Percentage Of Ki-67 Research Articles

Abstract 4979: Prevalence of proliferating CD8+cells in normal lymphatic tissues, inflammation and cancer

Abstract Expansion of CD8+ cytotoxic T lymphocytes is a prerequisite for anti-cancer immune activity. In the era of immune checkpoint therapy, profound knowledge of the dynamics of CD8+ has regained considerable interest. However, systematically acquired data on CD8+ proliferation in large sets of normal and diseased tissues are sparse. Here, we applied multiplex fluorescence immunohistochemistry to conventional large sections and tissue microarrays in order to quantitate Ki67+CD8+ cells in >20 different compartments of normal lymphoid tissues, 7 types of inflammatory diseases and 785 cancers. In most normal lymphoid tissues (tonsil, lymph node, thymus, Peyer's patches, spleen, colon, appendix) the percentage of Ki67+CD8+ cells typically did not exceed 3%. The percentage of Ki67+CD8+ cells was markedly higher (45%) in the immune-active cortex of the thymus, however. In inflammatory conditions (including Hashimoto thyroiditis, Lichen sclerosus of the penis, sarcoidosis, sialadenitis, IgG4 pancreatitis, Crohn's disease and eczema), the percentage of Ki67+CD8+ cells was much more variable and often sharply higher than in normal tissues. It ranged from 0.5% in one patient with sialadenitis to 19% in the intraepithelial compartment of Crohn's disease. In 765 colorectal cancers, the fraction of Ki67 positive CD8+ cytotoxic T cells ranged from 0 to 100% (mean: 20.6%). A high fraction of Ki67+CD8+ cells was significantly associated with microsatellite instability (p<0.0001), low pT stage (p<0.0001) and absence of nodal metastases (p=0.0005). In summary, our data show a variable increase of the fraction of proliferating CD8+ T cells in cancers and in inflammatory diseases as compared to healthy secondary lymphoid organs. The striking link with microsatellite instability and unfavorable tumor features suggest a potential clinical utility of assessing Ki67+CD8+ in colorectal cancer. Citation Format: Maximilian Lennartz, Raed Abu-Hashem, Niclas Christian Blessin, Tim Mandelkow, Elena Bady, Ronald Simon, Claudia Hube-Magg, Guido Sauter, Christoph Fraune, Katharina Möller, Till S. Clauditz, Andreas M. Luebke, Franziska Büscheck. Prevalence of proliferating CD8+cells in normal lymphatic tissues, inflammation and cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4979.

Abstract 6640: Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti-tumor activity of PD-1 blockade in mouse models of colorectal cancer

Abstract Despite remarkable responses to immune checkpoint blockade across multiple tumor types, the clinical benefit in colorectal cancer (CRC) is limited to microsatellite unstable tumors. PD-L1 expression is a negative prognostic marker in CRC but correlates with a better response to PD-1 blockade. Here, we investigated the role of PD-L1 in colorectal tumorigenesis and evaluated the utility of targeting myeloid-derived suppressor cells (MDSCs) in combination with PD-1 blockade in mouse models of CRC. We generated knockin mice that conditionally express the murine Pdl1 gene (R26-LSL-Pdl1-EGFP) and crossed them with LysM-Cre mice to overexpress PD-L1 specifically in the myeloid lineage. Mice received azoxymethane (AOM; 10 mg/kg i.p.) followed one week later with 2.5% dextran sodium sulfate (DSS) in the drinking water for 7 days. AOM/DSS-treated control mice formed tumors at 10 weeks and developed adenocarcinoma at 17 weeks post-AOM. LysM-Cre; R26-PD-L1 mice treated with AOM/DSS showed markedly enhanced colorectal tumorigenesis, with a significant increase in tumor number and size and enlarged spleen. In both groups, AOM/DSS treatment led to a significant expansion of myeloid cells, particularly CD11b+Gr-1+ MDSCs, in the colon and spleen, along with decreased NK cells compared with untreated mice. Notably, AOM/DSS-treated LysM-Cre; R26-PD-L1 mice showed decreased intratumoral CD8+ T cell infiltration compared to control tumors. Furthermore, there was a significant decrease in the percentage of Ki-67+ cells in intratumoral CD8+ T cells, indicating attenuated anti-tumor immunity. Trefoil factor 2 (TFF2), a secreted anti-inflammatory peptide, inhibits colon tumor growth by suppressing the expansion of CD11b+Gr-1+ MDSCs. TFF2 fused with two carboxyl-terminal peptide and three Flag motifs (TFF2-CTP-Flag) prolonged the circulation time in blood but retained bioactivity. We induced tumors in R26-PD-L1 mice with AOM/DSS, administered fusion recombinant TFF2-CTP-Flag (300 ug i.p.) and/or anti-PD-1 (RMP1-14; 200 ug i.p.) three times a week starting at 10 weeks and 14 weeks, respectively, and examined tumors at 18 weeks post-AOM. R26-PD-L1 mice treated with anti-PD-1 + TFF2-CTP showed a marked reduction in tumor growth while anti-PD-1 monotherapy failed to suppress growth. The combination of anti-PD-1 and TFF2-CTP significantly increased tumor-infiltrating CD8+ T cells and concomitantly decreased intratumoral regulatory T cells and CD11b+Gr-1+ myeloid cells. These early findings suggest that TFF2 augments the response rate of CRC to PD-1 blockade, possibly through suppressing MDSC expansion, supporting the potential of TFF2-CTP in combination I-O treatment for CRC. We are currently testing the efficacy of combined TFF2-CTP and anti-PD-1 therapy in the AOM/DSS model with PD-L1-overexpressing LysM-Cre; R26-PD-L1 mice. Citation Format: Woosook Kim, Na Fu, Phaneendra Duddempudi, Zinaida Dubeykovskaya, Steven Almo, Chandan Guha, Seth Lederman, Timothy Wang. Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti-tumor activity of PD-1 blockade in mouse models of colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6640.

