The effects of lymph node status on predicting outcome in ER+ /HER2- tamoxifen treated breast cancer patients using gene signatures.

BACKGROUND: Lymph node (LN) status is the most important prognostic variable used to guide ER positive (+) breast cancer treatment. While a positive nodal status is traditionally associated with a poor prognosis, a subset of these patients respond well to treatment and achieve long-term survival. Several gene signatures have been established as a means of predicting outcome of breast cancer patients, but the development and indication for use of these assays varies. Here we compare the capacity of two approved gene signatures and a third novel signature to predict outcome in distinct LN negative (-) and LN+ populations. We also examine biological differences between tumours associated with LN- and LN+ disease. METHODS: Gene expression data from publically available data sets was used to compare the ability of Oncotype DX and Prosigna to predict Distant Metastasis Free Survival (DMFS) using an in silico platform. A novel gene signature (Ellen) was developed by including patients with both LN- and LN+ disease and using Prediction Analysis of Microarrays (PAM) software. Gene Set Enrichment Analysis (GSEA) was used to determine biological pathways associated with patient outcome in both LN- and LN+ tumors. RESULTS: The Oncotype DX gene signature, which only used LN- patients during development, significantly predicted outcome in LN- patients, but not LN+ patients. The Prosigna gene signature, which included both LN- and LN+ patients during development, predicted outcome in both LN- and LN+ patient groups. Ellen was also able to predict outcome in both LN- and LN+ patient groups. GSEA suggested that epigenetic modification may be related to poor outcome in LN- disease, whereas immune response may be related to good outcome in LN+ disease. CONCLUSIONS: We demonstrate the importance of incorporating lymph node status during the development of prognostic gene signatures. Ellen may be a useful tool to predict outcome of patients regardless of lymph node status, or for those with unknown lymph node status. Finally we present candidate biological processes, unique to LN- and LN+ disease, that may indicate risk of relapse.

Overexpression of IL-15 promotes tumor destruction via NK1.1+ cells in a spontaneous breast cancer model.

BACKGROUND: Natural Killer (NK) cells play an important role in tumor prevention, but once tumors form, the numbers as well as the cytotoxic functions of NK cells are reduced. IL-15 is a cytokine that increases and activates NK cells. Here we will examine the anti-tumor role of IL-15 in a spontaneous breast cancer model. METHODS: To achieve this, Polyoma Middle T (MT) mice that form spontaneous breast cancer were crossed with mice that either overexpress IL-15 (IL-15 transgenic (TG)) or mice that lack IL-15 (IL-15 knockout (KO)). We compared survival curves and tumor formation in IL-15 KO/MT, MT and IL-15 TG/MT groups. In addition, the phenotype, activation and contribution of NK cells and CD8 T cells to tumor formation were examined in each of these mouse strains via flow cytometry, ELISA, adoptive transfer and antibody depletion experiments. RESULTS: IL-15KO/MT tumors formed and progressed to endpoint more quickly than MT tumors. These tumors displayed little apoptosis and poor CD8 T cell infiltration. In contrast, IL-15 TG/MT mice had increased survival and the tumors displayed extensive cell death, high proportions of activated NK cells and a higher infiltration of CD8 T cells than MT tumors. CD8 T cells in IL-15 TG/MT tumors were capable of secreting IFNγ, possessed markers of memory, did not display an exhausted phenotype and were frequently NK1.1+. Long-term antibody depletion studies in IL-15 TG/MT mice revealed that NK1.1+, but not CD8 T cells, were critical for tumor destruction. Lastly, human NK cells, when exposed to a similar cytokine environment as that found in IL-15TG/MT tumors, were capable of killing human breast cancer cells. CONCLUSIONS: This study reveals that high levels of IL-15 can promote tumor destruction and reduce metastasis in breast cancer via effects on NK1.1+ cells. Our results suggest that strategies aimed at increasing NK cell activation may be effective against solid epithelial cancers.

Genital HSV-2 infection induces short-term NK cell memory.

NK cells are known as innate immune cells that lack immunological memory. Recently, it has been shown that NK cells remember encounters with chemical haptens that induce contact hypersensitivity and cytomegalovirus infection. Here, we show the existence of NK cell memory following HSV-2 infection. Stimulation with HSV-2 Ags led to higher IFNγ production in NK cells that were exposed 30 days previously to HSV-2, compared to NK cells from naïve mice. More importantly, this increased production of IFNγ in NK cells was independent of B- and T- lymphocytes and specific for the HSV-2 Ags. We also showed that previously exposed NK cells in a B- and T-lymphocyte free environment mediate protection against HSV-2 infection and they are necessary for the protection of mice against HSV-2 infection. Collectively, NK cells remember prior HSV-2 encounters independent of B- and T- lymphocytes leading to protection against HSV-2 mediated morbidity and mortality upon re-exposure.

