Frequency Of Ki-67 Research Articles

Analysis of proliferating Treg cells as a predictor for immunotherapy response using systemic longitudinal immune profiling: Biomarker analysis of the phase 3 CONTINUUM trial.

140 Background: The discovery of predictive biomarkers for immunotherapy is hindered by the lack of control groups in most translational studies, making it impossible to discern whether the identified biomarkers are merely prognostic or truly predictive for treatment outcomes. This study reported on prospectively designed biomarker analyses of the phase 3 CONTINNUUM trial (NCT03700476), the first trial that demonstrated prolonged event-free survival (EFS) with the addition of anti-PD-1 (aPD1) to chemoradiotherapy (CRT) in locoregionally-advanced nasopharyngeal carcinoma (LA-NPC). Methods: Mass cytometry was performed to generate a dynamic single-cell atlas on longitudinal peripheral blood mononuclear cell (PBMC) samples from 12 pairs of matched patients with or without relapse in the aPD1-CRT arm. The machine-learning algorithm CellCnn was applied to identify a cell subset that was most strongly associated with disease relapse, which was further confirmed by flow cytometry (n = 120). Single-cell RNA sequencing data from matched PBMC and tumor samples were analyzed and multiplex immunohistochemistry was performed on baseline tumor samples (n = 249) to verify the predictive value of the cell subset within tumors. Results: Baseline circulating regulatory T cells (Tregs), with a Ki67+ proliferating phenotype, were found to display a higher frequency in LA-NPC patients who developed posttreatment relapse ( P < 0.001), which was further validated by flow cytometry. The intratumoral Ki67+ Tregs were also identified and correlated with an immunosuppressive tumor microenvironment. In patients with a low baseline level of Ki67+ Tregs, additional aPD1 significantly improved EFS compared with CRT alone (log-rank P = 0.011, HR = 0.22, 95% CI: 0.06−0.79), while EFS was almost identical in both treatment arms in patients with high Ki67+ Treg levels (log-rank P = 0.995, HR = 1.00, 95% CI: 0.49−2.05; interaction P = 0.047). This predictive value was further validated in datasets from two independent randomized trials in renal cancer. Conclusions: The frequency of Ki67+ Tregs can serve as a reliable predictive tool in selecting patients who are more likely to benefit from immunotherapy, potentially informing individualized immunotherapy strategies.

Pathological proliferation: a potential mechanism for poor CD4+ T cell recovery in people living with HIV.

People living with HIV (PLWH) fail to achieve normalization of CD4+ T cell counts and function, especially in immunological non-responders (INRs). The frequencies of Ki67+CD4+ T cells were inversely associated with CD4+ T cell counts in HIV infected patients. Early ART did not normalize CD4+ T cell proliferation. However, the features of the abnormal proliferation CD4+ T cell in INRs are far from known. PLWH were divided into INRs (n= 16) and immunological responders (IRs, n= 53) groups. Mass cytometry was applied to peripheral blood T cells to profile the immune cells and liquid chip technique was used to measure plasma levels of cytokines and chemokines. Correlation analyses were conducted to evaluate associations between the degree of CD4+ T cell proliferation and immune function. The percentage of Ki67+ CD4+ T cells were significant higher in INRs, and we defined these cells with significant higher level of Ki67, as over-proliferating cells. No significant difference of markers' expression (HLA-DR, CD38, CD57, PD-1, PD-L1, CD107a, perforin) was found between INRs and IRs. Compared with naïve CD4+ T cells in INRs, Ki67+ CD4+ T cells exhibited lower levels of CD57 and CD38. Whereas Ki67+ T cells exhibited higher levels of CD38 and CD57 and activation compared with differentiated mature central memory CD4+ T cells and effector memory CD4+ T cells. Ki67+ cells did not show higher levels of senescence and activation compared to certain Ki67- CD4+ central memory T cells in IRs. Furthermore, Ki67+ CD4+ Tcm cells exhibited positive correlations with pro-inflammatory cytokines. We proposed and validated the hypothesis of "pathological proliferation" in INRs: excessive proliferation of CD4+ T cells in INRs may be accompanied by aberrant activation, senescence and loss of immune function. Eventually, such over-proliferating but poor-quality cells in INRs result in incomplete recovery of both CD4+ T cell counts and function. An intervention that enhancing the proliferative capacity or functional ability or both of CD4+ T cell in INRs might therefore be beneficial.

