Immune responses of lung transplant recipients against SARS-CoV-2 and common respiratory coronaviruses: Evidence for pre-existing cross-reactive immunity.

Humoral and cellular immune responses to SARS-CoV-2 and other coronaviruses in lung transplant recipients are unknown. We measured antibodies and T cell responses against the SARS-CoV-2 spike S2 and nucleocapsid antigens and spike antigens from common respiratory coronaviruses (229E, NL63, OC43, and HKU1) after vaccination or infection of LTxRs. 148 LTxRs from single center were included in this study: 98 after vaccination and 50 following SARS-CoV-2 infection. Antibodies were quantified by enzyme-linked immunosorbent assay. The frequency of T cells secreting IL2, IL4, IL10, IL17, TNFα, and IFNγ were enumerated by enzyme-linked immunospot assay. Our results have shown the development of antibodies to SARS-CoV-2 spike protein in infected LTxRs (39/50) and vaccinated LTxRs (52/98). Vaccinated LTxRs had higher number of T cells producing TNFα but less cells producing IFNγ than infected LTxRs in response to the nucleocapsid antigen and other coronavirus spike antigens. We didn't find correlation between the development of antibodies and cellular immune responses against the SARS-CoV-2 spike protein after vaccination. Instead, LTxRs have pre-existing cellular immunity to common respiratory coronaviruses, leading to cross-reactive immunity against SARS-CoV-2 which likely will provide protection against SARS-Cov-2 infection.

Cytosolic EpCAM cooperates with H-Ras to regulate epithelial to mesenchymal transition through ZEB1.

Next generation sequencing of human cancer mutations has identified novel therapeutic targets. Activating Ras oncogene mutations play a central role in oncogenesis, and Ras-driven tumorigenesis upregulates an array of genes and signaling cascades that can transform normal cells into tumor cells. In this study, we investigated the role of altered localization of epithelial cell adhesion molecule (EpCAM) in Ras-expressing cells. Analysis of microarray data demonstrated that Ras expression induced EpCAM expression in normal breast epithelial cells. Fluorescent and confocal microscopy showed that H-Ras mediated transformation also promoted epithelial-to-mesenchymal transition (EMT) together with EpCAM. To consistently localize EpCAM in the cytosol, we generated a cancer-associated EpCAM mutant (EpCAM-L240A) that is retained in the cytosol compartment. Normal MCF-10A cells were transduced with H-Ras together with EpCAM wild-type (WT) or EpCAM-L240A. WT-EpCAM marginally effected invasion, proliferation, and soft agar growth. EpCAM-L240A, however, markedly altered cells and transformed to mesenchymal phenotype. Ras-EpCAM-L240A expression also promoted expression of EMT factors FRA1, ZEB1 with inflammatory cytokines IL-6, IL-8, and IL1. This altered morphology was reversed using MEK-specific inhibitors and to some extent JNK inhibition. Furthermore, these transformed cells were sensitized to apoptosis using paclitaxel and quercetin, but not other therapies. For the first time, we have demonstrated that EpCAM mutations can cooperate with H-Ras and promote EMT. Collectively, our results highlight future therapeutic opportunities in EpCAM and Ras mutated cancers.

Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

Accumulation of senescent cells contributes to age-related diseases including idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding proteins (IGFBPs) regulate many biological processes; however, the functional contributions of IGFBP2 in lung fibrosis remain largely unclear. Here, we report that intranasal delivery of recombinant IGFBP2 protects aged mice from weight loss and demonstrated antifibrotic effects after bleomycin lung injury. Notably, aged human-Igfbp2 transgenic mice reveal reduced senescence and senescent-associated secretory phenotype factors in alveolar epithelial type 2 (AEC2) cells and they ameliorated bleomycin-induced lung fibrosis. Finally, we demonstrate that IGFBP2 expression is significantly suppressed in AEC2 cells isolated from fibrotic lung regions of patients with IPF and/or pulmonary hypertension compared with patients with hypersensitivity pneumonitis and/or chronic obstructive pulmonary disease. Altogether, our study provides insights into how IGFBP2 regulates AEC2-cell-specific senescence and that restoring IGFBP2 levels in fibrotic lungs can prove effective for patients with IPF.