Is Ki-67 index overexpression in IDH wild type glioblastoma a predictor of shorter Progression Free survival? A clinical and Molecular analytic investigation

BackgroundKi-67 proliferation index is widely used for differentiating between high and low-grade gliomas, but differentiating between the same grade IV appears to be more problematic, and the point about its prognostic value for GBM patients remains unclear. To reduce the possibility to find a marked histological heterogeneity, and may contain areas that could be diagnosed as lower grade, in this study we considered a large group of patients with IDH wild-type Glioblastoma (IDH-WT GBM) and we have analyzed previously reported prognostic factors, in regards to their relationship with the Ki-67 expression index. MethodsWe explore the prognostic impact of ki-67 index status in 127 patients affected by IDH-WT GBM. We therefore analyzed clinical characteristics, tumor genetics, dimension and clinical outcomes. We selected a total of 127 patients affected by newly diagnosed IDH-WT GBM who underwent surgery, radiation, and chemotherapy in our Institution in the period ranging between January 2014 and December 2016 ResultsThe volume of the lesion had a strong association with the Ki67 overexpression. In particular lesions whose volume was greater than 45 cm3, presented a higher percentage of Ki67 expression demonstrating that greater tumors are more likely associated to higher values of Ki67 percentages. On a multivariate analysis, it was possible to outline that Ki67 was significant a predictor of shorter PFS independently from the age of the patients, the volume of the lesion and preoperative KPS. ConclusionsThere is a correlation between percentage staining of Ki-67 and OS in our cohort of patients with IDH-WT GBM. This is only the third observational study documenting a positive correlation between Ki-67 and overall survival in GBM and the first one demonstrates that percentage Ki-67 staining >20 % predicts poorer progression free survival in IDH-WT GBM.

Insulin Reduces the Efficacy of Vemurafenib and Trametinib in Melanoma Cells.

BackgroundDespite the progress made in the clinical management of metastatic melanoma, a patient’s response to treatment cannot be fully predicted, and intrinsic or acquired resistance that is developed in most melanoma patients warrants further research efforts. In addition to genetic factors, microenvironmental input should be considered to explain the diversity of response to treatment among melanoma patients. In this study, we evaluated the impact of insulin on patient-derived BRAFV600E melanoma cells, either untreated or treated with vemurafenib or trametinib, inhibitors of BRAFV600 and MEK1/2, respectively.MethodsCells were cultured in serum-free conditions, either with or without insulin. The activity of the MAPK/ERK and PI3K/AKT pathways was assessed by Western blotting, cell viability, and percentages of Ki-67- and NGFR-positive cells by flow cytometry. Transcript levels were analyzed using qRT-PCR, and γ-H2AX levels by immunoblotting and confocal microscopy. A luminescence-based assay was used to measure glutathione content.ResultsWhile insulin did not influence the MAPK/ERK pathway activity, it had a strong influence on melanoma cells, in which this pathway was suppressed by either vemurafenib or trametinib. In the presence of insulin, both drugs were much less efficient in 1) inhibiting proliferation and reducing the percentage of Ki-67-positive cells, and 2) inducing apoptosis and phosphorylation of histone H2AX in melanoma cells. Changes induced by vemurafenib and trametinib in glutathione homeostasis and DNA repair gene expression were also attenuated by insulin. Moreover, insulin impaired the combined effects of targeted drugs and doxorubicin in melanoma cells. In addition to insulin-induced PI3K/AKT activity, which was either transient or sustainable depending on the cell line, an insulin-triggered increase in the percentage of cells expressing NGFR, a marker of neural crest stem-like cells, may contribute to the reduced drug efficacy.ConclusionOur results demonstrate the role of insulin in reducing the efficacy of vemurafenib and trametinib. This needs clinical assessment.