The TOPK Inhibitor HI-TOPK-032 Enhances CAR T-cell Therapy of Hepatocellular Carcinoma by Upregulating Memory T Cells.

Chimeric antigen receptor (CAR) T cells are emerging as an effective antitumoral therapy. However, their therapeutic effects on solid tumors are limited because of their short survival time and the immunosuppressive tumor microenvironment. Memory T cells respond more vigorously and persist longer than their naïve/effector counterparts. Therefore, promoting CAR T-cell development into memory T cells could further enhance their antitumoral effects. HI-TOPK-032 is a T-LAK cell-originated protein kinase (TOPK)-specific inhibitor that moderately represses some types of tumors. However, it is unknown whether HI-TOPK-032 works on hepatocellular carcinoma (HCC) and whether it impacts antitumoral immunity. Using both subcutaneous and orthotopic xenograft tumor models of two human HCC cell lines, Huh-7 and HepG2, we found that HI-TOPK-032 significantly improved proliferation/persistence of CD8+ CAR T cells, as evidenced by an increase in CAR T-cell counts or frequency of Ki-67+CD8+ cells and a decrease in PD-1+LAG-3+TIM-3+CD8+ CAR T cells in vivo. Although HI-TOPK-032 did not significantly suppress HCC growth, it enhanced the capacity of CAR T cells to inhibit tumor growth. Moreover, HI-TOPK-032 augmented central memory CD8+ T cell (TCM) frequency while increasing eomesodermin expression in CD8+ CAR T cells in tumor-bearing mice. Moreover, it augmented CD8+ CAR TCM cells in vitro and reduced their expression of immune checkpoint molecules. Finally, HI-TOPK-032 inhibited mTOR activation in CAR T cells in vitro and in tumors, whereas overactivation of mTOR reversed the effects of HI-TOPK-032 on CD8+ TCM cells and tumor growth. Thus, our studies have revealed mechanisms underlying the antitumoral effects of HI-TOPK-032 while advancing CAR T-cell immunotherapy.

The ketone body β-hydroxybutyrate mitigates ILC2-driven airway inflammation by regulating mast cell function.

Ketone bodies are increasingly understood to have regulatory effects on immune cell function, with β-hydroxybutyrate (BHB) exerting a predominantly anti-inflammatory response. Dietary strategies to increase endogenous ketone body availability such as the ketogenic diet (KD) have recently been shown to alleviate inflammation of the respiratory tract. However, the role of BHB has not been addressed. Here, we observe that BHB suppresses group 2 innate lymphoid cell (ILC2)-mediated airway inflammation. Central to this are mast cells, which support ILC2 proliferation through interleukin-2 (IL-2). Suppression of the mast cell/IL-2 axis by BHB attenuates ILC2 proliferation and the ensuing type 2 cytokine response and immunopathology. Mechanistically, BHB directly inhibits mast cell function in part through GPR109A activation. Similar effects are achieved with either the KD or 1,3-butanediol. Our data reveal the protective role of BHB in ILC2-driven airway inflammation, which underscores the potential therapeutic value of ketone body supplementation for the management of asthma.

The effects of human pregnancy-specific \u03b21-glycoprotein preparation on Th17 polarization of CD4+ cells and their cytokine profile

BackgroundPregnancy-specific β1-glycoproteins are capable of regulating innate and adaptive immunity, exerting predominantly suppressive effects. In this regard, they are of interest in terms of their pharmacological potential for the treatment of autoimmune diseases and post-transplant complications. The effect of these proteins on the main pro-inflammatory subpopulation of T lymphocytes, IL-17-producing helper T cells (Th17), has not been comprehensively studied. Therefore, the effects of the native pregnancy-specific β1-glycoprotein on the proliferation, Th17 polarization and cytokine profile of human CD4+ cells were assessed.ResultsNative human pregnancy-specific β1-glycoprotein (PSG) at а concentration of 100 μg/mL was shown to decrease the frequency of Th17 (RORγτ+) in CD4+ cell culture and to suppress the proliferation of these cells (RORγτ+Ki-67+), along with the proliferation of other cells (Ki-67+) (n = 11). A PSG concentration of 10 μg/mL showed similar effect, decreasing the frequency of Ki-67+ and RORγτ+Ki67+ cells. Using Luminex xMAP technology, it was shown that PSG decreased IL-4, IL-5, IL-8, IL-12, IL-13, IL-17, MIP-1β, IL-10, IFN-γ, TNF-α, G-CSF, and GM-CSF concentrations in Th17-polarized CD4+ cell cultures but did not affect IL-2, IL-7, and MCP-1 output.ConclusionsIn the experimental model used, PSG had а mainly suppressive effect on the Th17 polarization and cytokine profile of Th17-polarized CD4+ cell cultures. As Th17 activity and a pro-inflammatory cytokine background are unfavorable during pregnancy, the observed PSG effects may play a fetoprotective role in vivo.

