Stimulating T cell responses against patient-derived breast cancer cells with neoantigen peptide-loaded peripheral blood mononuclear cells.

Breast cancer stands as a formidable global health challenge for women. While neoantigens exhibit efficacy in activating T cells specific to cancer and instigating anti-tumor immune responses, the accuracy of neoantigen prediction remains suboptimal. In this study, we identified neoantigens from the patient-derived breast cancer cells, PC-B-142CA and PC-B-148CA cells, utilizing whole-genome and RNA sequencing. The pVAC-Seq pipeline was employed, with minor modification incorporating criteria (1) binding affinity of mutant (MT) peptide with HLA (IC50 MT) ≤ 500 nm in 3 of 5 algorithms and (2) IC50 wild type (WT)/MT > 1. Sequencing results unveiled 2513 and 3490 somatic mutations, and 646 and 652 non-synonymous mutations in PC-B-142CA and PC-B-148CA, respectively. We selected the top 3 neoantigens to perform molecular dynamic simulation and synthesized 9-12 amino acid neoantigen peptides, which were then pulsed onto healthy donor peripheral blood mononuclear cells (PBMCs). Results demonstrated that T cells activated by ADGRL1E274K, PARP1E619K, and SEC14L2R43Q peptides identified from PC-B-142CA exhibited significantly increased production of interferon-gamma (IFN-γ), while PARP1E619K and SEC14L2R43Q peptides induced the expression of CD107a on T cells. The % tumor cell lysis was notably enhanced by T cells activated with MT peptides across all three healthy donors. Moreover, ALKBH6V83M and GAAI823T peptides from PC-B-148CA remarkably stimulated IFN-γ- and CD107a-positive T cells, displaying high cell-killing activity against target cancer cells. In summary, our findings underscore the successful identification of neoantigens with anti-tumor T cell functions and highlight the potential of personalized neoantigens as a promising avenue for breast cancer treatment.

HCC-derived EGFR mutants are functioning, EGF-dependent, and erlotinib-resistant.

BACKGROUND: Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target. Overexpression of EGFR is frequently observed in hepatocellular carcinoma (HCC) and EGFR activation has been proven to be a potential determinant of primary resistance of HCC cells to sorafenib. In our previous study, we found 13 missense mutations in EGFR exon 19-23 from hepatocellular carcinoma (HCC) tissues, but the functions of these mutations have not been determined. This study aims to determine the kinase activity and sensitivity to erlotinib, a 1st-generation EGFR-tyrosine kinase inhibitor (TKI), of seven HCC-derived mutants (K757E, N808S, R831C, V897A, P937L, T940A, and M947T). RESULTS: Using transduction of pBabe-puro retroviral vector with or without EGFR, we constructed and determined the function of EGFRs in NIH-3T3 cells stably harboring each of the seven mutants, as well as the erlotinib-sensitive L858R-mutant, the erlotinib-resistant T790M-mutant, and EGFR wild type (WT). Our results indicate that the seven mutants are functioning, EGF-dependent, EGFRs. Cells harboring six of the seven mutants could generate some level of EGFR phosphorylation in the absence of EGF, indicating some constitutive kinase activity, but all of the seven mutants remain primarily EGF-dependent. Our results demonstrate that erlotinib induces differential degree of apoptosis and autophagy among cells harboring different EGFRs: complete apoptosis and autophagy (cleavage of both caspase-3 and PARP, and marked LC3-II increment) in L858R-mutant; partial apoptosis and autophagy (only cleavage of caspase-3, and moderate LC3-II increment) in WT and HCC-derived mutants; and no apoptosis and minimal autophagy (no cleavage of caspase-3 and PARP, and minimal LC3-II increment) in T790M-mutant. The seven HCC-derived mutants are erlotinib-resistant, as treatment with erlotinib up to high concentration could only induce partial inhibition of EGFR phosphorylation, partial or no inhibition of AKT and ERK phosphorylation, and partial apoptosis and autophagy. CONCLUSION: The seven HCC-derived EGFR mutants in this study are functioning, EGF-dependent, and erlotinib-resistant. Erlotinib induces differential degree of apoptosis and autophagy among cells harboring different EGFRs. The degree of inhibition of EGFR phosphorylation by erlotinib is the determining factor for the degree of apoptosis and autophagy amongst cells harboring EGFR mutants. This study paves the way for further investigation into the sensitivity of these HCC-derived mutants to the 3rd-generation irreversible EGFR-TKI, osimertinib.

Droplet digital PCR using HER2/EIF2C1 ratio for detection of HER2 amplification in breast cancer tissues.