A study of Ki 67, p53 and their combination as immunomarkers in squamous cell carcinoma of oral cavity

Background: Oral cancer is the most common cancer and constitutes a major health problem. Tobacco and alcohol remains the most important etiologic factors. Molecular level changes in the lesion occur before clinical and histopathological changes. The prognostic evaluation of this neoplasia is given by histological and immunohistochemical findings, including staging, histological grade, tumor size, lymph node involvement, immunohistochemical expression of p53 and Ki-67.In squamous cell carcinomas of oral cavity, p53 and Ki-67 are related to large tumors, metastasis to lymph nodes and very likely to a worse prognosis. Aim and objectives: To assess and compare the expression p53, Ki-67 and their combination markers in squamous cell carcinoma of oral cavity in relation to lymph node status, degree of histological differentiation and pathological staging. Materials and Methods: The prospective study was conducted in N.I.M.S medical college carried out with 108 cases of oral cavity squamous cell carcinoma with lymph node resection. The p53 and Ki-67 labelling indices were expressed as the percentage of Ki-67 and p53 immunolabelled tumor nuclei per 1000 tumor nuclei counted. Results: The maximum numbers of cases were found to be in the age group of 40-60 years (61.8%), mostly being men (85.1%). Female comprised of only 14.9% of the patients. All cases were subjected to immunohistochemical study for Ki- 67 and p53. Positive expression of Ki 67 detected was in 92 cases and p53 was detected in 72 cases. Both Ki -67 and p53 were positive in 58 cases. Ki -67 marker was tested in all 108 cases. Out of total 108 cases 92 (85.2%) were found to be positive for Ki -67 and only 16(14.8%) were found negative. In the histological grading 48 (44.4%) were low grade (well differentiated), and 30 (55.6%), were high grade (moderately and poorly differentiated). Out of 48 low grade cases 14(29.2%) cases and out of 60 high grade cases 44(73.3%) were positive for p53and Ki 67 which was statist

Prognostic Value of Ki67 Percentage, WT-1 Expression and p16/CDKN2A Deletion in Diffuse Malignant Peritoneal Mesothelioma: A Single-Centre Cohort Study.

Diffuse malignant peritoneal mesothelioma (DMPM) is a rare malignant neoplasm with a poor survival. Although some advances in knowledge have been obtained for the pleural form, much less is known about DMPM. Advantages in terms of prognosis are still limited and strong efforts need to be made. The aim of our study was to correlate several histological and molecular factors with survival in a large cohort of 45 DMPMs. We evaluated histotype, nuclear grade, mitotic count, necrosis, inflammation, desmoplastic reaction, Ki67 percentage, WT-1 expression, p16 protein by immunohistochemistry and CDKN2A deletion by FISH. Our results showed that epithelioid histotype, nuclear grade 2, mitotic count ≤5 x mm2, absence of desmoplasia and p16/CDKN2A deletion, low Ki67 value, and high WT-1 expression were correlated with the most prolonged survival (p = 0.0001). Moreover, p16 loss in immunohistochemistry reflected CDKN2A deletion detected with FISH, and both were correlated with the worst survival (p = 0.0001). At multivariate analysis, Ki67 value, WT-1 expression and p16/CDKN2A deletion emerged as independent prognostic factors (p = 0.01, p = 0.0001 and p = 0.01, respectively). These parameters are easy to analyse at the time of DMPM diagnosis and may support better patient stratification, prediction of treatment effectiveness and therapeutic optimization.

Liver Tumor Burden in Pancreatic Neuroendocrine Tumors: CT Features and Texture Analysis in the Prediction of Tumor Grade and 18F-FDG Uptake.

Pancreatic neuroendocrine tumors (p-NETs) are a rare group of neoplasms that often present with liver metastases. Histological characteristics, metabolic behavior, and liver tumor burden (LTB) are important prognostic factors. In this study, the usefulness of texture analysis of liver metastases in evaluating the biological aggressiveness of p-NETs was assessed. Fifty-six patients with liver metastases from p-NET were retrospectively enrolled. Qualitative and quantitative CT features of LTB were evaluated. Histogram-derived parameters of liver metastases were calculated and correlated with the tumor grade (G) and 18F-fluorodeoxyglucose (18F-FDG) standardized uptake value (SUV). Arterial relative enhancement was inversely related with G (−0.37, p = 0.006). Different metastatic spread patterns of LTB were not associated with histological grade. Arterialentropy was significantly correlated to G (−0.368, p = 0.038) and to Ki67 percentage (−0.421, p = 0.018). The ROC curve for the Arterialentropy reported an area under the curve (AUC) of 0.736 (95% confidence interval 0.545–0.928, p = 0.035) in the identification of G1–2 tumors. Arterialuniformity values were correlated to G (0.346, p = 0.005) and Ki67 levels (0.383, p = 0.033). Arterialentropy values were directly correlated with the SUV (0.449, p = 0.047) which was inversely correlated with Arterialuniformity (−0.499, p = 0.025). Skewness and kurtosis reported no significant correlations. In conclusion, histogram-derived parameters may predict adverse histological features and metabolic behavior of p-NET liver metastases.

Progress in the research of p53 tumour suppressor activity controlled by Numb in triple-negative breast cancer.