Overview of obesity and breast cancer in Brazil: 24 year of follow up.

e13580 Background: Obesity is one of the major public health challenges of the 21st century. The prevalence of obesity is growing exponentially across all age groups and on all continents and it is associated with morbidities such as cardiovascular disease, diabetes mellitus type 2, dyslipidemia, hypertension and cancer. There is a higher risk of developing breast cancer in postmenopausal obese women, and with worse outcome for women of all ages. Objectives: To evaluate obesity prevalence in breast cancer patients and its association with recovering, survival, death, age, Human Epidermal Growth Factor Receptor 2 (HER2) and anti-human KI67 antibody(KI67). Methods: A retrospective cohort study was performed between 1994 and 2018 at the Breast Cancer Department, São Paulo State Government Women's Health Reference Center (Perola Byington Hospital), São Paulo, Brazil with 8824 breast cancer patients. From those, 2899 patients were excluded because they did not have description of weight and/or Body Mass Index (BMI). Survival analysis was performed according to BMI groups. Results: The participants had mean (SD) age of 54 (12.0) years and weight of 70 (15.0) kg. There were 769 (13%) deaths and the mean survival was 20 (2.0) years. 1787 (30%) participants were stratified in obese (BMI≥30Kg/m2) and 4138 (70%) non-obese Obese were older (p < 0,001) and had a higher frequency of HER2 negative (p < 0,04) than non-obese women. There were not a significant difference in frequencies of KI67, recovering and survival between groups. The prevalence of obesity was not different over 24 years. Conclusions: There was a relevant prevalence of obesity in breast cancer patients, which was associated with HER2 negative and age, but not with recovering or survival.

Systemic and Intra-Nodal Activation of NK Cells After Rituximab Monotherapy for Follicular Lymphoma.

Monotherapy with the anti-CD20 monoclonal antibody rituximab can induce complete responses (CR) in patients with follicular lymphoma (FL). Resting FcRγIII+ (CD16+) natural killer (NK) cells respond strongly to rituximab-coated target cells in vitro. Yet, the contribution of NK cells in the therapeutic effect in vivo remains unknown. Here, we followed the NK cell repertoire dynamics in the lymph node and systemically during rituximab monotherapy in patients with FL. At baseline, NK cells in the tumor lymph node had a naïve phenotype albeit they were more differentiated than NK cells derived from control tonsils as determined by the frequency of CD56dim NK cells and the expression of killer cell immunoglobulin-like receptors (KIR), CD57 and CD16. Rituximab therapy induced a rapid drop in NK cell numbers coinciding with a relative increase in the frequency of Ki67+ NK cells both in the lymph node and peripheral blood. The Ki67+ NK cells had slightly increased expression of CD16, CD57 and higher levels of granzyme A and perforin. The in vivo activation of NK cells was paralleled by a temporary loss of in vitro functionality, primarily manifested as decreased IFNγ production in response to rituximab-coated targets. However, patients with pre-existing NKG2C+ adaptive NK cell subsets showed less Ki67 upregulation and were refractory to the loss of functionality. These data reveal variable imprints of rituximab monotherapy on the NK cell repertoire, which may depend on pre-existing repertoire diversity.

Immunohistochemical Evaluation of Ki-67 and Comparison with Clinicopathologic Factors in Breast Carcinomas

