PD-L1 Protein Expression on Both Tumor Cells and Macrophages are Associated with Response to Neoadjuvant Durvalumab with Chemotherapy in Triple-negative Breast Cancer.

PURPOSE: In both the IMpassion 130 trial in the metastatic setting and in Keynote 522 in the neoadjuvant setting, patients with triple-negative breast cancer (TNBC) showed benefit from PD-1 axis immunotherapy. Here, we assess PD-L1 expression on both tumor and immune cells using quantitative immunofluorescence to assess association with benefit from neoadjuvant durvalumab concurrent with chemotherapy in TNBC. EXPERIMENTAL DESIGN: Pretreatment core needle biopsies (n = 69) were obtained from patients who participated in a phase I/II clinical trial (NCT02489448). The final analysis included 45 patients [pathologic complete response (pCR) = 18, non-pCR = 27] due to technical issues and insufficient tissue. Slides were stained using a previously validated Ultivue DNA-based Ultimapper kit (CD8, CD68, PD-L1, Cytokeratin/Sox10, and Hoechst counterstain). The PD-L1 expression was analyzed by molecular compartmentalization without segmentation using AQUA software (version 3.2.2.1) in three tissue compartments including tumor (cytokeratin-positive cells), CD68+ cells, and overall stroma. RESULTS: In patients with pCR, PD-L1 expression was significantly higher in tumor cells, in CD68+ cells and in the stroma compared with patients non-pCR. There was no difference in the amount of CD68+ cells in the tumor or stromal compartments between cases with pCR and non-pCR. CONCLUSIONS: Expression of PD-L1 in tumor cells, immune cells in stroma, and colocalized with CD68+ cells is associated with higher rates of pCR to durvalumab and chemotherapy in TNBC.

The InSituPlex® Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples.

Multiplexed immunohistochemistry (mIHC) enables the detection, quantification, and localization of many markers within cell or tissue samples, leading to a better understanding of the architecture of a disease at the cellular level. Current mIHC techniques involve long staining and assay times, require dedicated and/or captive instrumentation, and entail tedious assay optimization, hindering their establishment as routine methods. Here, we demonstrate the use of the InSituPlex® method for spatial profiling of immuno-oncology targets in FFPE tumor tissue with the UltiMapper™ I/O PD-L1 multiplex assay. The panel consists of five protein markers to profile immune infiltration and PD-L1 expression and includes CD8, CD68, PD-L1, pan CK, and SOX10 markers. The assay shows benefits of high and low expression of markers, coexpression and colocalization of proteins in single cells, and completion of staining and image acquisition in 5.5 h. Through the combination of multiplexed characterization of protein expression in whole tissue sections, fast staining workflow, and compatibility with existing instrumentation, the InSituPlex method provides a robust modality for deep phenotyping of the tumor and its microenvironment.

Respiratory Chlamydia Infection Induce Release of Hepoxilin A3 and Histamine Production by Airway Neutrophils.

Background: Hepoxilins are biologically active metabolites of arachidonic acid that are formed through the 12-lipoxygenase pathway. Hepoxilin A3 is now known to be an important regulator of mucosal inflammation in response to infection by bacterial pathogens and was recently identified as a potent neutrophil chemoattractant in the intestinal mucosa. Our goal in this study was to determine if airway infection with Chlamydia in a murine model of allergic airway disease (AAD) induces hepoxilin secretion along with airway neutrophilia. Methods: We utilized an AAD adult Balb/c mouse model to evaluate airway pathology and immune response by assaying bronchoalveolar lavage (BAL) fluid cytokine, cellularity, histidine decarboxylase (HDC) as well as histamine released in response to in-vivo chlamydial antigen stimulation of purified airway neutrophils. Hepoxilin A3 production was determined by Western blot identification of 12-lipoxygenase precursor (12-LO). Results: Chlamydial infection induced increased production of IL-2, IL-12, TNF-α, and IFN-γ in BAL fluid compared to uninfected animals. Chlamydia-infected mice responded with robust airway neutrophil infiltration and upon induction of AAD increased their production of IL-4, IL-5, and IL-13 by >3 fold compared to unsensitized groups. In addition, 12-LO mRNA was upregulated in infected, but not in uninfected AAD mice, suggesting the production of hepoxilin A3. mRNA expression of HDC was induced only in neutrophils from the airways of Chlamydia-infected mice, but was not seen in AAD only or uninfected controls. When purified neutrophils from infected animals were challenged with chlamydial antigen in vitro there was significant histamine release. Conclusions: Our data confirms the production and release of hepoxilin A3 in the murine airways concomitant with airway neutrophilia in response to chlamydial infection. We further confirmed that Chlamydia provokes the production and release of histamine by these neutrophils. These findings suggest that neutrophils, provoked by Chlamydia infection can synthesize and release histamine, thereby contributing directly to airway inflammation.

