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Background: We previously revealed that single nucleotide polymorphisms (SNPs) of CYP19A1, MAP3K1 rs889312, and ABCB1 rs2032582 were associated with overall survival (OS) in early-stage hormone receptor (HR)-positive breast cancer patients. Previous studies also showed that FGFR2 rs1219468 and TOX3 rs8051542 influenced OS in early-stage breast cancer patients. In this study, we assessed whether undiscovered SNPs of CYP19A1, MAP3K1, ABCB1, FGFR2, and TOX3 are associated with clinical outcome of patients with early-stage HR-positive breast cancer. Patients and Methods: We used next-generation sequencing (NGS) methods to explore unidentified SNPs of CYP19A1, MAP3K1, ABCB1, FGFR2, and TOX3 in 80 patients who had HR+, T1 to T2, and N0 to N1 breast cancer and had matched clinicopathological features and clinical outcomes (experimental cohort). The mapping selected SNPs identified from NGS were located in different chromosomes, such as chromosome 5 (MAP3K), 7 (ABCB1), 10 (FGFR2), 15 (CYP19A1), and 16 (TOX3), were analyzed. The multivariate-adjusted hazard ratio (aHR) of OS associated with the individual genotypes of the experimental cohort was assessed after adjusting for age, tumor size, tumor grade, HER2 status, menopausal status, and chemotherapy using Cox regression analyses. We further correlated identified significant novel SNPs with disease-free survival (DFS) and OS in 195 patients with HR-positive, T1-T2, and N0-1 breast cancer (validation cohort) using stepwise selection multiple Cox model analyses. Results: Through target region re-sequencing, we identified 69 novel SNPs, including 18 CYP19A1 SNPs, 16 MAP3K SNPs, 18 ABCB1 SNPs, 12 FGFR2 SNPs, and 5 TOX3 SNPs in the experimental cohort (n = 80). After multivariate-adjusted analyzing, FGFR2 rs2981460(C/C) (p = 0.0115), and MAP3K1 rs3822625 (A/A) (p = 0.0475), rs702689 (G/G) (p = 0.0312), rs832567 (C/C) (p = 0.0312), and rs9687226 (C/C) (p = 0.0475), and ABCB1 rs2032582 (T/T+T/C) (p = 0.0061) were significantly associated with the poorer OS of the experimental cohort. In a validated cohort (n =195) (Table 1), we found that MAP3K1 rs702689 (G/G) was significantly associated with poorer DFS (aHR = 2.3; 95% confidence interval [CI] = 1.1 to 5.1; p = 0.03) and OS (aHR = 4.9; 95% CI = 1.3 to 18.7; p = 0.02). In addition, MAP3K1 rs832567 (C/C) was significantly associated with poorer DFS (aHR = 2.5; 95% CI = 1.1 to 5.5; p = 0.02) and OS (aHR = 5.4; 95% CI = 1.4 to 21.6; p = 0.02). In premenopausal women of validation cohort (n = 108), ABCB1 rs2032582 (T/T+T/C) was significantly associated with a poor OS (aHR = 62.3; 95% CI = 2.0 to 1911.7; p = 0.02). Conclusions: Our results indicate that 3 novel SNPs, MAP3K1 rs702689, MAP3K1 rs832567, and ABCB1 rs2032582, may affect the prognosis of early-stage HR-positive breast cancer. Further validation of these SNPs in a large series of patients with early-stage HR-positive breast cancer who received adjuvant endocrine therapy alone is warranted. Table 1. The Associations of Hormone Receptor-Positive Breast Cancer Patients with Various Genotypes in multiple Cox model (validation cohort)Disease-free survivalOverall survivalGenotypeaHR (95%CI)PaHR (95%CI)PTotal (n = 195)FGFR2_ rs2981460 (C/C vs.C/T+T/T)0.4 (0.1-1.8)0.220.6 (0.1-5.4)0.68MAP3K1_ rs3822625 (A/A vs. G/A+GG)2.0 (0.7-5.3)0.192.7 (0.6-12.9)0.20MAP3K1_ rs702689 (G/G vs. A/A+A/G)2.3 (1.1-5.1)0.034.9 (1.3-18.7)0.02MAP3K1_rs832567 (C/C vs. A/A+A/C)2.5 (1.1-5.5)0.025.4 (1.4-21.6)0.02MAP3K1_rs9687226 (C/C vs. T/T+T/C)2.0 (0.7-5.3)0.192.7 (0.6-12.9)0.20Premenopausal (n=108)ABCB1_rs2032582 (T/T+T/C vs. C/C)2.0 (0.8-5.0)0.1562.3 (2.0-1911.7)0.02aHR: adjusted by age, estrogen receptor and progesterone receptor status, number of lymph nodes, menopausal status, adjuvant chemotherapy, lymphovascular invasion, and tumor size. Citation Format: Sung-Hsin Kuo, Shi-Yi Yang, Po-Han Lin, Chen-Hsin Chen, Hsin-Chou Yang, Chih-Yuan Hsu, Chiun-Sheng Huang. Identification of novel single nucleotide polymorphisms located in MAP3K1 and ABCB1 genes as markers for poor overall survival in early-stage hormone receptor-positive breast cancer using next-generation sequencing [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 P5-06-30.