Numb is known as a cell fate determinant as it identifies the direction of cell differentiation via asymmetrical partitioning during mitosis. It is considered as a tumour suppressor, and a frequent loss of Numb expression in breast cancer is noted. Numb forms a tri‐complex with p53 and E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing the ubiquitination and degradation of p53. In this study, we examined Numb expression in 125 patients with triple‐negative breast cancer (TNBC). The results showed that 61 (48.8%) patients presented with a deficient or decreased Numb expression. The percentage of Ki67 > 14% in the retained Numb group was significantly lower than that in the decreased and deficient Numb groups (86.00% vs. 98.40%, P = .0171). This study aimed to detect the expression and migration of Numb, HDM2 and p53 in the membrane, cytoplasmic and nuclear fractions of normal mammary epithelial cell line MCF‐10A and basal‐like TNBC cell line MDA‐MB‐231. We obtained the cell fractions to identify changes in these three protein levels after the re‐expression of NUMB in the MDA‐MB‐231 cells and the knocking down of NUMB in the MCF‐10A cells. Results showed that Numb regulates p53 levels in the nucleus where the protein levels of Numb are positively correlated with p53 levels, regardless if it is re‐expressed in the MDA‐MB‐231 cells or knocked down in the MCF‐10A cells. Moreover, HDM2 was remarkably decreased only in the membrane fraction of NUMB knock‐down cells; however, its mRNA levels were increased significantly. Our results reveal a previously unknown molecular mechanism that Numb can migrate into the nucleus and interact with HDM2 and p53.

248-LB: Spexin Promotes Insulin Secretion and ß-Cell Proliferation

Spexin was firstly discovered as a neuropeptide and was also detected in peripheral tissues including liver, fat and pancreatic islets. Recently, studies documents that diabetes patients had lower circulation level of Spexin. It is unclear whether Spexin exerts glucose homeostasis regulating effects. Here, we provide evidence that Spexin stimulates β cell proliferation and insulin secretion in mice and human islets. Acute administration of Spexin prior glucose challenge promoted insulin secretion and improved glucose tolerance in a dose dependent manner (35-200 μg/kg BW). Meanwhile, Spexin treatment had no effect on insulin tolerance. The addition of Spexin to mouse islets increased β cell proliferation demonstrated by 2-fold increase of BrdU(+) β-cells. Spexin promoted cell growth of INS-1 cells in a dose dependent manner with maximum effect at dose of 25 ng/ml. In a high-fat diet (HFD)-induced β cell proliferation and β cell mass expansion model, the percentage of Ki67(+) β-cells increased by 2-fold compared to chow diet (CD)-fed mice. Spexin provoked further 80% increase. Consistently, HFD feeding increased β-cell mass by 2-fold and Spexin made further 58% increase. To identify the mechanism of Spexin-stimulated β cell proliferation, we tested whether the expression of well-established β cell cycle regulators was controlled by Spexin. Spexin treatment of INS-1 cells for 24 h significantly increased mRNA levels of cyclin D1 and Cyclin E1 compared with controls. Spexin stimulated the phosphorylation of CREB (p-CREB) with a peak at 10 min. The addition of H89, a potent PKA inhibitor blocked the effect of Spexin on activation of p-CREB and stimulation of the expression of Ki67, Cyclin D1 and Cyclin E1. Furthermore, Spexin stimulated the p-CREB, promoted insulin secretion and increased β cell proliferation in human islets. Thus, Spexin is an important factor for islet β cell proliferation, and our study provides previously unrecognized evidence of a factor governing endocrine pancreas growth and function. Disclosure R. Liu: None. Funding National Natural Science Foundation of China (81200622, 81570518, 81630020)

Safety and tolerability of MEDI0562 in combination with durvalumab or tremelimumab in patients with advanced solid tumors.

3003 Background: We report safety and tolerability of MEDI0562, a humanized IgG1κ OX40 monoclonal antibody (mAb), in combination with durvalumab (durva; anti-PD-L1 mAb) or tremelimumab (treme; anti-CTLA-4 mAb) in patients (pts) with previously treated advanced solid tumors. Methods: In this phase 1, open-label study (NCT02705482), adult pts received escalating doses of MEDI0562 (2.25, 7.5 or 22.5 mg/kg) every 2 wks (Q2W) in combination with durva (1500 mg/kg) or treme (75 or 225 mg/kg) Q4W, until confirmed disease progression or unacceptable toxicity. Tumor assessments were performed Q8W with immune-related Response Evaluation Criteria in Solid Tumors. Results: In total, 27 and 31 pts received MEDI0562 + durva or treme, across 5 dose combination cohorts (3 + 3 design), with a maximum tolerated dose of 7.5 mg MEDI0652 + 1500 mg durva and maximum administered dose of 10 mg MEDI0562 + 225 mg treme. Median duration of exposure was 12.0 (range 2.0–80.9) and 8.0 (range 2.0–42.0) wks, respectively. Two (22.5 mg MEDI10562 + durva) and 3 (2.25 mg MEDI0652 + 225 mg treme, 22.5 mg MEDI0562 + 75 and 225 mg treme) dose limiting toxicities were observed. For MEDI0562 + durva and MEDI0562 + treme groups respectively, treatment-emergent adverse events (TEAEs) were reported in 96.3% and 100% of pts; most common TEAEs were fatigue (55.6%) and pruritus (45.2%), Gr 3/4 TEAEs occurred in 74.1% and 67.7%; and MEDI0562-related AEs were reported in 20 (74.1%) and 24 (77.4%) pts. Six TEAEs in each group led to MEDI0562 discontinuation (22.2% and 19.4%, respectively), 2 led to death (renal failure [7.5 mg MEDI0562 + durva], multiple organ dysfunction syndrome [22.5 mg MEDI0562 + 225 mg treme]). Three response evaluable pts had PR (11.5% [7.5 and 22.5 mg MEDI0562 + durva, n = 26]). Median overall survival was 17.4 and 11.9 mos for MEDI0562 + durva and MEDI0562 + treme, with stable disease seen in 9 pts from each group, 34.6% vs 29.0%, respectively. Serum exposure of MEDI0562 increased dose proportionally. Post treatment serum antidrug antibody (ADA) was detected in 20 pts from MEDI0562 + durva and MEDI0562 + treme (74.1% and 71.4%, respectively). The impact of ADA on MEDI0562 pharmacokinetics was seen at all doses. Mean percentage of Ki67+CD4+ and Ki67+CD8+ memory T cells increased, while mean percentage of OX40+CD4+ memory T cells decreased following the first dose of MEDI0562 + durva or treme. Conclusions: The safety profile of MEDI0562 in combination with durva or treme was similar between groups. Clinical activity was observed with MEDI0562 + durva in pts with advanced solid tumors. Clinical trial information: NCT02705482 .