Evidence of infectious asthma phenotype: Chlamydia-induced allergy and pathogen-specific IgE in a neonatal mouse model.

Asthma is a chronic respiratory disease whose etiology is poorly understood. Recent studies suggest that early-life respiratory infections with atypical bacteria may play an important role in the induction or exacerbation of chronic respiratory disease. The current study utilized a neonatal mouse ovalbumin (OVA) sensitization model of asthma to determine the course of early-life respiratory tract infection by Chlamydia. Neonatal (day 1) and adult (6 wks) BALB/c mice were infected intranasally with Chlamydia (MoPn) and 7 weeks later were sensitized and challenged with ovalbumin. Allergic airway disease was characterized by examination of serum and bronchoalveolar lavage fluid (BAL) cellularity, cytokine production and antibody response. The presence of Chlamydia was determined by PCR and culture. Ova-specific IgE was quantified by ELISA and Chlamydia-specific IgE was determined via Western blot analysis. Chlamydial infection in neonatal mice induced increased production of Th2 cytokines (IL-4, 5, 10, and 13) in both BAL and serum, while infected adult mice produced increased Th1 cytokines (IL-2, IFN-γ). The BAL from infected neonates contained significantly elevated levels of eosinophils compared to infected adult mice. Although adult mice cleared the infection ∼30 days post infection (pi), neonates were still infected 66 days after initial infection. Chlamydia-specific IgE was detected in both the BAL and serum of neonatal mice beginning 28 days post infection, however, infected adult mice did not produce Chlamydia-specific IgE antibodies over the course of the study. When allergic airway was induced using Ova, infected neonatal mice increased their production of IL-4, IL-5 and IL-13 by >2 fold compared to uninfected controls and infected adult groups. Our findings demonstrate that early-life Chlamydia infection induces a Th2-dominant cytokine response in the airways of neonatal mice, leading to chronic infection. More significantly, early life respiratory colonization with Chlamydia elicits pathogen-specific IgE production, which further supports an infectious asthma phenotype.

Chlamydia pneumoniae-specific IgE is prevalent in asthma and is associated with disease severity.

BACKGROUND: Several Chlamydia pneumoniae (Cp) biomarkers have been associated with asthma but Cp-specific IgE (Cp IgE) has not been investigated extensively. Our objective was to investigate Cp IgE in community adult asthma patients. METHODS: (1) Prevalence of Cp IgE (measured by immunoblotting) and Cp DNA (by polymerase chain reaction) in peripheral blood, and biomarker associations with asthma severity. (2) Case-control studies of Cp IgE association with asthma using healthy blood donor (study 1) and non-asthmatic clinic patient (study 2) controls. RESULTS: Of 66 asthma subjects (mean age 40.9 years, range 5-75, 59% male, 45% ever-smokers) 33 (50%) were Cp IgE positive and 16 (24%) were Cp DNA positive (P = 0.001 for association of Cp IgE and DNA). Cp IgE was detected in 21% of mild intermittent asthma v 79% of severe persistent asthma (test for trend over severity categories, P = 0.002). Cp IgE detection was significantly (P = 0.001) associated with asthma when compared to healthy blood donor controls but not when compared to clinic controls. CONCLUSIONS: Half of this sample of community asthma patients had detectable IgE against C. pneumoniae. Cp IgE was strongly and positively associated with asthma severity and with asthma when healthy blood donor controls were used. These results support the inclusion of Cp IgE as a biomarker in future studies of infectious contributions to asthma pathogenesis.

The prevalence and identity of Chlamydia-specific IgE in children with asthma and other chronic respiratory symptoms.

BACKGROUND: Recent studies have confirmed the presence of viable Chlamydia in the bronchoalveolar lavage (BAL) fluid of pediatric patients with airway hyperresponsiveness. While specific IgG and IgM responses to C. pneumoniae are well described, the response and potential contribution of Ag-specific IgE are not known. The current study sought to determine if infection with Chlamydia triggers the production of pathogen-specific IgE in children with chronic respiratory diseases which might contribute to inflammation and pathology. METHODS: We obtained BAL fluid and serum from pediatric respiratory disease patients who were generally unresponsive to corticosteroid treatment as well as sera from age-matched control patients who saw their doctor for wellness checkups. Chlamydia-specific IgE was isolated from BAL and serum samples and their specificity determined by Western blot techniques. The presence of Chlamydia was confirmed by species-specific PCR and BAL culture assays. RESULTS: Chlamydial DNA was detected in the BAL fluid of 134/197 (68%) patients. Total IgE increased with age until 15 years old and then decreased. Chlamydia-specific IgE was detected in the serum and/or BAL of 107/197 (54%) patients suffering from chronic respiratory disease, but in none of the 35 healthy control sera (p < 0.0001). Of the 74 BAL culture-positive patients, 68 (91.9%, p = 0.0001) tested positive for Chlamydia-specific IgE. Asthmatic patients had significantly higher IgE levels compared to non-asthmatics (p = 0.0001). Patients who were positive for Chlamydia DNA or culture had significantly higher levels of serum IgE compared to negative patients (p = 0.0071 and p = 0.0001 respectively). Only 6 chlamydial antigens induced Chlamydia-specific IgE and patients with C. pneumoniae-specific IgE had significantly greater levels of total IgE compared to C. pneumoniae-specific IgE negative ones (p = 0.0001). CONCLUSIONS: IgE antibodies play a central role in allergic inflammation; therefore production of Chlamydia-specific IgE may prove significant in the exacerbation of chronic, allergic airway diseases, thus highlighting a direct role for Chlamydia in asthma pathogenesis.