Breast cancers with amplification and overexpression of human epithelial growth factor receptor 2 (HER2) are associated with poor prognosis, and targeted for anti-HER2 therapy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently the recommended methods to asses HER2 overexpression/amplification. Droplet digital PCR (ddPCR), a highly accurate method to quantify DNA copy number, is potentially a robust alternative for HER2 diagnostics. In the FISH assay and most of previous ddPCR reports, chromosome 17 centromere (CEP17) has been used as the reference control to determine HER2/CEP17 ratio. Nevertheless, miss-classification could occur when HER2 is co-amplified with CEP17. To avoid this inherent defect, in the present study, we employed ddPCR assay using the human eukaryotic translation initiation factor 2C1 (EIF2C1) gene located at chromosome 1p34.3 as the reference control to quantify HER2 copy number in 31 frozen breast cancer tissues. HER2 status of these samples had been determined by FISH and classified as HER2-amplified and HER2-non-amplified breast cancers. The results showed that HER2 determined by ddPCR using HER2/EIF2C1 ratio was in good concordance with HER2 determined by FISH using HER2/CEP17 ratio, the concordance rate 87.1% (27/31), Kappa = 0.719. The sensitivity and specificity of ddPCR assay was 90% (9/10) and 85.7% (18/21), respectively. The median HER2/EIF2C1 copy number ratio in HER2-amplified cancers (6.55, range 1.3-17.3) was significantly higher than in HER2-non-amplified cancers (1.05, range 0.6-3.6, p < 0.001). This study demonstrated that ddPCR using HER2/EIF2C1 ratio could accurately assess HER2 status in frozen breast cancer tissues. Thus, our findings warrant further studies into breast cancer with HER2-equivocal by IHC/FISH.

Reduced HBV cccDNA and HBsAg in HBV-associated hepatocellular carcinoma tissues.

Approximately 50% of hepatocellular carcinoma (HCC) is attributable to chronic infection with hepatitis B virus (HBV). Serum hepatitis B surface antigen (HBsAg) is an important diagnostic marker of HBV infection, whereas intrahepatic HBV covalently closed circular DNA (cccDNA) is a surrogate marker of HBV persistence. This study aimed to investigate relationships between serum HBsAg, intrahepatic HBsAg, and intrahepatic cccDNA in HBV-associated HCC. Intrahepatic HBsAg was determined by immunohistochemistry in matched non-cancerous and HCC tissues from 88 patients; 56 patients (63.64%) were serum HBsAg positive. In serum HBsAg-positive group, intrahepatic HBsAg was positive staining in 73.2% of non-cancerous tissues, but only in 10.7% of HCC tissues. Significant correlation between serum HBsAg and intrahepatic HBsAg was observed in non-cancerous tissues (p < 0.001), but not in HCC tissues (p = 0.415). Absolute quantification of intrahepatic cccDNA was performed by droplet digital PCR in tissues from 30 patients; 18 patients (60%) were serum HBsAg positive. In serum HBsAg-positive group, intrahepatic cccDNA was detected in 66.66% of non-cancerous tissues, but only in 5.55% of HCC tissue; intrahepatic cccDNA levels in non-cancerous tissues were significantly higher than those in HCC tissues (p < 0.001), and correlated with serum HBsAg (p < 0.01). Significant correlations between intrahepatic HBsAg and intrahepatic cccDNA were found in both non-cancerous tissues (p < 0.01) and HCC tissues (p < 0.05). We concluded that HBV cccDNA and intrahepatic HBsAg in HBV-associated HCC tissues were significantly reduced, as compared with matched non-cancerous tissues. This warrants further investigation into the impacts and the cause(s) of cccDNA reduction in HBV-associated HCC tissues, which might yield novel immune-related therapy for HBV-associated HCC.

Blocking ERK1/2 signaling impairs TGF-ß1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells.

BACKGROUND: Transforming growth factor-ß (TGF-ß) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investigated the roles of non-Smad pathway (ERK1/2) and Smad in TGF-ß tumor promoting and suppressing activities in intrahepatic cholangiocarcinoma (ICC) cells. METHODS: TGF-ß1 effects on proliferation, invasion and migration of ICC cells, KKU-M213 and/or HuCCA-1, were investigated using MTT, colony formation, in vitro Transwell and wound healing assays. Levels of mRNAs and proteins/phospho-proteins were measured by quantitative (q)RT-PCR and Western blotting respectively. E-cadherin localization was examined by immunofluorescence and secreted MMP-9 activity was assayed by gelatin zymography. The role of ERK1/2 signaling was evaluated by treating cells with TGF-ß1 in combination with MEK1/2 inhibitor U0126, and that of Smad2/3 and Slug using siSmad2/3- and siSlug-transfected cells. RESULTS: h-TGF-ß1 enhanced KKU-M213 cell invasion and migration and induced epithelial-mesenchymal transition as shown by an increase in vimentin, Slug and secreted MMP-9 levels and by a change in E-cadherin localization from membrane to cytosol, while retaining the cytokine's ability to attenuate cell proliferation. h-TGF-ß1 stimulated Smad2/3 and ERK1/2 phosphorylation, and the MEK1/2 inhibitor U0126 attenuated TGF-ß1-induced KKU-M213 cell invasion and MMP-9 production but moderately enhanced the cytokine growth inhibitory activity. The latter effect was more noticeable in HuCCA-1 cells, which resisted TGF-ß-anti-proliferative activity. Smad2/3 knock-down suppressed TGF-ß1 ability to induce ERK1/2 phosphorylation, Slug expression and cell invasion, whereas Slug knock-down suppressed cell invasion and vimentin expression but marginally affected ERK1/2 activation and MMP-9 secretion. These results indicate that TGF-ß1 activated ERK1/2 through Smad2/3 but not Slug pathway, and that ERK1/2 enhanced TGF-ß1 tumor promoting but repressed its tumor suppressing functions. CONCLUSIONS: Inhibiting ERK1/2 activation attenuates TGF-ß1 tumor promoting effect (invasion) but retains its tumor suppressing role, thereby highlighting the importance of ERK1/2 in resolving the TGF-ß paradox switch.