Comparison of pathological features of estrogen receptor-positive/HER2-negative early breast cancer in the intermediate-risk group of the 21 gene recurrence score (Oncotype Dx) divided into low and high-risk groups by the 95 gene classifier (Curebest 95GC Breast).

e12535 Background: Oncotype Dx is becoming the standard assay for chemotherapy decision making for estrogen receptor positive / HER2 negative early breast cancer. Issues concerning the choice of therapy for the intermediate risk patients are currently being addressed. We are using a 95-gene classifier developed by Osaka University and Sysmex Corporation (Curebest 95 GC Breast) which divides patients into "low" and "high" risk groups, which obviates this issue. In this study, we analyzed the histopathological characteristics of low and high risk patients by the 95-gene classifier that would be classified as intermediate risk by 21-gene-recurrence score (21RS). Methods: Fresh tissue samples collected at surgery was sent for 95-GC analysis. Cel-files containing the gene expression data of each sample was uploaded to Recurrence Online (recurrenceonline.com) and the 21-RS was obtained. Histopathological analysis of the tumor specimens including the pathological tumor size, degree of tumor infiltrating lymphocytes ("TIL"s) (0, 1+, 2+, 3+), nuclear pleomorphism (NP score 1, 2, 3), and number of mitoses (M score 1, 2, 3) was done. The age of the patient and the Ki-67 percentage of the preoperative biopsy specimen was also noted. Results: There were a total of 38 cases. The cases with a matching 21RS and 95GC "Low" (Group L-L) was 10, a matching "High" (Group H-H) was 15, 21RS "Intermediate / 95GC "Low" (Group I-L) was 4, 21RS "Intermediate / 95GC "High" (Group I-H) was 7, and discordant 21RS "High" / 95GC "Low" (Group H-L) was 2, respectively. There was no age related differences (average 53.3 y.o.). There was a significant difference between the L-L and H-H groups in all categories (i.e. pT 14.5 mm v.s. 30.0 mm / p = 0.0262, NP score average 2.1 v.s. 2.5 / p = 0.287, M score average 1.2 v.s 2.4 / p < 0.001, TILs average 0.4+ v.s. 1.2+ /p = 0.0413, Ki67 25.0% v.s. 33.2%). The trend was the same between the I-L and I-H group with the exception of the mitosis score, which showed an inverse trend of a high of 2.25 in the I-L v.s. low of 1.43 in the I-H group (p = 0.9522). The H-L group had high NP score (average 3.0), low M score (average 1.0), and high TILs (average 2+). Conclusions: The 95-gene classifier might be more sensitive compared to 21RS in picking up histio-morphological information, and also may be able to differentiate the type of immune response of TILs. We are planning further immunohistological evaluation.

Immunosuppressive therapy of immune-mediated GI damage may impair epithelial regeneration

Abstract Glucocorticoids (GC) are a standard therapy of immune-mediated gastrointestinal (GI) damage. However, there is little understanding of the effects that GC use may have on intestinal epithelium and its regenerative function. Using ex vivo organoid models of immune-mediated intestinal injury, we sought to investigate direct effects of GC on intestinal epithelium and test the impact of GC on cytokine- and T-cell-mediated effects. GC suppressed proliferation of small intestine (SI) organoids, reducing organoid size and decreasing cell cycle progression of Lgr5+ intestinal stem cells. Consistently, GC treatment of C57BL/6J mice suppressed epithelial proliferation, decreasing crypt height and reducing the percentage of Ki67+ crypt cells in vivo. We next investigated the role of GC-mediated epithelial suppression in the setting of tissue damage, modeling immune-mediated GI pathology by culturing organoids in the presence of IFNγ and by co-culturing organoids with allogeneic T-cells. Culture with GC in addition to IFNγ significantly increased the toxicity of IFNγ leading to reduced efficiency of organoid formation. Consistently, GC pretreated organoids were more susceptible to allogeneic T-cells with reduced formation efficiency. Finally, we studied cytokine-mediated epithelial regeneration in the context of GC treatment. Culturing organoids with both GC and IL-22 indicated that IL-22 could promote regeneration of intestinal epithelium in the presence of GC. Taken together, our findings indicate that GC, which are necessary clinically for suppressing pathologic inflammatory immune responses, may have direct effects on the epithelium potentially impairing protective regeneration responses and augmenting toxicity.