FimH, a TLR4 ligand, induces innate antiviral responses in the lung leading to protection against lethal influenza infection in mice.

Fimbriae H protein (FimH) is a novel TLR4 ligand that has been shown to stimulate the innate immune system and elicits protective responses against bacterial and viral infections. Here, we evaluated the protective role of local delivery of FimH against influenza A infection in a mouse model. We show that intranasal delivery of FimH prior to lethal challenge with influenza A virus, resulted in decreased morbidity and mortality in wild-type, but not TLR4(-/-), mice. Importantly, FimH was able to reduce the early viral burden in the lung leading to minimal cell infiltration into the airway lumen and reduced pulmonary pathology following infection in wild type mice compared to TLR4(-/-) mice. Local delivery of FimH to C57BL/6, not TLR4(-/-), mice in a prophylactic manner increased the IL-12 and RANTES responses as well as neutrophil recruitment into the airway lumen. These effects correlate to the course of influenza infection. The FimH-mediated antiviral response against influenza virus appears to be partially dependent on alveolar macrophages. The antiviral effects are likely mediated by the innate mediators (TNF-α, IL-12 or RANTES) and/or by activation of a feedback inhibition loop to curtail the pulmonary inflammation possibly be the potential mechanisms involved in FimH-mediated protection. FimH thus holds promise to be a possible prophylactic mean of control against influenza viral infection.

Estradiol regulates susceptibility following primary exposure to genital herpes simplex virus type 2, while progesterone induces inflammation.

We report here that sex hormones modulate susceptibility to a sexually transmitted viral agent, herpes simplex virus type 2 (HSV-2), in a mouse model. Ovariectomized mice were administered either saline (control), estradiol (E(2)), progesterone (P(4)), or a combination of both estradiol and progesterone (E+P) and infected intravaginally with HSV-2. With an inoculation dose of 10(5) PFU, the saline- and P(4)-treated mice were found to be highly susceptible to genital HSV-2 infection. Both groups had extensive pathology and high viral titers in vaginal secretions, and 100% of mice succumbed by day 4 postinfection. E(2)-treated mice were protected from HSV-2 infection at the same dose and did not display any vaginal pathology or viral shedding. There was a slow progression of genital pathology in the combination hormone-treated group, along with prolonged viral shedding; 80% of animals succumbed by day 13. With lower inoculation doses of 10(3) and 10(2) PFU, 50 and 100%, respectively, of the combination hormone-treated mice survived. Localization of HSV-2 infection showed extensive infection in the vaginal epithelium of P(4)- and saline-treated animals within 24 h of inoculation. E(2)-treated animals were clear of infection, while the E+P-treated group had focal infection at 24 h that had progressed extensively by day 3. Infection was accompanied by persistent inflammation and infiltration of neutrophils in the P(4)-treated group. An analysis of the genes in the vaginal tissue showed that inflammation in the P(4)-treated group correlated with local induction of chemokines and chemokine receptors that were absent in the E(2)-treated mice and in uninfected P(4)-treated mice. The results show that sex hormones regulate initiation of infection and immune responses to genital HSV-2 infection.

Protection against genital herpes infection in mice immunized under different hormonal conditions correlates with induction of vagina-associated lymphoid tissue.

The present study was undertaken to examine the effect of the hormonal environment on immunization with an attenuated strain of herpes simplex virus type 2 (HSV-2 TK(-)) and subsequent protection against challenge. Ovariectomized mice were administered saline (S; control), estradiol (E(2)), progesterone (P(4)), or a combination of estradiol and progesterone (E+P) and immunized intravaginally (IVAG) with HSV-2 TK(-). Three weeks later, the immunized mice were challenged IVAG with wild-type HSV-2. Mice that were immunized following E treatment were not protected, whereas complete protection against the challenge was seen in mice from the S- and P(4)-treated groups. In the P(4)-treated group, 15% of mice developed chronic pathology following TK(-) immunization. Interestingly, about 40% of the E+P-treated mice were also protected. Upon examination of viral shedding in the vaginal secretions, it was clear that protection against challenge was dependent on the ability of the TK(-) virus to cause productive genital infection under different hormonal conditions. In the protected mice (the S and P groups and part of the E+P group), induced vagina-associated lymphoid tissues composed of CD11c(+) dendritic cells and CD3(+) and CD4(+) T cells were formed transiently in the vaginal lamina propria from day 2 to day 5 postchallenge. These aggregates were absent in the unprotected mice (the E group and part of the E+P group). Significant HSV-2-specific activation of lymphocytes was observed in the local draining lymph nodes of protected mice. This response was absent in the unprotected groups. High titers of gB-specific local immunoglobulin A (IgA) antibodies were present in the vaginal secretions of S- and P(4)-treated immunized mice following HSV-2 challenge. The S-treated group of mice also had high gB-specific IgG titers. These studies show that sex hormones modify the induction of protective immune responses following IVAG immunization.