Background:Patients primarily received tamoxifen based on their menopausal status due to the lack of immunohistochemistry. A recent study has shown that hormonal receptors were not correlated with menopausal status, and thus, indicating that they present limited therapeutic and prognostic significance in breast cancer management. This study aimed to evaluate Ki-67 value and analyze its association with clinicopathologic parameters in breast cancer patients.Methods:The formalin-fixed paraffin-embedded breast tissue blocks of 125 patients with primary breast carcinomas were subjected to immunohistochemical analysis using Ventana Benchmark® GX automated immunostainer. Analysis of variance and Chi-2 test were used to examine the relationship between Ki-67 and clinicopathologic variables.Results:The mean age of 125 patients included in the study was 47.7 years. The average score of Ki-67 was 56.0%. 84.8% of patients showed Ki-67 ≥ 14%. Mean scores of Ki-67 were correlated with grade (p = 0.006), PR (p = 0.026), histological type, ER, combined ER/RP, and molecular subtype (p < 0.001). Ki-67 was independent of HER2 (p = 0.402) and menopausal status (p = 0.471). The frequency of Ki-67 according to St Gallen 2011 was associated with histological type (p = 0.005), grade (p = 0.005), ER (p < 0.001), combined ER/PR (p = 0.004), and molecular subtype (p = 0.004). There was no significant relationship between the distribution of Ki-67 and the age of the patients (p = 0.859), menopausal status (p = 0.979), PR (p = 0.149), and HER2 (p = 0.597).Conclusion:Ki-67 is useful for treatment decisions in primary breast cancer patients. The high value of Ki-67 was associated with adverse clinicopathologic factors. The increased Ki-67 value should be carefully investigated in triple negative patients.

Incorporation of p-53 mutation status and Ki-67 proliferating index in classifying Her2-neu positive gastric adenocarcinoma

ABSTRACTHer2-neu overexpression has a pathogenetic, therapeutic and a controversial prognostic role in gastric cancer. p-53 mutation status and Ki-67 proliferation index are established prognostic markers in many tumors. In this study we evaluated p-53 and Ki-67 in relation to Her2-neu positive and negative gastric adenocarcinoma (GA). This cross-sectional study was carried out at King Fahd Hospital of Imam Abdulrahman bin Faisal University. Fifty cases of GA were retrieved from pathology archives. Clinico-pathological parameters were evaluated. Immunohistochemical protein analysis for Her2-neu, Ki-67 and p-53 was carried out. Fluorescent in situ hybridization (FISH) analysis was done for Her2-neu positive cases showing 2+ immunoexpression. Frequency of Ki-67 and p-53 positivity in Her2-neu positive cases was calculated and compared with those in Her2-neu negative cases. Correlation of clinicopatological parameters with Her2 positive and negative cases, p-53 mutation status and Ki-67 proliferation index was carried out. Her2-neu overexpression was present in 12% (n = 6) cases. A high Ki-67 was seen predominantly in Her2-neu positive cases (83%, n = 5). Her2-neu negative cases (n = 44) showed moderate (31.88%, n = 14) to low (34%, n = 15) Ki-67. Diffuse p-53 positivity was seen predominantly in Her2-neu positive cases (33.33%, n = 2). Focal p-53 was seen mainly in Her2-neu negative cases 56.8% (n = 25). Negative p-53 was seen to be independent of Her2-neu status. Her2-neu positivity is strongly associated with diffuse p-53 mutation status and high Ki-67 proliferation. Her 2-neu negative status is associated with focal p-53 positivity and low to moderate Ki-67 proliferation index. Such stratifications in prognostic markers could not only be predictive in patient’s prognostics but could also form a basis of molecular classification of gastric cancer.

Age dependent differences in the kinetics of γδ T cells after influenza vaccination.

Immunosenescence is a hallmark of the aging immune system and is considered the main cause of a reduced vaccine efficacy in the elderly. Although γδ T cells can become activated by recombinant influenza hemagglutinin, their age-related immunocompetence during a virus-induced immune response has so far not been investigated. In this study we evaluate the kinetics of γδ T cells after vaccination with the trivalent 2011/2012 northern hemisphere seasonal influenza vaccine. We applied multi-parametric flow cytometry to a cohort of 21 young (19–30 years) and 23 elderly (53–67 years) healthy individuals. Activated and proliferating γδ T cells, as identified by CD38 and Ki67 expression, were quantified on the days 0, 3, 7, 10, 14, 17, and 21. We observed a significantly lower number of activated and proliferating γδ T cells at baseline and following vaccination in elderly as compared to young individuals. The kinetics changes of activated γδ T cells were much stronger in the young, while corresponding changes in the elderly occurred slower. In addition, we observed an association between day 21 HAI titers of influenza A and the frequencies of Ki67+ γδ T cells at day 7 in the young. In conclusion, aging induces alterations of the γδ T cell response that might have negative implications for vaccination efficacy.