Downregulation of a tumor suppressor gene LKB1 in lung transplantation as a biomarker for chronic murine lung allograft rejection.

BACKGROUND: We recently demonstrated decreased tumor suppressor gene liver kinase B1 (LKB1) level in lung transplant recipients diagnosed with bronchiolitis obliterans syndrome. STE20-related adaptor alpha (STRADα) functions as a pseudokinase that binds and regulates LKB1 activity. METHODS: A murine model of chronic lung allograft rejection in which a single lung from a B6D2F1 mouse was orthotopically transplanted into a DBA/2J mouse was employed. We examined the effect of LKB1 knockdown using CRISPR-CAS9 in vitro culture system. RESULTS: Significant downregulation of LKB1 and STRADα expression was found in donor lung compared to recipient lung. STRADα knockdown significantly inhibited LKB1, pAMPK expression but induced phosphorylated mammalian target of rapamycin (mTOR), fibronectin, and Collagen-I, expression in BEAS-2B cells. LKB1 overexpression decreased fibronectin, Collagen-I, and phosphorylated mTOR expression in A549 cells. CONCLUSIONS: We demonstrated that downregulation of LKB1-STRADα pathway accompanied with increased fibrosis, results in development of chronic rejection following murine lung transplantation.

Novel role for tumor suppressor gene, liver kinase B1, in epithelial-mesenchymal transition leading to chronic lung allograft dysfunction.

Epithelial-mesenchymal transition (EMT) has been implicated to play a role in chronic lung allograft dysfunction (CLAD). Liver kinase B1 (LKB1), a tumor suppressor gene, can regulate EMT. However, its role in CLAD development following lung transplantation remains unknown. Using qRT-PCR, biopsies from lung transplant recipients with bronchiolitis obliterans syndrome (BOS) demonstrated significant downregulation of LKB1 (p = .0001), compared to stable biopsies. To determine the role of LKB1 in EMT development, we analyzed EMT in human bronchial epithelial cell line BEAS-2B. Knockdown of LKB1 by siRNA significantly dysregulated mesenchymal markers expression in BEAS-2B cells. Following incubation of human primary bronchial epithelial cell or BEAS-2B cells with exosomes isolated from BOS or stable lung transplant recipients, LKB1 expression was inhibited when incubated with BOS-exosome. Incubation with BOS-exosomes also decreased LKB1 expression and induced EMT markers in air-liquid interface culture method. Our results provide novel evidence that exosomes released from transplanted lungs undergoing chronic rejection are associated with inactivated tumor suppressor gene LKB1 and this loss induces EMT leading to the pathogenesis of CLAD following human lung transplantation.

Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer-BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.

Identification of Phospho-Tyrosine Targets as a Strategy for the Treatment of Esophageal Adenocarcinoma Cells.

INTRODUCTION: Esophageal cancer (EC) is an aggressive cancer type that is increasing at a high rate in the US and worldwide. Extensive sequencing of EC specimens has shown that there are no consistent driver mutations that can impact treatment strategies. The goal of this study was to identify activated tyrosine kinase receptors (TKRs) in EC samples as potential targets in the treatment of EC. METHODS: Activated tyrosine kinase receptors were detected using a dot-blot array for human TK receptors. Human esophageal cancer cell lines were transplanted into immunocompromised mice, and tumor xenografts were subjected to tyrosine kinase inhibitors based on the dot-blot array data. RESULTS: Using the OE33 esophageal cancer cell line, we identified activated EGF receptor (EGFR), as well as ErbB2 and ErbB3. Treatment of this cell line with erlotinib, a specific inhibitor of EGFR, did not impact the growth of this tumor cell line. Treating the OE33 cell line with afatinib, a pan-EGFR family inhibitor resulted in the growth inhibition of OE33, indicating that the ErbB2 and ErbB3 receptors were contributing to tumor cell proliferation. Afatinib treatment of mice growing OE33 tumors inhibited growth of the OE33 tumor cells. DISCUSSION: Activated tyrosine kinase receptors were readily detected in both cancer cell lines and human esophageal cancer samples. By identifying the activated receptors and then using the appropriate tyrosine kinase inhibitors, we can block tumor growth in vitro and in animal xenografts. We propose that identifying and targeting activated TKRs can be used as a personalized EC tumor treatment strategy.