Background: Immune checkpoint blockade has emerged as an important cancer treatment strategy. However, the cost of repeat injections of these antibodies limits its availability to many patients globally. We have been developing DNA vaccines targeting immune checkpoints as a potentially more affordable approach to cancer immunotherapy. We have reported this platform could induce endogenous anti-CTLA-4 antibody and anti-tumor immunity. Here, we tested a DNA vaccine targeting PD-1 for anti-tumor responses. Methods: Murine CTLA-4 or PD-1 DNA sequences were inserted into an expression plasmid, pVAC-1, forming DNA vaccines targeting CTLA-4 or PD-1 respectively. Mouse melanoma models were established by inoculating B16F10 cells in the flank of C57BL/6 mice. The mice were randomized into treatment groups, including vector (control), radiation therapy (RT), or RT plus DNA vaccinations targeting CTLA-4 and/or PD-1. Vaccinations were given weekly via intra-muscular injection of the DNA plasmids in conjunction with electroporation. RT was given in 3 daily fractions of 8 Gy during the second week of vaccination. Serum samples were collected and subjected to ELISA for antibody titers. Tumors were measured twice weekly. Tumor samples were collected and subjected to IHC staining for immune markers. Results: Vaccination with plasmids encoding CTLA-4 (pVAC-1-CTLA-4) and PD-1 (pVAC-1-PD-1) induced endogenous antibodies against CTLA-4 and PD-1, respectively. The antibodies were detected beginning 2 weeks after the first vaccination, reached peak concentrations of 3-15 μg/mL in the following 1-2 weeks, and declined within a week once vaccination stopped. Compared with vector alone, preliminary studies using vaccination with both pVAC-1-CTLA-4 and pVAC-1-PD-1 suggested enhanced B16F10 tumor response without evidence of toxicities such as body weight loss or organ damage. In further preliminary studies, adding of both pVAC-1-CTLA-4 and pVAC-1-PD-1 DNA vaccines to RT resulted in increased CD8+ tumor infiltrating lymphocytes compared to either RT or vaccine alone and suggested enhanced tumor response. Conclusions: Immune checkpoint DNA vaccines induced endogenous specific antibodies, demonstrated a favorable safety profile, and may confer anti-tumor immunity. This DNA vaccine platform holds potential as a cost-effective approach to immunotherapy. Further studies are warranted to compare the effects of these immune checkpoint DNA vaccines with those of exogenous immune checkpoint targeting antibodies. Citation Format: Keng-Hsueh Lan, Raghava Sriramaneni, Justin C. Jagodinsky, Claire Baniel, Amy Erbe-Gurel, Jacquelyn A. Hank, Sung-Hsin Kuo, Keng-Li Lan, Zachary S. Morris. Immune checkpoint DNA vaccines enhance anti-tumor immunity in murine melanoma model [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 4055.

