Low‐risk variants FGFR2, TNRC9 and LSP1 in German familial breast cancer patients

To validate common low‐risk variants predisposing for breast cancer (BC) in a large set of BRCA1/2 negative familial or genetically enriched cases from Germany, we genotyped 1,415 cases and 1,830 healthy women by MALDI‐TOF in 105 candidate SNPs. Significantly higher ORs than previously reported for heterozygous unselected cases were found for the minor allele in FGFR2 (OR = 1.43, 95% CI 1.30‐1.59, p‐value = 1.24 × 10⁻¹²) and for TNRC9 (OR = 1.33, 95% CI 1.19‐1.46, p‐value = 1.54 × 10⁻⁷). Most intriguing, however, were the ORs for homozygous carriers from high‐risk families for FGFR2 (OR = 2.05, 95% CI 1.68–2.51, LSP1 (OR = 0.49, 95% CI 0.28–0.86) and TNRC9 (OR = 1.62, 95% CI 1.27–2.07). Moreover, the additional validation of 99 CGEMS‐SNPs identified putative novel susceptibility alleles within the LSP1 gene (OR = 0.73, 95% CI 0.61–0.87, p‐value = 5.23 × 10⁻⁴⁾. Finally, we provide evidence for the first time that a low‐risk variant located at 6q22.33 (rs6569479) is associated with estrogen receptor negative BC in familial cases (OR = 1.33, 95% CI 1.06–1.66; p‐value = 0.012). Our data confirm the impact of the previously identified susceptibility loci and provide preliminary evidence for novel susceptibility loci in familial BC cases and correlate them to specific histopathological subtypes defined by estrogen receptor status.     

Afatinib versus cisplatin plus pemetrexed in Japanese patients with advanced non‐small cell lung cancer harboring activating EGFR mutations: Subgroup analysis of LUX‐Lung 3

In LUX‐Lung 3, afatinib significantly improved progression‐free survival (PFS) versus cisplatin/pemetrexed in EGFR mutation‐positive lung adenocarcinoma patients and overall survival (OS) in Del19 patients. Preplanned analyses in Japanese patients from LUX‐Lung 3 were performed. Patients were randomized 2:1 to afatinib or cisplatin/pemetrexed, stratified by mutation type (Del19/L858R/Other). Primary endpoint was PFS (independent review). Secondary endpoints included OS, objective response, and safety. Median PFS (data cut‐off: February 2012) for afatinib versus cisplatin/pemetrexed was 13.8 vs 6.9 months (hazard ratio [HR], 0.38; 95% confidence interval [CI], 0.20–0.70; P = 0.0014) in all Japanese patients (N = 83), with more pronounced improvements in those with common mutations (Del19/L858R; HR, 0.28; 95% CI, 0.15–0.52; P < 0.0001) and Del19 mutations (HR, 0.16; 95% CI, 0.06–0.39; P < 0.0001). PFS was also improved in L858R patients (HR, 0.50; 95% CI, 0.20–1.25; P = 0.1309). Median OS (data cut‐off: November 2013) with afatinib versus cisplatin/pemetrexed was 46.9 vs 35.8 months (HR, 0.75; 95% CI, 0.40–1.43; P = 0.3791) in all Japanese patients, with greater benefit in patients with common mutations (HR, 0.57; 95% CI, 0.29–1.12; P = 0.0966) and Del19 mutations (HR, 0.34; 95% CI, 0.13–0.87; P = 0.0181); OS was not significantly different in L858R patients (HR, 1.13; 95% CI, 0.40–3.21; P = 0.8212). Following study treatment discontinuation, most patients (93.5%) received subsequent anticancer therapy. The most common treatment‐related adverse events were diarrhea, rash/acne, nail effects and stomatitis with afatinib and nausea, decreased appetite, neutropenia, and leukopenia with cisplatin/pemetrexed. Afatinib significantly improved PFS versus cisplatin/pemetrexed in Japanese EGFR mutation‐positive lung adenocarcinoma patients and OS in Del19 but not L858R patients ( www.clinicaltrials.gov ; NCT00949650).     