Cancer-associated mutations reveal a novel role for EpCAM as an inhibitor of cathepsin-L and tumor cell invasion.

BACKGROUND: EpCAM (Epithelial cell adhesion molecule) is often dysregulated in epithelial cancers. Prior studies implicate EpCAM in the regulation of oncogenic signaling pathways and epithelial-to-mesenchymal transition. It was recently demonstrated that EpCAM contains a thyroglobulin type-1 (TY-1) domain. Multiple proteins with TY-1 domains are known to inhibit cathepsin-L (CTSL), a cysteine protease that promotes tumor cell invasion and metastasis. Analysis of human cancer sequencing studies reveals that somatic EpCAM mutations are present in up to 5.1% of tested tumors. METHODS: The Catalogue of Somatic Mutations in Cancer (COSMIC) database was queried to tabulate the position and amino acid changes of cancer associated EpCAM mutations. To determine how EpCAM mutations affect cancer biology we studied C66Y, a damaging TY-1 domain mutation identified in liver cancer, as well as 13 other cancer-associated EpCAM mutations. In vitro and in vivo models were used to determine the effect of wild type (WT) and mutant EpCAM on CTSL activity and invasion. Immunoprecipitation and localization studies tested EpCAM and CTSL protein binding and determined compartmental expression patterns of EpCAM mutants. RESULTS: We demonstrate that WT EpCAM, but not C66Y EpCAM, inhibits CTSL activity in vitro, and the TY-1 domain of EpCAM is responsible for this inhibition. WT EpCAM, but not C66Y EpCAM, inhibits tumor cell invasion in vitro and lung metastases in vivo. In an extended panel of human cancer cell lines, EpCAM expression is inversely correlated with CTSL activity. Previous studies have demonstrated that EpCAM germline mutations can prevent EpCAM from being expressed at the cell surface. We demonstrate that C66Y and multiple other EpCAM cancer-associated mutations prevent surface expression of EpCAM. Cancer-associated mutations that prevent EpCAM cell surface expression abrogate the ability of EpCAM to inhibit CTSL activity and tumor cell invasion. CONCLUSIONS: These studies reveal a novel role for EpCAM as a CTSL inhibitor, confirm the functional relevance of multiple cancer-associated EpCAM mutations, and suggest a therapeutic vulnerability in cancers harboring EpCAM mutations.

Optimized polyepitope neoantigen DNA vaccines elicit neoantigen-specific immune responses in preclinical models and in clinical translation.

BACKGROUND: Preclinical studies and early clinical trials have shown that targeting cancer neoantigens is a promising approach towards the development of personalized cancer immunotherapies. DNA vaccines can be rapidly and efficiently manufactured and can integrate multiple neoantigens simultaneously. We therefore sought to optimize the design of polyepitope DNA vaccines and test optimized polyepitope neoantigen DNA vaccines in preclinical models and in clinical translation. METHODS: We developed and optimized a DNA vaccine platform to target multiple neoantigens. The polyepitope DNA vaccine platform was first optimized using model antigens in vitro and in vivo. We then identified neoantigens in preclinical breast cancer models through genome sequencing and in silico neoantigen prediction pipelines. Optimized polyepitope neoantigen DNA vaccines specific for the murine breast tumor E0771 and 4T1 were designed and their immunogenicity was tested in vivo. We also tested an optimized polyepitope neoantigen DNA vaccine in a patient with metastatic pancreatic neuroendocrine tumor. RESULTS: Our data support an optimized polyepitope neoantigen DNA vaccine design encoding long (≥20-mer) epitopes with a mutant form of ubiquitin (Ubmut) fused to the N-terminus for antigen processing and presentation. Optimized polyepitope neoantigen DNA vaccines were immunogenic and generated robust neoantigen-specific immune responses in mice. The magnitude of immune responses generated by optimized polyepitope neoantigen DNA vaccines was similar to that of synthetic long peptide vaccines specific for the same neoantigens. When combined with immune checkpoint blockade therapy, optimized polyepitope neoantigen DNA vaccines were capable of inducing antitumor immunity in preclinical models. Immune monitoring data suggest that optimized polyepitope neoantigen DNA vaccines are capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine tumor. CONCLUSIONS: We have developed and optimized a novel polyepitope neoantigen DNA vaccine platform that can target multiple neoantigens and induce antitumor immune responses in preclinical models and neoantigen-specific responses in clinical translation.

