Downregulation of IRF8 in alveolar macrophages by G-CSF promotes metastatic tumor progression.

Tissue-resident macrophages (TRMs) are abundant immune cells within pre-metastatic sites, yet their functional contributions to metastasis remain incompletely understood. Here, we show that alveolar macrophages (AMs), the main TRMs of the lung, are susceptible to downregulation of the immune stimulatory transcription factor IRF8, impairing anti-metastatic activity in models of metastatic breast cancer. G-CSF is a key tumor-associated factor (TAF) that acts upon AMs to reduce IRF8 levels and facilitate metastasis. Translational relevance of IRF8 downregulation was observed among macrophage precursors in breast cancer and a CD68hiIRF8loG-CSFhi gene signature suggests poorer prognosis in triple-negative breast cancer (TNBC), a G-CSF-expressing subtype. Our data highlight the underappreciated, pro-metastatic roles of AMs in response to G-CSF and identify the contribution of IRF8-deficient AMs to metastatic burden. AMs are an attractive target of local neoadjuvant G-CSF blockade to recover anti-metastatic activity.

Validation of epidermal AMBRA1 and loricrin (AMBLor) as a prognostic biomarker for nonulcerated American Joint Committee on Cancer stage I/II cutaneous melanoma.

BACKGROUND: Combined expression of the autophagy-regulatory protein AMBRA1 (activating molecule in Beclin1-regulated autophagy) and the terminal differentiation marker loricrin in the peritumoral epidermis of stage I melanomas can identify tumour subsets at low risk of -metastasis. OBJECTIVES: To validate the combined expression of peritumoral AMBRA1 and loricrin (AMBLor) as a prognostic biomarker able to identify both stage I and II melanomas at low risk of tumour recurrence. METHODS: Automated immunohistochemistry was used to analyse peritumoral AMBRA1 and loricrin expression in geographically distinct discovery (n = 540) and validation (n = 300) cohorts of nonulcerated American Joint Committee on Cancer (AJCC) stage I and II melanomas. AMBLor status was correlated with clinical outcomes in the discovery and validation cohorts separately and combined. RESULTS: Analysis of AMBLor in the discovery cohort revealed a recurrence-free survival (RFS) rate of 95.5% in the AMBLor low-risk group vs. 81.7% in the AMBLor at-risk group (multivariate log-rank, P < 0.001) and a negative predictive value (NPV) of 96.0%. In the validation cohort, AMBLor analysis revealed a RFS rate of 97.6% in the AMBLor low-risk group vs. 78.3% in the at-risk group (multivariate log-rank, P < 0.001) and a NPV of 97.6%. In a multivariate model considering AMBLor, Breslow thickness, age and sex, analysis of the combined discovery and validation cohorts showed that the estimated effect of AMBLor was statistically significant, with a hazard ratio of 3.469 (95% confidence interval 1.403-8.580, P = 0.007) and an overall NPV of 96.5%. CONCLUSIONS: These data provide further evidence validating AMBLor as a prognostic biomarker to identify nonulcerated AJCC stage I and II melanoma tumours at low risk of disease recurrence.

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma.

INTRODUCTION: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. METHODS: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). RESULTS: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). CONCLUSION: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

Manipulating adrenergic stress receptor signalling to enhance immunosuppression and prolong survival of vascularized composite tissue transplants.

BACKGROUND: Vascularized composite tissue allotransplantation (VCA) to replace limbs or faces damaged beyond repair is now possible. The resulting clear benefit to quality of life is a compelling reason to attempt this complex procedure. Unfortunately, the high doses of immunosuppressive drugs required to protect this type of allograft result in significant morbidity and mortality giving rise to ethical concerns about performing this surgery in patients with non-life-threatening conditions. Here we tested whether we could suppress anti-graft immune activity by using a safe ß2 -adrenergic receptor (AR) agonist, terbutaline, to mimic the natural immune suppression generated by nervous system-induced signalling through AR. METHODS: A heterotopic hind limb transplantation model was used with C57BL/6 (H-2b) as recipients and BALB/c (H-2d) mice as donors. To test the modulation of the immune response, graft survival was investigated after daily intraperitoneal injection of ß2 -AR agonist with and without tacrolimus. Analyses of immune compositions and quantification of pro-inflammatory cytokines were performed to gauge functional immunomodulation. The contributions to allograft survival of ß2 -AR signalling in donor and recipient tissue were investigated with ß2 -AR-/- strains. RESULTS: Treatment with the ß2 -AR agonist delayed VCA rejection, even with a subtherapeutic dose of tacrolimus. ß2 -AR agonist decreased T-cell infiltration into the transplanted grafts and decreased memory T-cell populations in recipient's circulation. In addition, decreased levels of inflammatory cytokines (IFN-γ, IL-6, TNF-α, CXCL-1/10 and CCL3/4/5/7) were detected following ß2 -AR agonist treatment, and there was a decreased expression of ICAM-1 and vascular cell adhesion molecule-1 in donor stromal cells. CONCLUSIONS: ß2 -AR agonist can be used safely to mimic the natural suppression of immune responses, which occurs during adrenergic stress-signalling and thereby can be used in combination regimens to reduce the dose needed of toxic immunosuppressive drugs such as tacrolimus. This strategy can be further evaluated for feasibility in the clinic.

