pan-ERBB inhibitor afatinib inhibits lung tumor growth in Kmt2d-deficient LUSC murine models and in patient-derived xenografts (PDXs) harboring KMT2D mutations. Our study identifies KMT2D as a pivotal epigenetic modulator for LUSC oncogenesis and suggests that KMT2D loss renders LUSC therapeutically vulnerable to RTK-RAS inhibition.

In the version of this article initially published, Giampaolo Merlini (IRCCS Foundation Policlinico San Matteo, Pavia, Italy) was shown with an incorrect affiliation, which has now been corrected in the HTML and PDF versions of the article.

BACKGROUND:The attenuated, genetically engineered vaccinia virus has been shown to be a promising oncolytic virus for the treatment of patients with solid tumors, through both direct cytotoxic and immune-activating effects. Whereas systemically administered oncolytic viruses can be neutralized by pre-existing antibodies, locoregionally administered viruses can infect tumor cells and generate immune responses. We conducted a phase I clinical trial to investigate the safety, feasibility and immune activating effects of intrapleural administration of oncolytic vaccinia virus (NCT01766739). METHODS:Eighteen patients with malignant pleural effusion due to either malignant pleural mesothelioma or metastatic disease (non-small cell lung cancer or breast cancer) underwent intrapleural administration of the oncolytic vaccinia virus using a dose-escalating method, following drainage of malignant pleural effusion. The primary objective of this trial was to determine a recommended dose of attenuated vaccinia virus. The secondary objectives were to assess feasibility, safety and tolerability; evaluate viral presence in the tumor and serum as well as viral shedding in pleural fluid, sputum, and urine; and evaluate anti-vaccinia virus immune response. Correlative analyses were performed on body fluids, peripheral blood, and tumor specimens obtained from pre- and post-treatment timepoints. RESULTS:Treatment with attenuated vaccinia virus at the dose of 1.00E+07 plaque-forming units (PFU) to 6.00E+09 PFU was feasible and safe, with no treatment-associated mortalities or dose-limiting toxicities. Vaccinia virus was detectable in tumor cells 2-5 days post-treatment, and treatment was associated with a decrease in tumor cell density and an increase in immune cell density as assessed by a pathologist blinded to the clinical observations. An increase in both effector (CD8+, NK, cytotoxic cells) and suppressor (Tregs) immune cell populations was observed following treatment. Dendritic cell and neutrophil populations were also increased, and immune effector and immune checkpoint proteins (granzyme B, perforin, PD-1, PD-L1, and PD-L2) and cytokines (IFN-γ, TNF-α, TGFβ1 and RANTES) were upregulated. CONCLUSION:The intrapleural administration of oncolytic vaccinia viral therapy is safe and feasible and generates regional immune response without overt systemic symptoms. CLINICAL TRIAL REGISTRATION:https://clinicaltrials.gov/ct2/show/NCT01766739, identifier NCT01766739.