Single‐nucleotide polymorphisms in the p53 pathway genes modify cancer risk in BRCA1 and BRCA2 carriers of Jewish‐Ashkenazi descent

Germline mutations in the BRCA1 and BRCA2 genes are associated with a significantly increased lifetime risk for developing breast and/or ovarian cancer. However, incomplete penetrance and substantial variability in age of disease onset among carriers of the same mutation suggests the involvement of additional modifier genes and/or environmental factors. Somatic inactivating mutations in the p53 gene and genes of the p53 pathway often accompany BRCA1/2‐associated tumors. Therefore, we assessed whether these genes are modifiers of penetrance. We genotyped Jewish‐Ashkenazi women for functional single‐nucleotide polymorphisms (SNPs) in the AKT1 (C>T rs3730358) and the PERP (C>T rs2484067) genes that affect p53‐mediated apoptosis, as well as two tag‐SNPs in the CHEK2 (C>T rs743184) and the ZBRK1/ZNF350 (G>A rs2278414) genes that encode for proteins involved in growth arrest following DNA damage. The study population included 138 healthy women, 148 breast/ovarian cancer BRCA1/2 mutation carriers, 121 asymptomatic BRCA1/2 mutation carriers, and 210 sporadic noncarrier breast cancer patients. Utilizing λ² and Kaplan–Meier analysis revealed a hazard ratio (HR) of 3.23 (95% CI: 1.44–54, P = 0.0184) for the TT genotype of AKT (rs3730358), HR = 2.105 (95% CI: 1.049–7.434, P = 0.039) for CHEK2 CC genotype (rs743184), and HR = 2.4743 (95% CI: 1.205–11.53, P = 0.022) for the AG genotype of ZBRK1/ZNF350 (rs2278414). No significant association between PERP variants and cancer was identified HR = 0.662 (95% CI: 0.289–1.324, P = 0.261). Our results suggest that genes that act upstream of p53, or participate in the DNA damage response, may modify the risk of cancer in women with mutant BRCA1/2 alleles. © 2010 Wiley‐Liss, Inc.     

Expression of the SART‐1 tumor rejection antigen in breast cancer

We investigated in breast cancers the expression of the SART‐1 gene encoding tumor rejection antigens. SART‐1 mRNA was expressed in all of the samples tested. The SART‐1₈₀₀ antigen was detectable in 20 of 50 (40%) breast cancer tissues and all breast cancer cell lines tested, but not in normal breast tissues. The SART‐1₈₀₀⁺ breast cancer cells transfected with HLA‐A2601 or HLA‐A2402 cDNA were recognized by the HLA‐A26‐restricted and SART‐1‐specific cytotoxic T lymphocytes (CTLs) or the HLA‐A24‐restricted and SART‐1‐specific CTLs, respectively. Among the 20 SART‐1₈₀₀⁺ tumors, 9 or 8 tumors expressed estrogen receptor or progesterone receptor, respectively. Therefore, the patients with HLA‐A26 or ‐A24 haplotype might be appropriate candidates for specific immunotherapy with the SART‐1 peptides independently or in combination with hormone therapy. Int. J. Cancer 80:64–67, 1999. © 1999 Wiley‐Liss, Inc.     

Lifetime alcohol intake is associated with an increased risk of KRAS+ and BRAF‐/KRAS‐ but not BRAF+ colorectal cancer