Landscape of mutations in early stage primary cutaneous melanoma: An InterMEL study.

It is unclear why some melanomas aggressively metastasize while others remain indolent. Available studies employing multi-omic profiling of melanomas are based on large primary or metastatic tumors. We examine the genomic landscape of early-stage melanomas diagnosed prior to the modern era of immunological treatments. Untreated cases with Stage II/III cutaneous melanoma were identified from institutions throughout the United States, Australia and Spain. FFPE tumor sections were profiled for mutation, methylation and microRNAs. Preliminary results from mutation profiling and clinical pathologic correlates show the distribution of four driver mutation sub-types: 31% BRAF; 18% NRAS; 21% NF1; 26% Triple Wild Type. BRAF mutant tumors had younger age at diagnosis, more associated nevi, more tumor infiltrating lymphocytes, and fewer thick tumors although at generally more advanced stage. NF1 mutant tumors were frequent on the head/neck in older patients with severe solar elastosis, thicker tumors but in earlier stages. Triple Wild Type tumors were predominantly male, frequently on the leg, with more perineural invasion. Mutations in TERT, TP53, CDKN2A and ARID2 were observed often, with TP53 mutations occurring particularly frequently in the NF1 sub-type. The InterMEL study will provide the most extensive multi-omic profiling of early-stage melanoma to date. Initial results demonstrate a nuanced understanding of the mutational and clinicopathological landscape of these early-stage tumors.

Giant cutaneous squamous cell carcinoma of the scalp arising in the setting of folliculitis decalvans.

Squamous cell carcinoma (SCC) is uncommon in African Americans (AAs), with an incidence of approximately 0.003%. However, it is the most common skin cancer in that patient population. In AAs, SCC typically arises in sun-protected areas and mainly affects patients older than 50 years. We report a case of giant SCC in an AA man in his 40s with long-standing folliculitis decalvans on the scalp. Three previous skin biopsies were inconclusive. A wide excision was performed and the defect was reconstructed with an anterolateral thigh free flap. Histological analysis of the resected specimen revealed a well-moderately differentiated keratinising SCC with clear cell changes, severe mixed inflammation, folliculitis and dermal scar. He was discharged 2 weeks later and has been followed up closely. Four months later, the patient presents with metastatic SCC to an occipital lymph node.

Prognostic biomarkers of cutaneous melanoma.

BACKGROUND/PURPOSE: Melanomas account for only approximately 4% of diagnosed skin cancers in the United States but are responsible for the majority of deaths caused by skin cancer. Both genetic factors and ultraviolet (UV) radiation exposure play a role in the development of melanoma. Although melanomas have a strong propensity to metastasize when diagnosed late, melanomas that are diagnosed and treated early pose a low mortality risk. In particular, the identification of patients with increased metastatic risk, who may benefit from early adjuvant therapies, is crucial, especially given the advent of new melanoma treatments. However, the accuracy of classic clinical and histological variables, including the Breslow thickness, presence of ulceration, and lymph node status, might not be sufficient to identify such individuals. Thus, there is a need for the development of additional prognostic melanoma biomarkers that can improve early attempts to stratify melanoma patients and reliably identify high-risk subgroups with the aim of providing effective personalized therapies. METHODS: In our current work, we discuss and assess emerging primary melanoma tumor biomarkers and prognostic circulating biomarkers. RESULTS: Several promising biomarkers show prognostic value (eg, exosomal MIA (ie, melanoma inhibitory activity), serum S100B, AMLo signatures, and mRNA signatures); however, the scarcity of reliable data precludes the use of these biomarkers in current clinical applications. CONCLUSION: Further research is needed on several promising biomarkers for melanoma. Large-scale studies are warranted to facilitate the clinical translation of prognostic biomarker applications for melanoma in personalized medicine.

AT-RvD1 Mitigates Secondhand Smoke-Exacerbated Pulmonary Inflammation and Restores Secondhand Smoke-Suppressed Antibacterial Immunity.