Precision delivery of RAS-inhibiting siRNA to KRAS driven cancer via peptide-based nanoparticles.

Over 95% of pancreatic adenocarcinomas (PDACs), as well as a large fraction of other tumor types, such as colorectal adenocarcinoma, are driven by KRAS activation. However, no direct RAS inhibitors exist for cancer therapy. Furthermore, the delivery of therapeutic agents of any kind to PDAC in particular has been hindered by the extensive desmoplasia and resultant drug delivery challenges that accompanies these tumors. Small interfering RNA (siRNA) is a promising modality for anti-neoplastic therapy due to its precision and wide range of potential therapeutic targets. Unfortunately, siRNA therapy is limited by low serum half-life, vulnerability to intracellular digestion, and transient therapeutic effect. We assessed the ability of a peptide based, oligonucleotide condensing, endosomolytic nanoparticle (NP) system to deliver siRNA to KRAS-driven cancers. We show that this peptide-based NP is avidly taken up by cancer cells in vitro, can deliver KRAS-specific siRNA, inhibit KRAS expression, and reduce cell viability. We further demonstrate that this system can deliver siRNA to the tumor microenvironment, reduce KRAS expression, and inhibit pancreatic cancer growth in vivo. In a spontaneous KPPC model of PDAC, this system effectively delivers siRNA to stroma-rich tumors. This model has the potential for translational relevance for patients with KRAS driven solid tumors.

Development of an adenovirus vector vaccine platform for targeting dendritic cells.

Adenoviral (Ad) vector vaccines represent one of the most promising modern vaccine platforms, and Ad vector vaccines are currently being investigated in human clinical trials for infectious disease and cancer. Our studies have shown that specific targeting of adenovirus to dendritic cells dramatically enhanced vaccine efficacy. However, this was achieved using a molecular adapter, thereby necessitating a two component vector approach. To address the mandates of clinical translation of our strategy, we here sought to accomplish the goal of DC targeting with a single-component adenovirus vector approach. To redirect the specificity of Ad vector vaccines, we replaced the Ad fiber knob with fiber-fibritin chimeras fused to DC1.8, a single-domain antibody (sdAb) specific for murine immature DC. We engineered a fiber-fibritin-sdAb chimeric molecule using the coding sequence for DC1.8, and then replaced the native Ad5 fiber knob sequence by homologous recombination. The resulting Ad5 virus, Ad5FF1.8, expresses the chimeric fiber-fibritin sdAb chimera. Infection with Ad5FF1.8 dramatically enhances transgene expression in DC2.4 dendritic cells compared with infection with native Ad5. Ad5FF1.8 infection of bone marrow-derived DC demonstrates that Ad5FF1.8 selectively infects immature DC consistent with the known specificity of DC1.8. Thus, sdAb can be used to selectively redirect the tropism of Ad5 vector vaccines, providing the opportunity to engineer Ad vector vaccines that are specifically targeted to DC, or specific DC subsets.

Breast Cancer Neoantigens Can Induce CD8+ T-Cell Responses and Antitumor Immunity.

