Genomic and Immunological Tumor Profiling Identifies Targetable Pathways and Extensive CD8+/PDL1+ Immune Infiltration in Inflammatory Breast Cancer Tumors.

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer that remains poorly understood at the molecular level. Comprehensive tumor profiling was performed to understand clinically actionable alterations in IBC. Targeted next-generation sequencing (NGS) and IHC were performed to identify activated pathways in IBC tumor tissues. siRNA studies examined the impact of IBC genomic variants in cellular models. IBC tumor tissues were further characterized for immune infiltration and immune checkpoint expression by IHC. Genomic analysis identified recurrent alterations in core biologic pathways, including activating and targetable variants in HER/PI3K/mTOR signaling. High rates of activating HER3 point mutations were discovered in IBC tumors. Cell line studies confirmed a role for mutant HER3 in IBC cell proliferation. Immunologic analysis revealed a subset of IBC tumors associated with high CD8(+)/PD-L1(+) lymphocyte infiltration. Immune infiltration positively correlated with an NGS-based estimate of neoantigen exposure derived from the somatic mutation rate and mutant allele frequency, iScore. Additionally, DNA mismatch repair alterations, which may contribute to higher iScores, occurred at greater frequency in tumors with higher immune infiltration. Our study identifies genomic alterations that mechanistically contribute to oncogenic signaling in IBC and provides a genetic basis for the selection of clinically relevant targeted and combination therapeutic strategies. Furthermore, an NGS-based estimate of neoantigen exposure developed in this study (iScore) may be a useful biomarker to predict immune infiltration in IBC and other cancers. The iScore may be associated with greater levels of response to immunotherapies, such as PD-L1/PD-1-targeted therapies. Mol Cancer Ther; 15(7); 1746-56. ©2016 AACR.

Expression of a 28-kilodalton glutathione S-transferase antigen of Schistosoma mansoni on the surface of filamentous phages and evaluation of its vaccine potential.

A cloning and expression system that allows display of proteins on the surface of filamentous phages was exploited to display a 28-kDa glutathione S-transferase (Sm28GST) antigen of the human parasite Schistosoma mansoni. The phage-displayed Sm28GST (pdGST) was immunoreactive and was recognized by immune sera, suggesting that the Sm28GST protein displayed on the surface of phages potentially maintains native conformation. Subsequent immunization studies showed that mice can develop high titers of antibodies against pdGST and do not require any additional adjuvant for immunization. Isotype analysis suggested that the pdGST immunization predominantly induced immunoglobulin G2b (IgG2b), IgG3, and IgM anti-GST antibodies in mice. Furthermore, the pdGST immunization was found to confer about 30% protection after a challenge infection with 100 cercariae of S. mansoni in BALB/c mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against infectious agents.

Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres.

Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5'-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.

Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1.

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.

Skin-stage schistosomula of Schistosoma mansoni produce an apoptosis-inducing factor that can cause apoptosis of T cells.

Skin-stage schistosomula of Schistosoma mansoni were found to secrete molecules that are pro-apoptotic for skin T lymphocytes as measured by annexin V staining, caspase-3 activity, caspase-8 activities, and DNA fragmentation. Caspase-8 activities in lymphocytes peaked approximately 8 h and caspase-3 activity peaked approximately 16 h after exposure to the parasite secretions. Subset analysis showed that mainly CD4(+) and CD8(+) cells (but not B cells) were susceptible to the parasite-induced pro-apoptotic effect. In situ staining confirmed the presence of apoptotic T cells around challenge parasites in the skin of naive or immunized animals. Analysis of T cells to identify the potential molecular pathway of the parasite-induced apoptosis showed increases in the expression of Fas, FasL, and the Fas-associated death domain. Blocking of FasL with a fusion protein reversed the parasite-induced apoptosis, suggesting a role for the Fas/FasL-mediated pathway in the parasite-induced T cell apoptosis. Subsequent analyses of the secretions of skin-stage schistosomula identified the pro-apoptotic activity as being associated with a protein of approximately 23 kDa. This protein was termed S. mansoni-derived apoptosis-inducing factor.

Cloning and characterization of a calcium-binding, histamine-releasing protein from Schistosoma mansoni.

A homologue of the mammalian translationally controlled tumor protein (TCTP) was cloned from the human parasite Schistosoma mansoni (SmTCTP). Sequence analysis showed that SmTCTP differed from other reported TCTPs in having only one signature sequence. Subsequently, SmTCTP was cloned in a T7 expression system and expressed as a histidine-tagged fusion protein. Recombinant SmTCTP (rSmTCTP) has a molecular mass of approximately 23 kDa with the histidine tag. Further analysis showed that SmTCTP transcripts and protein are expressed in all life cycle stages of the parasite within the vertebrate hosts. Interestingly, antibodies to SmTCTP were present in the sera of mice 9 weeks after infection with S. mansoni. Characterization studies showed that rSmTCTP is a calcium-binding protein that can cause histamine release from basophil/mast cells and induce eosinophil infiltration. These findings suggest that SmTCTP may have an important role in the development of allergic inflammatory responses associated with schistosomiasis and may be a target for new drug development.