Cigarette smoke is a potent proinflammatory trigger contributing to acute lung injury and the development of chronic lung diseases via mechanisms that include the impairment of inflammation resolution. We have previously demonstrated that secondhand smoke (SHS) exposure exacerbates bacterial infection-induced pulmonary inflammation and suppresses immune responses. It is now recognized that resolution of inflammation is a bioactive process mediated by lipid-derived specialized proresolving mediators that counterregulate proinflammatory signaling and promote resolution pathways. We therefore hypothesized that proresolving mediators could reduce the burden of inflammation due to chronic lung infection following SHS exposure and restore normal immune responses to respiratory pathogens. To address this question, we exposed mice to SHS followed by chronic infection with nontypeable Haemophilus influenzae (NTHI). Some groups of mice were treated with aspirin-triggered resolvin D1 (AT-RvD1) during the latter half of the smoke exposure period or during a period of smoking cessation and before infection. Treatment with AT-RvD1 markedly reduced the recruitment of neutrophils, macrophages, and T cells in lung tissue and bronchoalveolar lavage and levels of proinflammatory cytokines in the bronchoalveolar lavage. Additionally, treatment with AT-RvD1 improved Ab titers against the NTHI outer membrane lipoprotein Ag P6 following infection. Furthermore, treatment with AT-RvD1 prior to classically adjuvanted immunization with P6 increased Ag-specific Ab titers, resulting in rapid clearance of NTHI from the lungs after acute challenge. Collectively, we have demonstrated that AT-RvD1 potently reverses the detrimental effects of SHS on pulmonary inflammation and immunity and thus could be beneficial in reducing lung injury associated with smoke exposure and infection.

Cutaneous Leiomyosarcoma: A SEER Database Analysis.

BACKGROUND: Cutaneous leiomyosarcoma is a rare dermal neoplasm usually arising from the pilar smooth muscle. It is considered a relatively indolent neoplasm, and there is debate whether designation as sarcoma is appropriate. Owing to some conflicting data in the literature, however, its behavior warrants further clarification. OBJECTIVE: To determine the clinical behavior and demographic and pathologic characteristics of cutaneous leiomyosarcoma. MATERIALS AND METHODS: The Surveillance, Epidemiology and End Results database was used to collect data on cutaneous leiomyosarcoma and 2 reference populations: cutaneous angiosarcoma (aggressive) and atypical fibroxanthoma (indolent). Demographic and oncologic characteristics were examined, and overall survivals (OS) and disease-specific survivals were compared. RESULTS: Leiomyosarcoma and atypical fibroxanthoma displayed lower stage (localized: 69.7% and 66.8% respectively), smaller size (<3 cm: 90.5% and 72%), and lower rates of disease-specific mortality (2.9% and 7.8%) compared with angiosarcoma. Patients with leiomyosarcoma had a 5-year disease-specific survival rate of 98% and OS rate of 85%. CONCLUSION: Cutaneous leiomyosarcoma shows outcomes similar to atypical fibroxanthoma. It is nearly always indolent and should be distinguished from more aggressive cutaneous and subcutaneous sarcomas. Clear communication of the biologic potential may be best achieved using alternate diagnostic terminology such as "atypical intradermal smooth-muscle neoplasm."

Recombinant Sialyltransferase Infusion Mitigates Infection-Driven Acute Lung Inflammation.

Inappropriate inflammation exacerbates a vast array of chronic and acute conditions with severe health risks. In certain situations, such as acute sepsis, traditional therapies may be inadequate in preventing severe organ damage or death. We have previously shown cell surface glycan modification by the circulating sialyltransferase ST6Gal-1 regulates de novo inflammatory cell production via a novel extrinsic glycosylation pathway. Here, we show that therapeutic administration of recombinant, bioactive ST6Gal-1 (rST6G) mitigates acute inflammation in a murine model mimicking acute exacerbations experienced by patients with chronic obstructive pulmonary disease (COPD). In addition to suppressing proximal neutrophil recruitment at onset of infection-mediated inflammation, rST6G also muted local cytokine production. Histologically, exposure with NTHI, a bacterium associated with COPD exacerbations, in rST6G-treated animals revealed consistent and pronounced reduction of pulmonary inflammation, characterized by smaller inflammatory cuffs around bronchovascular bundles, and fewer inflammatory cells within alveolar walls, alveolar spaces, and on pleural surfaces. Taken together, the data advance the idea that manipulating circulatory ST6Gal-1 levels has potential in managing inflammatory conditions by leveraging the combined approaches of controlling new inflammatory cell production and dampening the inflammation mediator cascade.