Increased CD4+ T cell proliferation upon treatment correlates with improved overall survival of patients with metastatic melanoma upon anti-CTLA-4 therapy

Abstract Blockade of cytotoxic T-lymphocyte antigen 4 (CTLA-4) augments anti-tumor immunity and has shown success in treating patients with metastatic melanoma. Mechanisms of anti-CTLA-4 efficacy have been proposed in animal models, yet the mechanisms in humans remain to be elucidated. Increased frequency of ICOS+ CD4+ T cells has been shown to be a marker of drug bioactivity, but discovery of prognostic indicators and baseline indicators of which patients are most likely to benefit from CTLA-4 blockade remains a priority. PBMCs from 29 metastatic melanoma patients in a multicenter compassionate use trial of anti-CTLA-4 antibody were evaluated using flow cytometry. At baseline and 12 weeks following initiation of treatment, CTLA-4, HLA-DR, ICOS, PD-L1, PD-1, and Ki-67 frequencies were assessed on CD4+ and CD8+ T cells, as well as CD3+CD4+ regulatory T cells. Independent of survival, anti-CTLA-4 treatment significantly increased the frequency of ICOS+ CD4+ T cells, corroborating previous findings. Interestingly, this increase was also observed in ICOS+ Tregs, but not in CD8+ T cells. Increased proliferative Ki67+ CD4+ T cells and Ki67+ Tregs upon CTLA-4 blockade were both associated with prolonged overall survival. Additionally, when the frequency of Ki67+ CD4+ T cells post-treatment was compared to the baseline frequency, patients with a ratio greater than 1.5 had greater overall survival. Increased post-treatment absolute lymphocyte count—a known prognostic biomarker—also correlated with clinical outcome. Our results suggest that anti-CTLA-4 therapy may be mediated by proliferation of CD4+ T cells, and we identify this proliferation as a novel prognostic biomarker in metastatic melanoma patients treated with anti-CTLA-4 antibody.

The Proliferative Activity of Mammary Epithelial Cells in Normal Tissue Predicts Breast Cancer Risk in Premenopausal Women

The frequency and proliferative activity of tissue-specific stem and progenitor cells are suggested to correlate with cancer risk. In this study, we investigated the association between breast cancer risk and the frequency of mammary epithelial cells expressing p27, estrogen receptor (ER), and Ki67 in normal breast tissue. We performed a nested case-control study of 302 women (69 breast cancer cases, 233 controls) who had been initially diagnosed with benign breast disease according to the Nurses' Health Studies. Immunofluorescence for p27, ER, and Ki67 was performed on tissue microarrays constructed from benign biopsies containing normal mammary epithelium and scored by computational image analysis. We found that the frequency of Ki67(+) cells was positively associated with breast cancer risk among premenopausal women [OR = 10.1, 95% confidence interval (CI) = 2.12-48.0]. Conversely, the frequency of ER(+) or p27(+) cells was inversely, but not significantly, associated with subsequent breast cancer risk (ER(+): OR = 0.70, 95% CI, 0.33-1.50; p27(+): OR = 0.89, 95% CI, 0.45-1.75). Notably, high Ki67(+)/low p27(+) and high Ki67(+)/low ER(+) cell frequencies were significantly associated with a 5-fold higher risk of breast cancer compared with low Ki67(+)/low p27(+) and low Ki67(+)/low ER(+) cell frequencies, respectively, among premenopausal women (Ki67(hi)/p27(lo): OR = 5.08, 95% CI, 1.43-18.1; Ki67(hi)/ER(lo): OR = 4.68, 95% CI, 1.63-13.5). Taken together, our data suggest that the fraction of actively cycling cells in normal breast tissue may represent a marker for breast cancer risk assessment, which may therefore impact the frequency of screening procedures in at-risk women. Cancer Res; 76(7); 1926-34. ©2016 AACR.

Abstract P2-10-01: The BRCA-1 polymorphism (major homozygous rs5820483) is associated with higher expression of phosphorylated -IGF-1 receptor