Colonization of paediatric lower respiratory tract with genital Mycoplasma species.

BACKGROUND AND OBJECTIVE: Recently, much attention has been given to the possible role played by pathogens that colonize neonatal or paediatric airway and their potential involvement in chronic respiratory disease. The goal of the current study was to evaluate the prevalence of Mycoplasma organisms in the BAL fluid of paediatric patients suffering from a variety of chronic respiratory diseases to determine if there was any clear disease association with bacterial presence. METHODS: We examined 319 paediatric BAL samples for the presence of M.genitalium, M.hominis, U.urealyticum, U.parvum and M.pneumoniae DNA with species-specific PCR. RESULTS: Mycoplasma DNA was found in 32.6% (104/319) of patient samples; 10% (32/319) for M.pneumoniae, 8.8% for U.parvum, 2.8% for U.urealyticum; 4.7% for M.hominis and 9.1% for M.genitalium. There were no significant clinical and laboratory differences except serum IgE in asthmatics according to Mycoplasma colonization or not. Elevated levels of IgE were found more often in Mycoplasma DNA-negative patients than patients with bacterial DNA, 85/109 versus 24/109 respectively (P<0.0001). There was no difference in the frequency of Mycoplasmas between the asthmatics and the non-asthmatics; 30.6% (69/225) versus 37.2% (35/94) for Mycoplasma 16S DNA, and 8% versus 14.9% for M.pneumoniae, respectively. CONCLUSIONS: Our data indicate that in addition to M.pneumoniae, urogenital Mycoplasma species may colonize the airway of patients with chronic respiratory diseases. There was, however, no association between chronic asthma diagnosis and Mycoplasma colonization in this study.

Infectious Chlamydia pneumoniae is associated with elevated interleukin-8 and airway neutrophilia in children with refractory asthma.

BACKGROUND: Neutrophilic asthma is thought to be less responsive than eosinophilic asthma to anti-inflammatory therapies including corticosteroids. Chlamydia pneumoniae has been implicated in asthma, possibly by induction of interleukin (IL-8). We hypothesized that IL-8 is increased in the bronchoalveolar lavage (BAL) fluid from children with asthma and C. pneumoniae. METHODS: BAL fluid was analyzed for C. pneumoniae and IL-8 using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay from 2 asthma patient populations in the Bronx, NY and Massachusetts with an average age of 8 and 8.7 years old, respectively. For comparison, samples were also analyzed for C. trachomatis and Mycoplasma 16s DNA. RESULTS: Of 18 Bronx samples analyzed, 6 (33%) were PCR-positive for C. pneumoniae, 10 (56%) for C. trachomatis, and 8 (44%) for Mycoplasma 16s DNA. IL-8 from C. pneumoniae-positive samples was 3.3-fold higher compared with negative samples (P = 0.003). There was no difference between patients tested for C. trachomatis or Mycoplasma. Of 84 Massachusetts samples analyzed, 42 (50%) were PCR-positive for C. pneumoniae, 42 (50%) for C. trachomatis, and 13 (16%) for Mycoplasma. IL-8 concentration from C. pneumoniae-positive samples was 10.49-fold higher compared with negative samples (P = 0.0001). As in the Bronx cohort, there were no differences between patients tested for C. trachomatis or Mycoplasma. Lastly, BAL neutrophilia predicted the presence of C. pneumoniae but not Mycoplasma or C. trachomatis. CONCLUSIONS: Children with asthma who were PCR-positive for C. pneumoniae demonstrated elevated concentrations of IL-8 and neutrophils in BAL fluid compared with similar patients who were positive for C. trachomatis or Mycoplasma organisms, but PCR-negative for C. pneumoniae. Undiagnosed C. pneumoniae infection in children may therefore contribute to poorly controlled asthma via induction of IL-8.