Ethanol in alcoholic beverages is a causative agent for colorectal cancer. Colorectal cancer is a biologically heterogeneous disease, and molecular subtypes defined by the presence of somatic mutations in BRAF and KRAS are known to exist. We examined associations between lifetime alcohol intake and molecular and anatomic subtypes of colorectal cancer. We calculated usual alcohol intake for 10‐year periods from age 20 using recalled frequency and quantity of beverage‐specific consumption for 38,149 participants aged 40–69 years from the Melbourne Collaborative Cohort Study. Cox regression was performed to derive hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between lifetime alcohol intake and colorectal cancer risk. Heterogeneity in the HRs across subtypes of colorectal cancer was assessed. A positive dose‐dependent association between lifetime alcohol intake and overall colorectal cancer risk (mean follow‐up = 14.6 years; n = 596 colon and n = 326 rectal cancer) was observed (HR = 1.08, 95% CI: 1.04–1.12 per 10 g/day increment). The risk was greater for rectal than colon cancer (p _homogeneity = 0.02). Alcohol intake was associated with increased risks of KRAS + (HR = 1.07, 95% CI: 1.00–1.15) and BRAF ?/KRAS ? (HR = 1.05, 95% CI: 1.00–1.11) but not BRAF + tumors (HR = 0.89, 95% CI: 0.78–1.01; p _homogeneity = 0.01). Alcohol intake is associated with an increased risk of KRAS + and BRAF ?/KRAS‐ tumors originating via specific molecular pathways including the traditional adenoma‐carcinoma pathway but not with BRAF+ tumors originating via the serrated pathway. Therefore, limiting alcohol intake from a young age might reduce colorectal cancer originating via the traditional adenoma‐carcinoma pathway.     

Tumor‐reactive CD8+ T‐cell responses after vaccination with NY‐ESO‐1 peptide,CpG 7909 and Montanide® ISA‐51: association with survival

Peptide‐based vaccines have led to the induction of antigen‐specific CD8⁺ T‐cell responses in patients with NY‐ESO‐1 positive cancers. However, vaccine‐induced T‐cell responses did not generally correlate with improved survival. Therefore, we tested whether a synthetic CpG 7909 ODN (deoxycytidyl‐deoxyguanosin oligodeoxy‐nucleotides) mixed with NY‐ESO‐1 peptide p157‐165 and incomplete Freund's adjuvants (Montanide® ISA‐51) led to enhanced NY‐ESO‐1 antigen‐specific CD8⁺ immune responses in patients with NY‐ESO‐1 or LAGE‐1 expressing tumors. Of 14 HLA‐A2+ patients enrolled in the study, 5 patients withdrew prematurely because of progressive disease and 9 patients completed 1 cycle of immunization. Nine of 14 patients developed measurable and sustained antigen‐specific CD8⁺ T‐cell responses: Four had detectable CD8+ T‐cells against NY‐ESO‐1 after only 2 vaccinations, whereas 5 patients showed a late‐onset but durable induction of NY‐ESO‐1 p157‐165 specific T‐cell response during continued vaccination after 4 months. In 6 patients, vaccine‐induced antigen‐specific T‐cells became detectable ex vivo and reached frequencies of up to 0.16 % of all circulating CD8⁺ T‐cells. Postvaccine T‐cell clones were shown to recognize and lyse NY‐ESO‐1 expressing tumor cell lines in vitro. In 6 of 9 patients developing NY‐ESO‐1‐specific immune responses, a favorable clinical outcome with overall survival times of 43+, 42+, 42+, 39+, 36+ and 27+ months, respectively, was observed.     

Interleukin‐10 gene transfer activates interferon‐γ and the interferon‐γ‐inducible genes Gbp‐1/Mag‐1 and Mig‐1 in mammary tumors

Expression of IL‐10 as a transgene inhibits murine mammary tumor growth and metastasis. Using differential display methodology, we sought genes whose expression was modulated by IL‐10. We compared mRNA isolated from parental murine mammary 66.1 tumors, as well as tumors derived from neo^r‐transfected cells and 6 different IL‐10‐expressing cell lines. We identified 2 cDNA products that were up‐regulated in all 6 IL‐10‐expressing tumors in comparison to parental and 66‐neo tumors. One cDNA corresponds to the murine guanylate‐binding protein gene Gbp‐1/Mag‐1. The other cDNA corresponds to the chemokine Mig‐1 (monokine induced by IFN‐γ). Both genes were originally identified in IFN‐γ‐activated macrophages or macrophage cell lines. We now report that cultured mammary epithelial tumor cell lines also express both genes in response to treatment with IFN‐γ and LPS. Furthermore, IFN‐γ mRNA is elevated in IL‐10‐expressing tumors in comparison with parental or neo‐transfected tumors. Thus, high‐level expression of IL‐10 as a transgene results in activation rather than suppression of IFN‐γ as well as 2 IFN‐γ‐inducible genes. Up‐regulation of host IFN‐γ is critical to anti‐tumor activity since IL‐10 no longer inhibits tumor growth in hosts with a deletion in the IFN‐γ gene. Additionally, Gbp‐1/Mag‐1 and Mig‐1 gene induction no longer occur in IFN‐γ mutant mice. Int. J. Cancer 80:624–629, 1999. © 1999 Wiley‐Liss, Inc.     