Backgrounds: The treatment of HNSCC in Taiwan is still very challenging and might be related to betel-nuts use. Betel-nuts chewing might contribute to (1)strong inflammation, invasion, and angiogenesis; (2)poor response to chemotherapy , radiation, and epidermal growth factor receptor inhibitors. In our previous studies, betel-nuts related TW2.6(p53 defective mutation, p16 loss, BCL2+) was resistant to traditional chemotherapies, radiation, and EGFR inhibition. In addition to PI3K/mTOR dual inhibition, polo-like kinase inhibitor with radiation, CDK4/6 inhibitor, WEE1 inhibitor, ALK/IGF-1R inhibitor, Bcl2 inhibitor, and eribulin, FGFR inhibitor & VEGFR2/FGFR/PDGFR or VEGFR2/c-MET/Axl triple blockage might be effective on TW2.6 and reverse treatment refractoriness, maybe through the inhibition of mesenchymal transformation & PI3K/Akt/mTOR signaling and the modulation of stemness & PD1/PDL1 pathway. PI3Kalpha inhibitor, Bcl2 inhibitor, and ALK/IGF-1R inhibitor also have roles to reverse treatment refractoriness. Astragalus polysaccharides is a kind of traditional Chinese medicine with immuno-modulatory effects(enhancing mature DC, NK cells, & effector CD8 cytolytic activity; adjusting chronic inflammation in tumor microenvironment; reversing M2 polarization; suppressing Treg/MDSC) and maybe anti-tumor efficacy. APS has also been manufactured to treat cancer-related fatigue in Taiwan. Purpose: We try to find out whether APS has anti-tumor effects on betel-nuts related HNSCC in Taiwan. Methods: APS was tested on TW2.6 to evaluate (1)the in vitro drug sensitivity; (2)synergistic effects with some target therapies by MTT assay, colon formation assay, flow cytometry, and western blot assay; (3) HUVEC response and invasion capacity by wound healing. Results: APS has minimal cytotoxic effect on TW2.6; however, it could inhibit tumor migration, suppress signals of epithelial-mesenchymal transformation & PI3K/AKT/mTOR pathway, and induce PDL1 drop. APS re-sensitizes TW2.6 to respond to afatinib & volasertib and could enhance further response of TW2.6 to BYL719, foretinib, BGJ398, and eribulin. APS and eribulin combinations might drop BMI-1. Conclusions: APS could possibly reverse TW2.6 treatment refractoriness to EGFR inhibitor & polo-like kinase inhibitor and further enhance PI3Kalpha inhibitor, VEGFR/c-MET/Axl triple inhibitor, FGFR inhibitor, & eribulin effects on TW2.6. APS can be further investigated to be combined with these targeted therapies and even immunotherapy in betel-nuts related HNSCC in Taiwan in the future, by modulating EMT, PI3K/AKT/mTOR pathway, immune escape, & cancer stemness. Citation Format: Jo-Pai Chen, Jui-Ying Chang, Sung-Hsin Kuo, Ruey-Long Hong. Astragalus polysaccharides (APS) effects on betel-nuts related head and neck squamous cell carcinoma (HNSCC) in Taiwan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 197.

Background: The treatment of head and neck squamous cell carcinoma(HNSCC) in Taiwan is very challenging and might be related to betel-nuts use. Betel-nuts chewing might contribute to (1)strong inflammation, invasion, and angiogenesis; (2)easy recurrence or metastasis; (3) poor response to chemotherapy, radiation, and epidermal growth factor receptor(EGFR) inhibitors. Purpose: We try to prove different kinds of anti-angiogenesis treatments will lead to different response on betel-nuts related HNSCC in Taiwan. Methods: Different anti-angiogenesis treatments, such as axitinib(VEGFR2 inhibitor), nintedanib(VEGFR2/FGFR inhibitor), and regorafenib(VEGFR2/FGFR/ more other signals inhibitor) were first used to treat (1)HUVEC; (2)HNSCC cell lines(SCC4, SCC9, SCC15, SCC25, FaDu, SAS, KB, Cal27, and TW2.6, betel-nuts related) to evaluate (a) invasion capacity by wound healing; (b) drug sensitivity by MTT assay; (c)synergistic effect with chemotherapy, EGFR inhibitor, and polo-like kinase inhibitor. Western blotting was also used to test signal change by treatments. TW2.6 had already been proved to possess defective p53, p16 loss, and increased Bcl2. Results: In our previous study, TW2.6 was resistant to chemotherapy, radiation, EGFR inhibitors, and ani-angiogenesis treamtents, such as axitinib and sunitinib. Axitinib, pure VEGFR2 inhibitor, was found to suppress HUVEC more prominently than nintedanib or regorafenib did. Invasion capacity of all HNSCC cell lines were all blocked by the three drugs but regorafenib did mostly well. However, axitinib had no effect on TW2.6; but nintedanb and regorafenib had moderate response on TW2.6. Besides, nintedanib and regorafenib both would resensitize TW2.6 to respond to chemotherapy, EGFR inhibitor, and polo-like kinase inhibitor again. Western blotting showed mesenchaml differentiation markers(slug, Twist, snail, Axl, c-MET, Vimentin) decreased after nintedanib and regorafenib use, too. Conclusion: TW2.6 might reflect treatment refractoriness of betel-nuts related HNSCC in Taiwan. In addition to PI3K/mTOR dual inhibition and polo-like kinase inhibitor with radiation(our studies shown in AACR and ESMO2015), VEGFR2/FGFR dual blockage might be effective on TW2,6 and resensitize TW2.6 to EGFR inhibitor, polo-like kinase inhibitor, & chemotherapy, maybe through the inhibition of mesenchymal transformation. VEGFR2/FGFR dual blockage will be one future combination backbone of betel-nuts related HNSCC. Citation Format: Jo-Pai Chen, Jui-Ying Chang, Sung-Hsin Kuo, Ruey-Long Hong. Differential effects of antiangiogenesis treatments on betel-nuts-related head and neck squamous cell carcinoma in Taiwan. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3260.