Next-generation sequencing technologies have provided insights into the biology and mutational landscape of cancer. Here, we evaluate the relevance of cancer neoantigens in human breast cancers. Using patient-derived xenografts from three patients with advanced breast cancer (xenografts were designated as WHIM30, WHIM35, and WHIM37), we sequenced exomes of tumor and patient-matched normal cells. We identified 2,091 (WHIM30), 354 (WHIM35), and 235 (WHIM37) nonsynonymous somatic mutations. A computational analysis identified and prioritized HLA class I-restricted candidate neoantigens expressed in the dominant tumor clone. Each candidate neoantigen was evaluated using peptide-binding assays, T-cell cultures that measure the ability of CD8+ T cells to recognize candidate neoantigens, and preclinical models in which we measured antitumor immunity. Our results demonstrate that breast cancer neoantigens can be recognized by the immune system, and that human CD8+ T cells enriched for prioritized breast cancer neoantigens were able to protect mice from tumor challenge with autologous patient-derived xenografts. We conclude that next-generation sequencing and epitope-prediction strategies can identify and prioritize candidate neoantigens for immune targeting in breast cancer. Cancer Immunol Res; 5(7); 516-23. ©2017 AACR.

Paired-like Homeodomain Transcription factor 2 expression by breast cancer bone marrow disseminated tumor cells is associated with early recurrent disease development.

The presence of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer patients is prognostic for early relapse. In the present study, we analyzed the gene expression profiles from BM cells of breast cancer patients to identify molecular signatures associated with DTCs and their relevance to metastatic outcome. We analyzed BM from 30 patients with stage II/III breast cancer by gene expression profiling and correlated expression with metastatic disease development. A candidate gene, PITX2, was analyzed for expression and phenotype in breast cancer cell lines. PITX2 was knocked down in the MDAMB231 cell lines for gene expression analysis and cell invasiveness. Expression of various signaling pathway molecules was confirmed by RT-PCR. We found that the expression of Paired-like Homeobox Transcription factor-2 (PITX2) is absent in the BM of normal healthy volunteers and, when detected in the BM of breast cancer patients, is significantly correlated with early metastatic disease development (p = 0.0062). Suppression of PITX2 expression significantly reduced invasiveness in MDAMB231 cells. Three genes-NKD1, LEF1, and DKK4-were significantly downregulated in response to PITX2 suppression. Expression of PITX2 in BM of early-stage breast cancer patients is associated with risk for early disease recurrence. Furthermore, PITX2 likely plays a role in the metastatic process through its effect on the expression of genes associated with the Wnt/beta-Catenin signaling pathway.

Searching for metastases in ovarian tissue before autotransplantation: a tailor-made approach.

OBJECTIVE: To exclude minimal residual disease in remaining ovarian tissue after harvesting the ovarian cortex for cryopreservation, by means of a tailor-made approach. DESIGN: Retrospective case series. SETTING: Hospital laboratory. PATIENT(S): We evaluated the ovarian and tubal tissue from 47 cancer patients (breast cancer, [non-]Hodgkin lymphoma; osteo-, Ewing, myxoid lipo-, and oropharyngeal synovial sarcoma; cervical, rectal, and esophageal cancer), who had stored ovarian tissue for fertility preservation. INTERVENTION(S): Immunohistochemistry (IHC) with tumor-related antibodies and genetic mutation analysis were performed to detect micrometastases by multiple sectioning at three levels of the paraffin-embedded formalin-fixed material. Molecular assays were performed with the use of tissue between these three levels of sectioning. MAIN OUTCOME MEASURE(S): Detection of micrometastases in ovaries. RESULT(S): We analyzed 847 ovarian slides to detect isolated tumor cells (ITCs) or micrometastases by IHC. In only one case (1/47) were ITCs detected in the fallopian tube. That patient had an intra-abdominal metastatic esophageal carcinoma. Additional DNA analyses of breast and rectal cancer, Ewing sarcoma, and human papilloma virus in cervical patients did not show evidence of micrometastases in the ovarian tissue. CONCLUSION(S): The tailor-made approach consisted of patient-specific tumor markers which were used to search for ovarian micrometastases. We found evidence of metastatic disease within the fallopian tube of a patient with intraperitoneal metastatic esophageal adenocarcinoma.

Safety and preliminary evidence of biologic efficacy of a mammaglobin-a DNA vaccine in patients with stable metastatic breast cancer.