Prolonged exposure to progesterone prevents induction of protective mucosal responses following intravaginal immunization with attenuated herpes simplex virus type 2.

Depo-Provera (Depo) is a long-acting progestational formulation that is a popular form of contraception for women. In animal models of sexually transmitted diseases, it is used to facilitate infection. Here we report that treatment with Depo, in a mouse model of genital herpes simplex virus type 2 (HSV-2), altered immune responses depending on the length of time that animals were exposed to Depo prior to immunization. Mice immunized intravaginally (i.vag.) with an attenuated strain (TK(-)) of HSV-2 following longer (15 days) exposure to Depo (Depo 15 group) failed to show protection when challenged with wild-type HSV-2. In contrast, mice that were immunized shortly after Depo treatment (5 days; Depo 5 group) were fully protected and showed no genital pathology after HSV-2 challenge. High viral titers were detected in the vaginal washes of the Depo 15 group up to 6 days postchallenge. In contrast, no viral shedding was observed beyond day 3 postchallenge in the Depo 5 group. Following i.vag. TK(-) immunization, high levels of gamma interferon (IFN-gamma) were detected locally in vaginal washes of the Depo 5 group but not the Depo 15 group. After HSV-2 challenge, an early peak of IFN-gamma in the Depo 5 group coincided with clearance of the virus. In Depo 15 animals IFN-gamma was present throughout the 6 days postinfection. HSV-2-specific T-cell cytokine responses measured in the lymph node cells of Depo 5 TK(-)-immunized mice indicated a significantly higher Th1 response than that of Depo 15 TK(-)-immunized mice. The protection after HSV-2 challenge in the Depo 5 group correlated with increased local HSV-2 glycoprotein B (gB)-specific immunoglobulin G (IgG) and IgA responses seen in the vaginal secretions. The Depo 15 group had poor gB-specific antibody responses in the genital tract after HSV-2 challenge. These results indicate that longer exposure to Depo leads to poor innate and adaptive immune responses to HSV-2 that fail to protect mice from subsequent genital challenges.

Pathway pathology: histological differences between ErbB/Ras and Wnt pathway transgenic mammary tumors.

To study phenotype-genotype correlations, ErbB/Ras pathway tumors (transgenic for ErbB2, c-Neu, mutants of c-Neu, polyomavirus middle T antigene (PyV-mT), Ras, and bi-transgenic for ErbB2/Neu with ErbB3 and with progesterone receptor) from four different institutions were histopathologically compared with Wnt pathway tumors [transgenes Wnt1, Wnt10b, dominant-negative glycogen synthase kinase 3-beta, beta-Catenin, and spontaneous mutants of adenomatous polyposis coli gene (Apc)]. ErbB/Ras pathway tumors tend to form solid nodules consisting of poorly differentiated cells with abundant cytoplasm. ErbB/Ras pathway tumors also have scanty stroma and lack myoepithelial or squamous differentiation. In contrast, Wnt pathway tumors exhibit myoepithelial, acinar, or glandular differentiation, and, frequently, combinations of these. Squamous metaplasia is frequent and may include transdifferentiation to epidermal and pilar structures. Most Wnt pathway tumors form caricatures of elongated, branched ductules, and have well-developed stroma, inflammatory infiltrates, and pushing margins. Tumors transgenic for interacting genes such as protein kinase CK2alpha (casein kinase IIalpha), and the fibroblast growth factors (Fgf) Int2/Fgf3 or keratinocyte growth factor (Kgf/Fgf7) also have the Wnt pathway phenotype. Because the tumors from the ErbB/Ras and the Wnt pathway are so distinct and can be readily identified using routine hematoxylin and eosin sections, we suggest that pathway pathology is applicable in both basic and clinical cancer research.