Infection by cagA-positive Helicobacter pylori (H. pylori) is strongly associated with gastric carcinomas and gastric mucosa–associated lymphoid tissue (MALT) lymphomas. H. pylori translocates the bacterial protein CagA into gastric epithelial cells, and the translocated CagA deregulates intracellular signaling pathways and thereby initiates pathogenesis. This in turn raised the possibility that H. pylori is associated with the development of MALT lymphomas during persistent infection by direct interaction with B lymphocytes. In this work, we showed that CagA can be directly translocated into human B lymphoid cells by H. pylori, and the translocated CagA undergoes tyrosine phosphorylation and binds to intracellular SH-2. Meanwhile, the translocated CagA induces activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in human B lymphoid cells, and upregulates the expressions of Bcl-2 and Bcl-XL, which prevents apoptosis. These results provide the first direct evidence for the role of CagA as a bacterium-derived oncoprotein that acts in human B cells, and further implies that CagA is directly delivered into B cells by H. pylori and is associated with the development of MALT lymphomas. Cancer Res; 70(14); 5740–8. ©2010 AACR.

Purpose: The treatments of HNSCC in Taiwan is still very challenging and might be related to betel nuts use. Betel nuts chewing might contribute to (1)strong inflammation and angiogenesis; (2)strong invasion and queer metastatic sites; (3)easy recurrence or metastasis, usually accompanied with hypercalcemia; (4)not so good response to chemotherapy and epidermal growth factor receptor inhibitors;(5)relatively good response to vascular endothelial growth factor inhibitors combined with chemotherapy; (6)difficult wound healing and repair. We try to prove the difference of betel nuts-related HNSCC cell lines in several aspects reflecting clinical characteristics of HNSCC in Taiwan and design effective treatment combinations for future clinical trials and treatment decisions. Methods: HNSCC cell lines(SCC4, SCC9, SCC15, SCC25, FaDu, KB, Cal27, SAS, and TW2.6, betel-nuts related) were used to evaluate (1) the in vitro drug sensitivity(several chemotherapies and target agents); (2) synergistic effect of some target therapies and irradiation with MTT assay, colony formation assay, flow cytometry, and western blot assay. TW2.6 had already been proved to possess defective p53, p16 loss, and increased Bcl2. Results: The MTT assay showed betel-nuts related TW2.6 was resistant to tradiational chemotherapies, radiation, EGFR inhibition, or anti-angiogenesis alone. TW2.6 was sensitive to high dose docetaxel and dual PI3K/mTOR inhibitor, BEZ235. Western blotting showed (1) p-AKT level was prominent in TW2.6 and correlated to BEZ235 response; (2) High PDL1 and VEGF levels in TW2.6 may confer to immunotherapy or anti-angiogenesis response; (3) Almost p16 loss in all cell lines; (4) Prominent Gli-1 expression in HPV-related FaDu and SCC25. SCC25, SAS, KB, and Cal27 showed good chemosensitivity. Well-differentiated cell lines(SCC4, 9, 15, 25), Cal27, and FaDu responded well to EGFR inhibitors. The cell viability assay showed that volasertib(polo like kinase inhibitor) did not decrease TW2.6 proliferation significantly. But clonogenic survival showed that combination of volasertib and radiation significantly enhanced radiation-induced death. The flow cytometry revealed that Plk1 inhibition by volasertib and radiation resulted in a significant G2/M cell cycle arrest. Western blotting also showed volasertib and radiation combination increased cyclin B levels and induced apoptosis. Conclusions: These findings suggested that TW2.6 might reflect treatment refractoriness of betel-nuts related HNSCC in Taiwan. PI3K/mTOR dual inhibition, anti-angiogenesis with chemotherapy, and immunotherapy are important strategies in our experiments. Besides, polo like kinase inhibition is a good radiosensitizer for TW2.6. Further animal studies, genetic studies, and array analysis will further clarify these points of views. Mesenchymal(like Axl) and invasion(such as c-MET) pathways will also be considered as future targets. Note: This abstract was not presented at the meeting. Citation Format: Jo-Pai Chen, Ling-Yu Chen, Sung-Hsin Kuo, Ruey-Long Hong. Special characteristics of betel-nuts related head and neck squamous cell carcinoma (HNSCC) in Taiwan. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3549. doi:10.1158/1538-7445.AM2015-3549