Dietary administration of alpha-tocopheryloxyacetic acid induces CD4+ T cell activation and prolongs survival in murine rhabdomyosarcoma

Abstract Relapsed sarcomas remain essentially incurable for most children with cancer. Alpha-Tocopheryloxyacetic acid (αTEA) is a scalable semi-synthetic derivative of alpha-tocopherol that has shown promising activity preclinically and clinically in adult cancer, but has not yet been studied in pediatric sarcoma. We hypothesized that αTEA would mediate anti-tumor effects in rhabdomyosarcoma (RMS) by modulating the immunosuppressive milieu. Treatment of murine M3-9-M RMS in vitro with αTEA (2.5–100uM) resulted in increased apoptosis by flow cytometry (Annexin V+/7AAD+) and Incucyte analysis (Annexin V+) within 24 hours (p < 0.05). Using an established orthotopic luciferase+ M3-9-M in vivo RMS model, αTEA-supplemented chow administered for 21 days significantly reduced tumor volume (p < 0.001) and bioluminescence (p < 0.05) by 28 days, resulting in a significant prolongation of survival compared to matched control chow (p < 0.05), up to 4 weeks after the feed was stopped. Analysis of spleens from RMS mice fed αTEA-supplemented diets showed an increased percentage of Ki-67+ and IFNγ+CD4+ T cells (p < 0.01) at the end of treatment as compared to recipients of the control diet. In addition, a decrease in the percentage of CD11b+Arg-1+ (p < 0.01) and PD-L1+ (p < 0.05) cells within the spleen were observed in αTEA-treated RMS tumor-bearing mice. RNA-Seq analysis of RMS tumors is underway to explore changes in αTEA vs. control-treated mice. These results suggest that αTEA causes direct induction of apoptosis of RMS in vitro and in vivo, leading to prolonged survival through enhanced CD4+ T cell stimulation and reduced myeloid cell-mediated immunosuppression in RMS-bearing mice. Further investigation of αTEA as an immunomodulating agent is warranted.

Clinical Treatment and Prognosis of 61 Patients with Extranodal NK/T Cell Lymphoma

To investigate the pathological features, clinical characteristics, treatment outcomes and prognosis influcing factors in patients with extranodal NK/T cell lymphoma(ENKTL). Clinical data of 61 patients with ENKTL treated in oncology and hematology department of Southwest Medical University Affiliated Hospital from April 2013 to August 2018 were restrospectively analyzied, the pathological features, clinical characteristics, therapeutic strategy and prognosis-related factors were explored. The median follow-up was 20 months（range 3 to 81 months）and median age was 48 years old（range 15 to 70 years old）, the proportion of male and female patients was 1.5∶1. The overall survival(OS) rate of 1,3 years was 67.2%,26.2% respectively and progression-free survival(PFS) rate was 63.9%,24.5% respectively. Univariate analysis showed that the percentage of Ki-67 affected efficacy of initial treatment; Ann Arbor stage, LDH level, EBER test, anemia, IPI score, B-symptoms, bone marrow involvement, lymphnodes involvement, monotherapy, initial treatment effect were factors associated with prognosis. According to multivariate analysis, the initial coure remission was the independent factors for prognosis. More survival benefit could be obtained by receiving radiotherapy especially for early stage patients. Initial course remission is independent factors influencing prognosis. For patients in early stage(IE to IIE), radiotherapy or combined radiotherapy with chemotherapy is superior to chemotherapy alone; The patients diagnosed with IIIE or IVE show rapid progression of disease and poor outcome, whether they are treated with combined treatment or monotherapy. New methods such as hematopoietic stem cell infusion and CAR-T can be considered.

Abstract P1-10-26: Gene expression levels of DTX3, CACNA1G, IL11, ETV4 and TSPAN7 selected by LASSO penalty regression could predict pCR after neoadjuvant chemotherapy in breast cancer tumors