PURPOSE: Mammaglobin-A (MAM-A) is overexpressed in 40% to 80% of primary breast cancers. We initiated a phase I clinical trial of a MAM-A DNA vaccine to evaluate its safety and biologic efficacy. EXPERIMENTAL DESIGN: Patients with breast cancer with stable metastatic disease were eligible for enrollment. Safety was monitored with clinical and laboratory assessments. The CD8 T-cell response was measured by ELISPOT, flow cytometry, and cytotoxicity assays. Progression-free survival (PFS) was described using the Kaplan-Meier product limit estimator. RESULTS: Fourteen subjects have been treated with the MAM-A DNA vaccine and no significant adverse events have been observed. Eight of 14 subjects were HLA-A2(+), and the CD8 T-cell response to vaccination was studied in detail. Flow cytometry demonstrated a significant increase in the frequency of MAM-A-specific CD8 T cells after vaccination (0.9% ± 0.5% vs. 3.8% ± 1.2%; P < 0.001), and ELISPOT analysis demonstrated an increase in the number of MAM-A-specific IFNγ-secreting T cells (41 ± 32 vs. 215 ± 67 spm; P < 0.001). Although this study was not powered to evaluate progression-free survival (PFS), preliminary evidence suggests that subjects treated with the MAM-A DNA vaccine had improved PFS compared with subjects who met all eligibility criteria, were enrolled in the trial, but were not vaccinated because of HLA phenotype. CONCLUSION: The MAM-A DNA vaccine is safe, capable of eliciting MAM-A-specific CD8 T-cell responses, and preliminary evidence suggests improved PFS. Additional studies are required to define the potential of the MAM-A DNA vaccine for breast cancer prevention and/or therapy.

Targeting an IKBKE cytokine network impairs triple-negative breast cancer growth.

Triple-negative breast cancers (TNBCs) are a heterogeneous set of cancers that are defined by the absence of hormone receptor expression and HER2 amplification. Here, we found that inducible IκB kinase-related (IKK-related) kinase IKBKE expression and JAK/STAT pathway activation compose a cytokine signaling network in the immune-activated subset of TNBC. We found that treatment of cultured IKBKE-driven breast cancer cells with CYT387, a potent inhibitor of TBK1/IKBKE and JAK signaling, impairs proliferation, while inhibition of JAK alone does not. CYT387 treatment inhibited activation of both NF-κB and STAT and disrupted expression of the protumorigenic cytokines CCL5 and IL-6 in these IKBKE-driven breast cancer cells. Moreover, in 3D culture models, the addition of CCL5 and IL-6 to the media not only promoted tumor spheroid dispersal but also stimulated proliferation and migration of endothelial cells. Interruption of cytokine signaling by CYT387 in vivo impaired the growth of an IKBKE-driven TNBC cell line and patient-derived xenografts (PDXs). A combination of CYT387 therapy with a MEK inhibitor was particularly effective, abrogating tumor growth and angiogenesis in an aggressive PDX model of TNBC. Together, these findings reveal that IKBKE-associated cytokine signaling promotes tumorigenicity of immune-driven TNBC and identify a potential therapeutic strategy using clinically available compounds.

Src homology phosphotyrosyl phosphatase-2 expression is an independent negative prognostic factor in human breast cancer.

AIMS: Src homology phosphotyrosyl phosphatase-2 (SHP2) is a ubiquitously expressed phosphatase that plays an essential role in the downstream signalling pathways of multiple growth factor receptors, thus representing a potential target for cancer therapy. Recent studies suggest that SHP2 contributes to tumour initiation, progression and metastasis in breast cancer, yet the impact of SHP2 expression on prognosis in human breast cancer has not been evaluated. METHODS AND RESULTS: To explore further the role of SHP2 in breast cancer, we conducted an immunohistochemical study using a tissue microarray encompassing 1401 formalin-fixed breast cancer specimens with detailed clinical annotation and outcome data. Of 1401 evaluable breast cancers, 651 (46%) were positive for SHP2. SHP2 expression was associated positively with tumour grade, lymph node status and tumour stage. In univariate survival analysis, cases with SHP2 expression had a significantly worse overall survival (OS). In multivariate analysis, SHP2 remained an independent negative prognostic factor for OS. SHP2 expression was a negative prognostic factor for OS in the luminal A and the luminal B HER2(-) intrinsic subtypes. CONCLUSIONS: Our data demonstrate for the first time that SHP2 is an independent predictor of survival in breast cancer, suggesting that SHP2 may be a potential target for therapy.