Alterations of T‐cell surface markers in older women with persistent human papillomavirus infection

We previously reported decreased lymphocyte proliferative responses among older women with persistent human papillomavirus (HPV) infection. To characterize the phenotype of peripheral lymphocytes associated with persistent HPV infection, we evaluated the expression of different cell surface markers in peripheral blood mononuclear cells (PBMCs) from a case–control study within a 10,049 woman population‐based cohort study in Guanacaste, Costa Rica. Women in the cohort aged 46–74 and with HPV results at their 5th year anniversary visit were considered, and all women (n = 87) with persistent HPV infections, all women (n = 196) with transient HPV infections and a random sample of HPV DNA‐negative women (n = 261) frequency‐matched to cases on age were selected for this study. A median of 3 years after the case–control matching visit, cervical cells were collected for liquid‐based cytology and repeat HPV DNA genotyping. Blood was obtained from which PBMCs were extracted and cryopreserved for immunological phenotyping via flow cytometry. Significant increases in risk of HPV persistence were observed for 3 marker subsets indicative of immune cell activation/differentiation. Relative risk estimates were 5.4 (95% CI = 2.2–13.3) for CD69⁺CD4⁺, 2.6 (95% CI = 1.2–5.9) for HLADR⁺CD3⁺CD4⁺ and 2.3 (95% CI = 1.1–4.7) for CD45RO⁺CD27^?CD8⁺. A significant decrease in HPV persistence was observed for a subset marker indicative of an immature, undifferentiated memory state CD45RO⁺CD27⁺CD4⁺ (OR = 0.36; 95% CI = 0.17–0.76). Adjustment for these markers only partially explained the previously reported association between decreased lymphoproliferative responses and persistent HPV infection. Whether phenotypic alterations observed predispose to HPV persistence or result from it should be the focus of future studies.     

Children with endemic Burkitt lymphoma are deficient in EBNA1‐specific IFN‐γ T cell responses

Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in equatorial Africa and is linked to Epstein–Barr virus (EBV) and Plasmodium falciparum coinfections early in life. Epstein–Barr nuclear antigen 1 (EBNA1) is the sole viral latent antigen expressed in BL tumors. Loss of EBNA1‐specific immune surveillance could allow eBL emergence. Therefore, EBNA1‐specific T cell responses were analyzed by IFN‐γ ELISPOT in Kenyan children with eBL and compared to healthy children with divergent malaria exposure. Significantly fewer children with eBL, 16% (7/44) had EBNA1‐specific IFN‐γ responses in contrast to healthy children living in a malaria holoendemic area or in an area with sporadic malaria transmission, 67% (40/60) and 72% (43/60) responders, respectively (p < 0.003). Children with eBL maintained IgG₁ dominated antibody responses to EBNA1 similar to healthy children suggesting a selective loss of IFN‐γ secreting EBNA1‐specific T cells in the presence of intact humoral immunity. CD8⁺ T cell responses to EBV lytic and latent antigens not expressed in the tumors were similarly robust in eBL patients compared to healthy children. In addition, CD4⁺ T cell responses to a malaria protein, merozoite surface protein 1, were present in lymphoma patients. This study demonstrates a selective loss of EBNA1‐specific T cell responses in children with eBL and suggests a potential immunotherapeutic target for this EBV‐associated lymphoma. © 2008 Wiley‐Liss, Inc.     