Abstract Introduction: While pCR has been amply associated with improved treatment outcome in patients treated with NAC and surgery, there are only few studies that explore the molecular determinants of pCR. Given the discrete percentage of patients who achieve pCR after NAC, it is of upmost need to identify biomarkers that can discern between responder and non-responder patients before NAC and therefore determine the best therapeutic approach in advance. Our aim is to find the best combination of variables to predict pCR in our cohort. Patients: To evaluate if there is a gene expression profile that could predict pathological complete response (pCR) to NAC, we selected a cohort of 69 patients treated with neoadjuvant chemotherapy consisting of anthracyclines + taxanes; HER2+ patients were further treated with trastuzumab. The median age was 48 years, and 71% of patients were premenopausal. The median of mammographic tumor size was 3,7. Most patients (89%) had invasive ductal carcinoma, and the histological grade was mainly 2 (55,7% Grade 1+2; 44,3% Grade 3). ER and PR positivity were seen in 63% and 53% of cases respectively. HER2 overexpression was seen in 20% of tumors. Methods: Gene expression was evaluated in 770 genes involved in 13 canonical cancer pathways as screened with the nCounter® PanCancer Pathways panel in FFPE pre-treatment tumor biopsies and analyzed with the nSolverTM software. The collected clinical and pathologic variables were age, mammographic tumor size, nodal status, tumor grade, HR status, HER2-overexpression, Ki67 percentage, IHQ subtype and PAM50 subtype. Finally, LASSO regression was used to predict pCR based on normalized gene expression levels and clinical and pathological data. Results: Out of the 69 enrolled patients, 27 achieved pCR (39%) and 42 did not (determined by Miller and Payne criteria). High-quality RNA was isolated from each patient for doing Nanostring nCounter experiment. Based on the gene expression levels of the PanCancer Pathways panel, Lasso regression identified a subset of genes as predictive of pCR. Therefore, the combination of expression levels of DTX3, CACNA1G, IL11, ETV4 and TSPAN7 genes, predicted pCR with an average 70% of Area Under the Curve ROC (AUC) on the test set in a cross-validation scheme. DTX3 is a member of Notch pathway, IL11 from JAK-STAT pathway, CACNA1G from MAPK pathway, ETV4 and TSPAN7 from Transcriptional misregulation pathway. DTX3, CACNA1G and TSPAN7 are down-regulated and IL11 and ETV4 are up-regulated in patients who achieved pCR. Regarding the clinical and pathological data, the best model for predict pCR obtained an 60% of AUC and the selected variables were age, mammographic tumor size, nodal status, tumor grade and Ki67 percentage. Conclusions: In our cohort, the combination of gene expression levels of DTX3, CACNA1G, IL11, ETV4 and TSPAN7 can predict tumor response to NAC even better than any clinical or pathological variable. Although validation in a separate cohort is necessary, our results indicate that this combination could constitute a predictive gene signature to discriminate resistant patients to NAC before the treatment in breast cancer tumors. Citation Format: Maria Rosario Chica-Parrado, Julio Montes-Torres, Cynthia Robles-Podadera, Martina Alvarez, Jose Jerez, Luis Vicioso, Lidia Pérez-Villa, Begoña Jimenez, Nuria Ribelles, Ana Godoy, Isabel Barragán-Mallofret, Emilio Alba. Gene expression levels of DTX3, CACNA1G, IL11, ETV4 and TSPAN7 selected by LASSO penalty regression could predict pCR after neoadjuvant chemotherapy in breast cancer tumors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-26.

Effects of anastrozole on Ki-67 antigen expression in the vaginal epithelium of female rats in persistent estrus

OBJECTIVES:Aromatase inhibitors are the first-choice drugs for the treatment of hormone sensitive breast cancer. However, in addition to the scarcity of studies, there are controversies about their effects on vaginal epithelial cell proliferation in rats, especially those in persistent estrus.METHODS:To investigate vaginal epithelial cell proliferation by Ki-67 antigen expression, persistent estrus was induced in 42 randomly selected rats. These rats were randomly divided into 2 groups: group I (control, n=21), which received 0.1 mL of propylene glycol (vehicle) daily, and group II (experimental, n=21), which received 0.5 mg/kg or 0.125 mg/day of anastrozole diluted with 0.1 mL of propylene glycol.RESULTS:Light microscopy showed a higher concentration of cells with brown Ki-67 stained nuclei in the control compared to the experimental group. The mean percentage of Ki-67 stained nuclei per 500 cells in the vaginal epithelium was 68.64±2.64 and 30.46±2.00 [mean±standard error of the mean (SEM)] in the control and experimental groups, respectively (p<0.003).CONCLUSION:This study showed that anastrozole, at the dose and treatment duration selected, significantly decreased cell proliferation in the vaginal mucosa of the rats in persistent estrus.

Immune-related adverse events are clustered into distinct subtypes by T-cell profiling before and early after anti-PD-1 treatment

ABSTRACT Although anti-programmed death-1 (PD-1) treatment has shown remarkable anti-tumor efficacy, immune-related adverse events (irAEs) develop with heterogeneous clinical manifestations. However, the immunological understanding of irAEs is currently limited. In the present study, we analyzed peripheral blood T cells obtained from cancer patients who received anti-PD-1 treatment to determine the immunological characteristics of irAEs. This study included 31 patients with refractory thymic epithelial tumor (TET) who were enrolled in a phase II trial of pembrolizumab (NCT02607631) and 60 patients with metastatic non-small cell lung cancer (NSCLC) who received pembrolizumab or nivolumab. T-cell profiling was performed by multicolor flow cytometry using peripheral blood obtained before treatment and 7 days after the first dose of anti-PD-1 antibodies. irAEs developed in 21 TET patients and 24 NSCLC patients. Severe (≥ grade 3) irAEs occurred in 7 TET patients (22.6%) and 6 NSCLC patients (10.0%). Patients with severe irAEs exhibited a significantly lower fold increase in the frequency of effector regulatory T (eTreg) cells after anti-PD-1 treatment, a higher ratio of T helper-17 (Th17) and T helper-1 cells at baseline, and a higher percentage of Ki-67+ cells among PD-1+CD8+ T cells posttreatment. In clustering analysis using the T-cell parameters, patients with irAEs were grouped into four distinct subtypes: Th17-related, TNF-related, CD8-related Treg-compensated, and CD8-related Treg-uncompensated. The T-cell parameters showed a predictive value for the development of each subtype of severe irAEs. In conclusion, severe irAEs after anti-PD-1 treatment were clustered into four immunological subtypes, and potential biomarkers for early prediction of severe irAEs were proposed.