EpCAM modulates NF-κB signaling and interleukin-8 expression in breast cancer.

The epithelial cell adhesion molecule (EpCAM) is a 40-kD type I transmembrane protein that is overexpressed in human epithelial cancers and is currently the target of molecular therapy based on its overexpression at the cell surface. Recently, we and others have shown a role for EpCAM in cell signaling and carcinogenesis, and EpCAM expression seems to promote breast cancer invasion. Interleukin-8 (IL-8/CXCL-8) is an inflammatory cytokine that has recently been shown to modulate breast cancer invasion and angiogenesis. In preliminary experiments, we identified a correlation between EpCAM and IL-8 expression in primary human breast cancers. Specific ablation of EpCAM in breast cancer cell lines results in decreased IL-8 expression, and IL-8 contributes to EpCAM-dependent breast cancer invasion. Specific ablation of EpCAM is also associated with decreased NF-κB transcription factor activity, decreased phosphorylation of the NF-κB family member RELA, and increased IκBα protein expression. EpCAM modulates IL-8 expression at baseline, and following IL-1ß stimulation, which is known to be a potent inducer of NF-κB in breast cancer. In functional rescue experiments, specific ablation of RELA or forced expression of the NF-κB inhibitor protein IκBα prevented EpCAM-dependent rescue of IL-8 promoter activity. These studies show for the first time that EpCAM can modulate NF-κB transcription factor activity and IL-8 expression in breast cancer and confirm the role of EpCAM signaling in modulating breast cancer invasion. Further study is required to define the molecular mechanism(s) of EpCAM signaling in breast cancer and to direct the rational development of molecular therapies targeting EpCAM.

Detection of disseminated tumor cells in the bone marrow of breast cancer patients using multiplex gene expression measurements identifies new therapeutic targets in patients at high risk for the development of metastatic disease.

Disseminated tumor cells (DTCs) detected in the bone marrow (BM) of breast cancer patients identify women at high risk of recurrence. DTCs are traditionally detected by immunocytochemical staining for cytokeratins or single gene expression measurements, which limit both specificity and sensitivity. We evaluated the Nanostring nCounter™ platform for multi-marker, gene expression-based detection and classification of DTCs in the BM of breast cancer patients. Candidate genes exhibiting tumor cell-specific expression were identified from microarray datasets and validated by qRT-PCR analysis in non-malignant human BM and identical samples spiked with predefined numbers of molecularly diverse breast tumor cell lines. Thirty-eight validated transcripts were designed for the nCounter™ platform and a subset of these transcripts was technically validated against qRT-PCR measurements using identical spiked BM controls. Bilateral iliac crest BM aspirates were collected and analyzed from twenty breast cancer patients, prior to neoadjuvant therapy, using the full 38-gene nCounter™ code set. Tumor cell-specific gene expression by nCounter™ was detected with a sensitivity of one cancer cell per 1 × 10(6) nucleated BM cells after optimization. Measurements were quantitative, log linear over a 20-fold range, and correlated with qRT-PCR measurements. Using the nCounter™ 38-gene panel, 6 of 8 patients (75 %) who developed metastatic disease had detectable expression of at least one transcript. Notably, three of these patients had detectable expression of ERBB2 in their BM, despite the fact that their corresponding primary tumors were HER2/ERBB2 negative and therefore did not receive trastuzumab therapy. Four of these patients also expressed the PTCH1 receptor, a newly recognized therapeutic target based on hedgehog signaling pathway inhibition. The presumptive detection and classification of DTCs in the BM of breast cancer patients, based on sensitive and quantitative multi-marker detection of gene expression using the nCounter™ platform, provide an opportunity to both predict early distant recurrence and, more importantly, identify opportunities for preventing the spread of disease based on the expression of unique, therapeutically actionable gene targets. This study demonstrates the application of a new technology for multiplexed gene expression-based detection of DTCs in the BM of breast cancer patients and identifies at least two therapeutically targetable genes that are frequently expressed in the BM of patients who develop metastatic disease.