Abstract Background: Data from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) indicates that common variation in the BRCA1 locus modifies the penetrance of mutations. The rs5820483 variation is one such polymorphism in which patients homozygous for the major allele have a greater risk of breast cancer. The BRCA1 gene contains 24 exons that generate a full-length (FL) protein. Major alternatively spliced isoforms include skipping of exon 11 (Δ11) and a truncated form (IRIS), each of which appear to have distinct roles in cellular processes. rs5820483 is the only variant that maps to intron 10, flanking exon 11. Experimental models suggest that deregulation of the FL/D11 isoform ratio can have profound functional consequences. We have shown that cells from patients with the major rs5820483 allele express more FL relative to Δ11 compared to cells from patients with the minor allele. Breast cancer exhibit increased IGF-1 and IGF-1 activity in patients with BRCA1 mutations relative to breast cancer in women without BRCA1 mutations. IGF-1 produced in the mammary stroma mediates estrogen dependent proliferation. Here we test whether the rs5820483 allele affects IGF-1 activity in breast tissue of BRCA1 mutation carriers. Design: We assessed the zygosity of the rs5820483 alleles in 28 BRCA1 mutation carriers. Phosphorylated IGF-1 receptor (p-IGF-1R rabbit polyclonal Ab39398, Abcam, MA) was measured by immunofluorescence and Ki-67 was measured by immunohistochemistry in sections from histologically normal breast tissue from these cases were compared to heterozygous controls. Image analysis was used to assess the intensity of the p-IGF-1R immunofluorescence at the epithelial cell membrane with appropriate controls. Ki67 mmunohistochemistry was also performed on these specimens. Results:The rs5820483 allele was heterozygous in 10 specimens, homozygous for the major allele in 11 and homozygous for the minor allele in 7. The intensity of p-IGF-1R was higher in major homozygous cases (140±54 SD) than in either the minor homozygous (99±24) or heterozygous cases (95±44). The frequency of Ki-67+ cells was higher in the major homozygous case (3.1±2.9 SD) than in either the minor homozygous (1.5±1.9 SD) or heterozygous cases (2.2±2.5 SD). However, neither p-IGF-IR immunoreactivity nor frequency of Ki-67+ cells was statistically different between groups. Conclusion: Breast cancer risk in BRCA1 mutation carriers is modified by common genetic variation at the corresponding locus. We have identified a variation at rs5820483 that affects the isoform ratio. Our preliminary analysis suggests that IGF-1 activity increases in those women homozygous for the major allele, concomitant with increased Ki-67. Corroboration of this analysis in a larger series is ongoing. A statistically significant difference might have fundamental implications for cancer prevention in those carriers. Citation Format: Singh B, Bochaca II, Ruiz de Garibay G, Damiola F, Mazoyer S, Antoniou A, Chenevix-Trench G, Pujana MA, Barcellos-Hoff MH, Kleinberg D. The BRCA-1 polymorphism (major homozygous rs5820483) is associated with higher expression of phosphorylated -IGF-1 receptor. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-10-01.

TOX3 as a novel biomarker in luminal B breast cancer.

130 Background: A breast cancer (BC)-associated SNP is found in an enhancer region of the TOX3 gene, which encodes a nuclear protein expressed in ER+ mammary epithelial cells but with unknown biological function. As the disease-risk allele reduces TOX3 expression, we proposed that a decrease in TOX3 might favor a progenitor cell state that is more susceptible to tumorigenesis. However, in the context of BC, our data has implicated TOX3 as a tumor promoter. Indeed, high TOX3 gene expression is significantly associated with poor outcome among patients with luminal B (LumB) disease. The Oncotype DX assay predicts likelihood of distant recurrence in ER+ early stage BC, improving patient selection for adjuvant chemotherapy. However, LumB subtypes often have an intermediate recurrence score (RS), where the absolute benefit of chemotherapy is unclear. Thus, we investigated whether TOX3 expression might provide additional discriminatory power. Methods: Tumor specimens were obtained from Cedars-Sinai Medical Center in a retrospective study. Tissue microarrays of 200 histologically-defined LumB breast tumors (ER+Her2-/+ Ki67 &gt; 14%), half of which with associated Oncotype DX data, were stained with an anti-TOX3 monoclonal antibody, to correlate with RS and clinicopathologic outcomes. Results: TOX3 protein was expressed in a significant subset of LumB tumors. Using image analysis to create a histoscore, we found no correlation between TOX3 expression and RS. TOX3 expression was also not associated with frequency of Ki67+ cells, patient age, or tumor size. Further in-depth clinico-pathologic evaluation and prognosis data will be reported. Conclusions: Despite overall efficacy of adjuvant chemotherapy in early stage BC, many patients are exposed to the associated risks without deriving significant benefit. A number of lines of evidence suggest that TOX3 renders tumors more aggressive and metastatic. The lack of correlation of TOX3 expression and RS may suggest a facet of the biology of ER+ aggressive cancers that may not be addressed by the Oncotype DX assay. Thus, prospective studies examining recurrence risk and chemotherapy benefit may need to be expanded to integrate TOX3 expression as an adjunct novel biomarker to guide optimal personalized therapy for BC.