A novel glycobiomarker,Wisteria floribunda agglutinin macrophage colony‐stimulating factor receptor,for predicting carcinogenesis of liver cirrhosis

Recently, we identified a novel liver fibrosis glycobiomarker, Wisteria floribunda agglutinin (WFA)‐reactive colony stimulating factor 1 receptor (WFA⁺‐CSF1R), using a glycoproteomics‐based strategy. The aim of this study was to assess the value of measuring WFA⁺‐CSF1R levels for the prognosis of carcinogenesis and outcome in liver cirrhosis (LC) patients with hepatitis C virus (HCV). WFA⁺‐CSF1R and Total‐CSF1R levels were measured in serum samples from 214 consecutive HCV‐infected patients to evaluate their impact on carcinogenesis and the survival of LC patients. Serum WFA⁺‐CSF1R levels were significantly higher in LC patients than chronic hepatitis (CH) patients (p < 0.001). The AUC of WFA⁺‐CSF1R for predicting overall survival, calculated by time‐dependent ROC analysis, was 0.691 and the HR (per 1‐SD increase) was 1.80 (95% CI, 1.23–2.62, p < 0.001). Furthermore, the survival rate of LC patients with high WFA⁺‐CSF1R levels (≥310 ng/ml) was significantly worse than those with lower levels (p < 0.01). The AUC of WFA⁺/total‐CSF1R percentage (WFA⁺‐CSF1R%) for predicting the cumulative carcinogenesis rate was 0.760, with an HR of 1.66 (95% CI 1.26–2.20, p < 0.001). In fact, the carcinogenesis rate was significantly higher in LC patients with a high WFA⁺‐CSF1R% (≥ 35%, p = 0.006). Assessing serum levels of WFA⁺‐CSF1R has diagnostic value for predicting carcinogenesis and the survival of LC patients.     

EGFR exon 19 in‐frame deletion and polymorphisms of DNA repair genes in never‐smoking female lung adenocarcinoma patients

We explored potential associations between genetic polymorphisms in genes related to DNA repair and detoxification metabolism and epidermal growth factor receptor (EGFR) mutations in a cohort of 410 never‐smoking patients with lung adenocarcinoma. Multivariate‐adjusted odds ratios (aORs) and corresponding 95% confidence intervals (CI) of EGFR mutation status in association with the genotypes of DNA repair and detoxification metabolism genes were evaluated using logistic regression analysis. We found an association between in‐frame deletion in EGFR exon 19 and a single nucleotide polymorphism (SNP) rs1800566C/T located in NQO1 (aOR, 2.2 with 95% CI, 1.0–4.8) in female never‐smokers. The SNP rs744154C/G in ERCC4 was also associated with the EGFR exon 19 in‐frame deletion both in never‐smokers (aOR, 1.7 with 95% CI, 1.0–3.0) and female never‐smokers (aOR, 1.9 with 95% CI, 1.0–3.6). Although the association was marginally significant in multivariate logistic regression analysis, the A/A genotype of rs1047840 in EXO1 was associated with a 7.6‐fold increase in the occurrence of the EGFR exon 19 in‐frame deletion in female never‐smokers. Moreover, risk alleles in NQO1, ERCC4 and EXO1 were associated with an increasing aOR of the EGFR exon 19 in‐frame deletion both in never‐smokers (p = 0.007 for trend) and female never‐smokers (p = 0.002 for trend). Our findings suggest that the in‐frame deletion in EGFR exon 19 is associated with polymorphisms in DNA repair and detoxification metabolism genes in never‐smoking lung adenocarcinoma patients, especially in females.     

Comparison of tumor‐infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients

The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2⁺, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8⁺ and CD4⁺ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2⁺ and TN breast cancers have a lower percentage of TILs and CD8⁺ and CD4⁺ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.     

Protease‐activated receptor‐2 accelerates intestinal tumor formation through activation of nuclear factor‐κB signaling and tumor angiogenesis in ApcMin/+ mice