Inhibition of Siah2 ubiquitin ligase ameliorates monocrotaline-induced pulmonary arterial remodeling through inactivation of YAP

AimsIt has been shown that up-regulation of E3 ubiquitin ligase seven-in-absentia-homolog 2 (Siah2) and activation of Hippo signaling pathway effector yes-associated protein (YAP) are involved in the development of pulmonary arterial hypertension (PAH). However, it is still unclear whether Siah2 activates YAP in monocrotaline (MCT)-induced PAH rat models. Main methodsIntraperitoneal injection of MCT was used to induce PAH rat models. The right ventricular systolic pressure (RVSP), right ventricle hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-SMA, Ki-67 and TUNEL staining were performed to evaluate the development of PAH. Protein levels of Siah2, Lats1/2, YAP phosphorylation and total YAP, and the subcellular localization of YAP were examined using immunoblotting. Proteasome activity was measured by an assay kit. Key findingsThe protein level of Siah2 was significantly increased in MCT-induced PAH rats, this was accompanied with the proteasome-dependent degradation of Lats1/2 and subsequent up-regulation and dephosphorylation of YAP and its nuclear localization. Administration of PAH rats with Siah2 inhibitor Vitamin K3 or proteasome inhibitor MG-132 dramatically suppressed MCT-induced down-regulation of Lats1/2 and activation of YAP, finally reduced RVSP, RVHI, %MT, pulmonary arterial muscularization, pulmonary arterial smooth muscle cells (PASMCs) proliferation and enhanced PASMCs apoptosis in PAH rats. SignificanceSiah2 contributes to the development of MCT-induced PAH by destabilizing Lats1/2 and subsequently stimulating YAP activation. Inhibition of Siah2 or proteasome alleviates pulmonary arterial remodeling through inactivation of YAP, indicating Siah2 ubiquitin ligase as a novel target might have potential value in the management of PAH.

Plaque stage folliculotropic mycosis fungoides: histopathologic features and prognostic factors in a series of 40 patients.

BackgroundFolliculotropic mycosis fungoides (FMF) is a distinct variant of mycosis fungoides. Recent studies recognized indolent and aggressive subgroups of FMF, but there is controversy how patients presenting with plaques should be classified. The present study describes the histopathologic features of 40 FMF plaques. The aim of the study was to identify risk factors for disease progression and poor outcome in this group.MethodsClinical, histopathological, and immunophenotypical data from 40 patients with plaque stage FMF were reviewed and analysed for risk factors for disease progression and survival.ResultsAfter a median follow‐up of 80 months, disease progression occurred in 20 of 40 patients. Percentage of atypical cells, cell size, percentage of Ki‐67+ cells, and co‐existent interfollicular epidermotropism, but not the extent of perifollicular infiltrates, were associated with disease progression and reduced survival, while extensive follicular mucinosis was associated with increased survival.ConclusionsThis study underlines that FMF patients presenting with plaques represent a heterogeneous group and that a subgroup of these patients may have an indolent clinical course. It further shows that histological examination is a valuable tool to differentiate between indolent and aggressive disease.

Correlation Between Digital and Manual Determinations of Ki-67/MIB-1 Proliferative Indices in Human Meningiomas.

Objective. Proliferative activity in tumor tissues is assessed as the percentage of Ki-67/MIB-1-positive cells, or the proliferative index (PI). The PI is routinely assessed manually. However, the subjectivity of manual assessments might result in poor reproducibility. We hypothesized that digital assessments might reduce the error. Method. In our study, we assessed Ki-67/MIB-1 PIs, both manually and digitally, with tissue microarrays constructed from 141 human meningioma samples. Spearman-rank correlation and κ statistics were applied for correlation and agreement analyses, respectively. Mann-Whitney U tests were used to compare MIB-1 PIs between groups. Prognostic ability was assessed with Kaplan-Meier and Cox regression analyses. Results. We found a significant, high correlation (Spearman ρ = 0.832, P < .01) and moderate agreement (κ coefficient = 0.617, observed agreement = 80.9%) between the 2 methods. Both methods found significantly different Ki-67/MIB-1 PIs for different World Health Organization grades (P < .05). Neither method showed significant prognostic value. Conclusion. Digital determinations of Ki-67/MIB-1 PIs in human meningiomas are feasible for the daily routine.