Correlations between clinical and pathological features in 17 cases of mycosis fungoides before and after transformation

Mycosis fungoides (MF) may progress to transformed MF (T-MF), a condition with aggressive behavior. This study was designed to compare the clinical and pathological features of biopsies in 17 cases of MF before and after transformation. During a revision of primary cutaneous T cell lymphomas, 53 cases of MF were identified, including 17 cases of T-MF. Clinical, pathological, and immunohistochemical data for the MF patients were evaluated. Cases of T-MF and intermediate transformed (IT) MF were diagnosed according to previous criteria. The histological and immunohistochemical features of T-MF biopsies were compared with those of MF/IT-MF biopsies taken before or concomitant with transformation. At the initial diagnosis, three patients were found to have more advanced stages of disease: two had MF and T-MF simultaneously, and another had only oral T-MF. Four patients considered to show histological transformation maintained disease stages Ia and Ib and all remain alive. Of five patients with IT-MF at first diagnosis, all progressed to complete histological transformation, three developed tumors, and two died of disease. Four patients progressed to CD30+ large cell lymphoma, and three of these died of disease. In one of these patients, the MF biopsy showed a high level of expression of CD30 in the epidermis and dermis. No correlation between advanced MF and expression of CD25 and CD30, or frequency of Ki-67+ cells was found. The frequency of transformation among patients with initially non-transformed MF was high. Our findings support the emphasis given by other authors to IT-MF, a pattern of MF which is generally not considered in many studies.

Paternal antigen-specific proliferating regulatory T cells are increased in uterine-draining lymph nodes just before implantation and in pregnant uterus just after implantation by seminal plasma-priming in allogeneic mouse pregnancy

Paternal antigen-specific regulatory T (PA-specific Treg) cells play an important role in feto-maternal tolerance. To detect the PA-specific Tregs, female BALB/c mice were mated with male DBA/2 mice. Mls Ia antigen on DBA/2 mice is recognized by the T-cell receptor Vβ6; thus, CD4+Foxp3+Vβ6+ cells are recognized as PA-specific Treg cells. CD4+CD25+Vβ6+ cells effectively suppressed the allo-reactive proliferation of lymphocytes compared with that of CD4+CD25+Vβ6− cells. Vβ6+ PA-specific Treg cells expressed CCR4 and CCR5 on their surface. The frequency of Ki67+ PA-specific Treg cells among Treg cells was significantly increased in draining lymph nodes on day 3.5 post-coitus (pc; 6.8±1.1%, p<0.05) and day 5.5 pc (7.2±1.1%, p<0.05) in allogeneic pregnant mice compared with that in nonpregnant mice (2.7±0.2%). The frequency of Ki67+ PA-specific Treg cells in the uterus increased significantly after day 5.5 pc in allogeneic pregnant mice compared with that in nonpregnant mice (8.8±2.8% vs. 1.2±1.3%, p<0.05). However, Ki67-PA-specific Tregs did not change during pregnancy. To analyze the role of seminal fluid or sperm in Treg expansion, female BALB/c mice were mated with vasectomized DBA/2 male mice (VAS) or seminal vesicle-excised DBA/2 male mice (SVX). The frequency of Ki67+ PA-specific Treg cells did not increase in draining lymph nodes or uterus in BALB/c×DBA/2 (SVX) allogeneic mating mice. These findings suggest that the priming by seminal fluid is important for the induction of proliferating PA-specific Tregs in uterine-draining lymph nodes just before implantation and pregnant uterus after implantation, resulting in successful implantation and the maintenance of allogeneic pregnancy.

Programmed death-1 (PD-1)-dependent functional impairment of CD4+ T cells in recurrent genital papilloma

Genital papilloma is caused by human papilloma virus (HPV) infection and recurs frequently. Although T cells are known to play a critical role in the control of HPV infection and papilloma development, the function and phenotype of these cells in the lesion remain to be elucidated. In the present study, we examined the function and phenotype of CD4(+) T cells isolated from the lesions of primary (n = 9) and recurrent (n = 11) genital papillomas. In recurrent papillomas, the frequency of proliferating (Ki-67(+)) CD4(+) T cells was significantly reduced compared with primary papillomas. Cytokine production was evaluated by intracellular cytokine staining in anti-CD3/anti-CD28-stimulated CD4(+) T cells. CD4(+) T cells from recurrent lesions showed impaired production of IL-2, IFN-γ, and TNF-α. Of interest, the frequency of cytokine-producing CD4(+) T cells significantly correlated with the frequency of Ki-67(+)CD4(+) T cells. We also studied expression of programmed death-1 (PD-1), a T-cell exhaustion marker. The frequency of PD-1(+)CD4(+) T cells was significantly increased in recurrent lesions and inversely correlated with the frequency of cytokine-producing CD4(+) T cells. The functional significance of PD-1 expression was determined in blocking assays with anti-PD-L1, which restored cytokine production of CD4(+) T cells from recurrent lesions. Taken together, in recurrent genital papilloma lesions, proliferation, and cytokine production by CD4(+) T cells are impaired and the PD-1/PD-L1 interaction is responsible for the functional impairment of CD4(+) T cells.