Hepatocyte growth factor activator inhibitor‐1 (HAI‐1), encoded by the SPINT1 gene, is a membrane‐bound protease inhibitor expressed on the surface of epithelial cells. Hepatocyte growth factor activator inhibitor‐1 regulates type II transmembrane serine proteases that activate protease‐activated receptor‐2 (PAR‐2). We previously reported that deletion of Spint1 in Apc^Min/+ mice resulted in accelerated formation of intestinal tumors, possibly through enhanced nuclear factor‐κB signaling. In this study, we examined the role of PAR‐2 in accelerating tumor formation in the Apc^Min/+ model in the presence or absence of Spint1. We observed that knockout of the F2rl1 gene, encoding PAR‐2, not only eliminated the enhanced formation of intestinal tumors caused by Spint1 deletion, but also reduced tumor formation in the presence of Spint1. Exacerbation of anemia and weight loss associated with HAI‐1 deficiency was also normalized by compound deficiency of PAR‐2. Mechanistically, signaling triggered by deregulated protease activities increased nuclear translocation of RelA/p65, vascular endothelial growth factor expression, and vascular density in Apc^Min/+‐induced intestinal tumors. These results suggest that serine proteases promote intestinal carcinogenesis through activation of PAR‐2, and that HAI‐1 plays a critical tumor suppressor role as an inhibitor of matriptase, kallikreins, and other PAR‐2 activating proteases.     

BRCA1‐like profile predicts benefit of tandem high dose epirubicin‐cyclophospamide‐thiotepa in high risk breast cancer patients randomized in the WSG‐AM01 trial

BRCA1 is an important protein in the repair of DNA double strand breaks (DSBs), which are induced by alkylating chemotherapy. A BRCA1‐like DNA copy number signature derived from tumors with a BRCA1 mutation is indicative for impaired BRCA1 function and associated with good outcome after high dose (HD) and tandem HD DSB inducing chemotherapy. We investigated whether BRCA1‐like status was a predictive biomarker in the WSG AM 01 trial. WSG AM 01 randomized high‐risk breast cancer patients to induction (2× epirubicin‐cyclophosphamide) followed by tandem HD chemotherapy with epirubicin, cyclophosphamide and thiotepa versus dose dense chemotherapy (4× epirubicin‐cyclophospamide followed by 3× cyclophosphamide‐methotrexate‐5‐fluorouracil). We generated copy number profiles for 143 tumors and classified them as being BRCA1‐like or non‐BRCA1‐like. Twenty‐six out of 143 patients were BRCA1‐like. BRCA1‐like status was associated with high grade and triple negative tumors. With regard to event‐free‐survival, the primary endpoint of the trial, patients with a BRCA1‐like tumor had a hazard rate of 0.2, 95% confidence interval (CI): 0.07–0.63, p = 0.006. In the interaction analysis, the combination of BRCA1‐like status and HD chemotherapy had a hazard rate of 0.19, 95% CI: 0.067–0.54, p = 0.003. Similar results were observed for overall survival. These findings suggest that BRCA1‐like status is a predictor for benefit of tandem HD chemotherapy with epirubicin‐thiotepa‐cyclophosphamide.     

Endogenous conversion of ω‐6 to ω‐3 polyunsaturated fatty acids in fat‐1 mice attenuated intestinal polyposis by either inhibiting COX‐2/β‐catenin signaling or activating 15‐PGDH/IL‐18

Omega‐3 polyunsaturated fatty acids (ω‐3PUFAs) have inhibitory effects in various preclinical cancer models, but their effects in intestinal polyposis have never been examined. As attempts have been made to use nutritional intervention to counteract colon cancer development, in this study we evaluated the effects of ω‐3 PUFAs on intestinal polyposis in the Apc^Min/+ mouse model. The experimental groups included wild‐type C56BL/6 mice, Apc^Min/+ mice, fat‐1 transgenic mice expressing an n‐3 desaturase to enable ω‐3 PUFA synthesis, and Apc^Min/+ × fat‐1 double‐transgenic mice; all mice were 20 weeks of age. Small intestines were collected for gross and pathologic evaluation, including assessment of polyp number and size, followed by immunohistochemical staining and Western blotting. After administration of various concentrations of ω‐3 PUFAs, PUFA levels were measured in small intestine tissue by GC/MS/MS analysis to compare with PUFA synthesis of between C57BL6 and fat‐1mice. As a result, ω‐3 PUFAs significantly attenuated Apc mutation–induced intestinal polyposis accompanied with significant inhibition of Wnt/β‐catenin signaling, COX‐2 and PGE_2, but induced significant levels of 15‐PGDH. In addition, significant induction of the inflammasome‐related substrates as IL‐1β and IL‐18 and activation of caspase‐1 was observed in Apc^Min/+ × fat‐1 mice. Administration of at least 3 g/60 kg ω‐3 PUFAs was equivalent to ω‐3 PUFAs produced in fat‐1 mice and resulted in significant increase in the expression of IL‐1β, caspase‐3 and IL‐18, as seen in Apc^Min/+ × fat‐1 mice. We conclude that ω‐3PUFAs can prevent intestinal polyp formation by inhibition of Wnt/β‐catenin signaling, but increased levels of 15‐PGDH and IL‐18.     