Background: We previously reported that activation of TNF-α results in upregulation of NF-κB and nuclear translocation of BCL3 and BCL10 in breast cancer cell line and lymphoma cell line (J Biol Chem 2006;281:167-75, Blood 2008;112:2927-34). Furthermore, we recently demonstrated that BCL10 plays an important role in controlling the growth of cervical cancer cells through NF-κB dependent cyclin D1 regulation (Gynecol Oncol 2012;126:245-51). Since NF-κB activation has been shown to occur in the pathogenesis of pancreatic cancer, we sought to investigate whether BCL10-signaling possesses clinical significance in relation to pancreatic cancer. Methods: Three pancreatic cancer cell lines (PANC-1, AsPC-1, and BxPC-3) were used in this study. Cell cycle was analyzed by flow cytometry. The expression of protein and mRNA of BCL10- and NF-κB- related signaling was assessed by immunoblotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The DNA-binding activity of NF-κB was determined by the luciferase assay. The expression of BCL10, and NF-κB in tumor cells from recurrent, locally advanced, and metastatic pancreatic patients who received chemotherapy was examined using immunohistochemical staining. Results: We transfected three pancreatic cancer cell lines with BCL10 small interfering RNA (siRNA), and discovered that the down-regulation of BCL10 inhibited the viability of these pancreatic cancer cells through the G1 arrest. BCL10 siRNA treatment inhibited the expression of p-IKKβ and p-IκB, and nuclear BCL3 translocation, and also down-regulated NF-κB activation and its downstream cell cycle proteins, c-Myc, and cyclin B1. The results of qRT-PCR showed that BCL10 siRNA treatment inhibited the mRNA expression levels of BCL10, BCL3, c-Myc, and cyclin B1. Our results reveal that patients with nuclear BCL10 expression had a worse median overall survival than those without (7.9 months versus 16.9 months, p = 0.007). Furthermore, nuclear expression of BCL10 was closely associated with nuclear BCL3 expression (p < 0.001), and NF-κB activation (p < 0.001). Conclusions: Our findings indicate that nuclear BCL10 plays an important role in controlling the growth and progression of pancreatic cancer cells through NF-κB-related signaling pathway. Citation Format: Sung-Hsin Kuo, Shih-Hung Yang, Kun-Huei Yeh, Pei-Yen Yeh, Yu-Wen Tien, Hsiao-Wei Lee, Li-Tzong Chen, Ann-Lii Cheng. Nuclear expression of BCL10 has a role in the regulation of cell growth and progression of pancreatic cancer through the activation of NF-κB-related signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4217. doi:10.1158/1538-7445.AM2014-4217

Background We previously reported that H. pylori-positive gastric DLBCL, with or without evidence of MALT origin, may respond to H. pylori eradication therapy. In this study, we further investigated the clinicopathologic features of H. pylori-positive DLBCL without histologic evidence of MALT origin. Methods We reviewed consecutive cases of gastric DLBCL without histologic evidence of MALT origin. H. pylori infection was documented by histologic examination, a urease test or bacterial culture. H. pylori-positive cases were further examined for CagA protein expression. Results A group of 45 patients positive for H. pylori were not histomorphologically different from a group of 48 patients negative for H. pylori. However, patients with H. pylori infection had a better International Prognostic Index score (0-1, 70% vs 30%, P = ·001), a lower clinical stage (I-IIE1, 73% vs 33%, P < [[Unable to Display Character: ∙]]001), a better tumor response to chemotherapy (complete response, 80% vs 50%, P = .01), and significantly better 5-year event-free survival (EFS) (71% vs 24%, P < [[Unable to Display Character: ∙]]001) and overall survival (OS) (73% vs 30%, P < [[Unable to Display Character: ∙]]001). CagA expression, as defined by more than 5% of tumor cells with CagA staining, was identified in 23 (68%) of 34 H. pylori-positive tumors. Compared with the 11 H. pylori-positive but CagA-negative cases, the 23 H. pylori-positive, CagA-positive cases were associated with significantly better 5-year EFS (73% vs 15%, P = [[Unable to Display Character: ∙]]002) and OS (91% vs 27%, P = [[Unable to Display Character: ∙]]001). Conclusion H. pylori-positive gastric DLBCL, particularly with CagA expression in tumor cells, appears to be a distinct tumor entity, sharing some key clinicopathologic features with H. pylori-associated gastric MALT lymphoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1747.