Characterization of regulatory T cells in decidua of miscarriage cases with abnormal or normal fetal chromosomal content

Decreased regulatory T (Treg) cells have been reported in cases of recurrent pregnancy loss. To understand the role of Treg cells in human pregnancy, we have studied the frequency, localization and characterization of Treg cells in the decidua. The frequency of Foxp3+ cells among CD3+CD8− cells at the decidua basalis in cases of miscarriage with a normal embryo karyotype (n=10) was significantly lower than in normally progressing pregnancies (n=10). However, those frequencies in miscarriage with an abnormal embryo karyotype were similar to normally progressing pregnancies. Next, we used flow cytometry to study Treg cell expression of the proliferation marker Ki67 and functional Treg marker CCR5. The frequency of Foxp3+CD4+ T cells in miscarriage with a normal embryo (n=10) was significantly lower than those in normally progressing pregnancies (n=15) and in miscarriage with an abnormal embryo (n=14). In miscarriage with a normal embryo, the population of Ki67−Foxp3+CD4+ T cells was significantly smaller than in normal pregnancy. However, the frequencies of Ki67+Foxp3+CD4+ cells and CCR5+Foxp3+CD4+ cells were not different between the three groups. These data suggest that increased Ki67− Treg cells in the decidua basalis may play an important role in the induction of immune tolerance, and that immune-medicated pregnancy loss may be caused by decreased Ki67− Treg cells in the implantation site.

Frequency of Ki-67 (MIB-1) and P53 expressions among patients with prostate cancer

Prostate cancer is the most common malignant tumor in men. Tumor grade is one of the most important prognostic factors of prostate cancer. P53 and Ki-67 expressions have also been considered to be prognostic factors. This study was performed to investigate the frequency of these proteins expression and compare the obtained results with Gleason's grading. In this cross-sectional study, 49 paraffin blocks of prostate cancers were assessed. Tumor grade was determined according to the Gleason's criteria. Ki-67 and P53 expressions were determined by immunohistochemical staining. The obtained results were analyzed and evaluated using Spearman's statistical test (SPSS version 15). Three out of 49 (6.1%) cases were well differentiated, 21 (43%) moderately differentiated and 25 (51%) were poorly differentiated. P53 was negative in all well-differentiated cases. Ki-67 was negative in 14 cases (28%) including all well-differentiated tumors. Among moderately and poorly differentiated tumors Ki-67 was negative in eight (38%) and three (12%) of cases, respectively. A statistically significant relation was observed between the increased Ki-67 labeling index (LI) and increased Gleason's grade. Conversely, no statistically significant relation was found between P53 expression and increased Gleason's grade. According to the findings of this study, it seems that Ki-67 can be used as a prognostic factor for prostate cancer. On the other hand, the probable relation between P-53 and prostate cancer prognosis requires further studies.

Genomic instability and proliferative activity as risk factors for distant metastases in breast cancer

The role of genomic instability and proliferative activity for development of distant metastases in breast cancer was analysed, and the relative contribution of these two risk factors was quantified. A detailed quantitative comparison was performed between Ki67 and cyclin A as proliferative markers. The frequency of Ki67 and cyclin A-positive cells was scored in the same microscopic areas in 428 breast tumours. The frequency of Ki67-positive cells was found to be highly correlated with the frequency of cyclin A-positive cells, and both proliferation markers were equally good to predict risk of distant metastases. The relative contribution of degree of aneuploidy and proliferative activity as risk markers for developing distant metastases was studied independently. Although increased proliferative activity in general was associated with an increased risk of developing distant metastases, ploidy level was found to be an independent and even stronger marker when considering the group of small (T1) node negative tumours. By combining proliferative activity and ploidy level, a large group of low risk breast tumours (39%) could be identified in which only a few percentage of the tumours (5%) developed distant metastases during the 9-year follow-up time period.