Therapy‐induced antitumor vaccination in neuroblastomas by the combined targeting of IL‐2 and TNFα

L19‐IL2 and L19TNFα are fusion proteins composed of L19(scFv), specific for the angiogenesis‐associated ED‐B containing fibronectin isoform and IL‐2 or TNFα. Because of the tumor targeting properties of L19, IL‐2 and TNFα concentrate at therapeutic doses at the tumor vascular level. To evaluate the therapeutic effects of L19‐IL2 and L19mTNFα in neuroblastoma (NB)‐bearing mice, A/J mice bearing Neuro2A or NIE115 NB were systemically treated with L19‐IL2 and L19mTNFα, alone or in combination protocols. Seventy percent of Neuro2A‐ and 30% of NIE115‐bearing mice were cured by the combined treatment with L19‐IL2 and L19mTNFα, and further rejected a homologous tumor challenge, indicating specific antitumor immune memory. The immunological bases of tumor cure and rejection were studied. A highly efficient priming of CD4⁺ T helper cells and CD8⁺ CTL effectors was generated, paralleled by massive infiltration in the tumor tissue of CD4⁺ and CD8⁺ T cells at day 16 after tumor cell implantation, when, after therapy, tumor volume was drastically reduced and tumor necrosis reached about 80%. The curative treatment resulted in a long‐lasting antitumor immune memory, accompanied by a mixed Th1/Th2 type of response. Concluding, L19‐IL2 and L19mTNFα efficiently cooperate in determining a high percentage of NB cure that, in our experimental models, is strongly associated to the generation of adaptive immunity involving CD4⁺ and CD8⁺ T cells.     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

JmjC‐domain containing histone demethylase 1B‐mediated p15Ink4b suppression promotes the proliferation of leukemic progenitor cells through modulation of cell cycle progression in acute myeloid leukemia

The histone demethylase JHDM1B has been implicated in cell cycle regulation and tumorigenesis. In addition, it has been reported that JHDM1B is highly expressed in various human tumors, including leukemias. However, it is not clearly understood how JHDM1B contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of JHDM1B in AML cells. In AML cell lines and AML‐derived ALDH^hi (high aldehyde dehydrogenase activity)/CD34⁺ cells, the levels of JHDM1B mRNA were significantly higher than in normal ALDH^hi/CD34⁺ cells. Reduction of JHDM1B expression in AML cells inhibited cell proliferation compared to control cells, through induction of G1 cell cycle arrest, an increase in the p15^Ink4b mRNA and protein expression. JHDM1B mRNA was overexpressed in all 133 AML clinical specimens tested (n = 22, 57, 34, and 20 for M1, 2, 4, and 5 subtypes respectively). Compared to normal ALDH^hi/CD34⁺ cells, JHDM1B gene expression was 1.57‐ to 1.87‐fold higher in AML‐derived ALDH^hi/CD34⁺cells. Moreover, the JHDM1B protein was more strongly expressed in AML‐derived ALDH^hi/CD34⁺ cells from compared to normal ALDH^hi/CD34⁺ cells. In addition, depletion of JHDM1B reduced colony formation of AML‐derived ALDH^hi/CD34⁺ cells due to induction of p15^Ink4b expression through direct binding to p15^Ink4b promoter and loss of demethylation of H3K36me2. In summary, we found that JHDM1B mRNA is predominantly expressed in AML‐derived ALDH^hi/CD34⁺ cells, and that aberrant expression of JHDM1B induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of JHDM1B expression represents an attractive target for AML therapy. © 2011 Wiley Periodicals, Inc.