Comprehensive pharmacogenomic characterization of gastric cancer.

BACKGROUND: Gastric cancer is among the most lethal human malignancies. Previous studies have identified molecular aberrations that constitute dynamic biological networks and genomic complexities of gastric tumors. However, the clinical translation of molecular-guided targeted therapy is hampered by challenges. Notably, solid tumors often harbor multiple genetic alterations, complicating the development of effective treatments. METHODS: To address such challenges, we established a comprehensive dataset of molecularly annotated patient derivatives coupled with pharmacological profiles for 60 targeted agents to explore dynamic pharmacogenomic interactions in gastric cancers. RESULTS: We identified lineage-specific drug sensitivities based on histopathological and molecular subclassification, including substantial sensitivities toward VEGFR and EGFR inhibition therapies in diffuse- and signet ring-type gastric tumors, respectively. We identified potential therapeutic opportunities for WNT pathway inhibitors in ALK-mutant tumors, a significant association between PIK3CA-E542K mutation and AZD5363 response, and transcriptome expression of RNF11 as a potential predictor of response to gefitinib. CONCLUSIONS: Collectively, our results demonstrate the feasibility of drug screening combined with tumor molecular characterization to facilitate personalized therapeutic regimens for gastric tumors.

Systematic Evaluation of Gastric Tumor Cell Index and Two-Drug Combination Therapy via 3-Dimensional High-Throughput Drug Screening.

Tumor heterogeneity greatly limits personalized treatment of cancer. Patient-derived tumor cell (PDC) models precisely recapitulate the molecular properties and biology of the disease, making them effective preclinical tools for assessing anti-cancer drug activities. Accurate estimation of tumor purity is essential for performing high-throughput drug screening (HTS). In the present study, we measured and predicted the tumor population index in PDC models for two-drug combinational strategies using HTS system. Gastric cancer cell-lines and PDCs were subjected to multi-color immunofluorescence analysis against EpCAM and vimentin to evaluate the tumor cell index based on EpCAM expression levels. We generated a tumor purity prediction model using five different gastric cancer cell-lines (AGS, KATO-III, MKN-45, NCI-N87, SNU-216) with fluorescence intensity-based techniques. Afterwards, stage IV gastric cancer PDC models were evaluated using a micropillar/microwell chip-based HTS system. HER2/CCNE1-amplified PDCs were considerably resistant to an HER2 inhibitor, while combinational treatment consisting of an HER2 inhibitor with anti-WEE1 compound substantially suppressed tumor cellular growth. Moreover, PDCs with BRCA1/2 mutations were synergistically sensitive to HER2 and PARP inhibition therapy. Finally, somatic mutations in TP53 and CDKN2A with MYC amplification rendered PDCs susceptible to the drug combination of WEE1 and HER2. Collectively, our systematic method of high-throughput drug sensitivity screening is an integral pre-clinical platform for evaluating potential two-drug combinational approaches for personalized treatment of cancer.

Selective colony area method for heterogeneous patient-derived tumor cell lines in anti-cancer drug screening system.

We aimed to establish a fluorescence intensity-based colony area sweeping method by selecting the area of highest viability among patient-derived cancer cells (PDC) which has high tumor heterogeneity. Five gastric cancer cell lines and PDCs were screened with 24 small molecule compounds using a 3D micropillar/microwell chip. 100 tumor cells per well were immobilized in alginate, treated with the compounds, and then stained and scanned for viable cells. Dose response curves and IC50 values were obtained based on total or selected area intensity based on fluorescence. Unlike homogeneous cell lines, PDC comprised of debris and low-viability cells, which resulted in an inaccurate estimation of cell viability using total fluorescence intensity as determined by high IC50 values. However, the IC50 of these cells was lower and accurate when calculated based on the selected-colony-area method that eliminated the intensity offset associated with the heterogeneous nature of PDC. The selected-colony-area method was optimized to accurately predict drug response in micropillar environment using heterogeneous nature of PDCs.

Pharmacogenomic landscape of patient-derived tumor cells informs precision oncology therapy.

Outcomes of anticancer therapy vary dramatically among patients due to diverse genetic and molecular backgrounds, highlighting extensive intertumoral heterogeneity. The fundamental tenet of precision oncology defines molecular characterization of tumors to guide optimal patient-tailored therapy. Towards this goal, we have established a compilation of pharmacological landscapes of 462 patient-derived tumor cells (PDCs) across 14 cancer types, together with genomic and transcriptomic profiling in 385 of these tumors. Compared with the traditional long-term cultured cancer cell line models, PDCs recapitulate the molecular properties and biology of the diseases more precisely. Here, we provide insights into dynamic pharmacogenomic associations, including molecular determinants that elicit therapeutic resistance to EGFR inhibitors, and the potential repurposing of ibrutinib (currently used in hematological malignancies) for EGFR-specific therapy in gliomas. Lastly, we present a potential implementation of PDC-derived drug sensitivities for the prediction of clinical response to targeted therapeutics using retrospective clinical studies.

Effects of ADAM10 and ADAM17 Inhibitors on Natural Killer Cell Expansion and Antibody-dependent Cellular Cytotoxicity Against Breast Cancer Cells In Vitro.

BACKGROUND/AIM: The inhibition of a disintegrin and metalloproteinase (ADAM) has the potential to become a novel approach for natural killer (NK) cell-based cancer immunotherapy. Thus, the aim of this study was to investigate the influence of ADAM10 and ADAM17 inhibitors on expanded NK cell to enhance antibody-dependent cellular cytotoxicity (ADCC) in breast cancer cell lines. MATERIALS AND METHODS: NK cells were expanded in medium supplemented with an ADAM10 or ADAM17 inhibitor to prevent the shedding of soluble CD16/FcγRIII. The expression level of CD16 and production of interferon-gamma (IFN-γ) was detected by flow cytometry using specific antibodies. ADCC activity of expanded NK cells was estimated in trastuzumab treated breast cancer cell lines such as MCF-7, MDA-MB-231, SKBR3, and BT-474 cells. RESULTS: The ADAM17 inhibitor increased the purity of expanded NK cells to 90% after 14 days at 5 and 10 µM in vitro (p=0.043). However, the expansion rate of NK cells was decreased at 10 µM of the ADAM 17 inhibitor (p=0.043). Inhibition of ADAM10 suppressed the expansion of NK cells, although the NK purity was increased at 1 µM of the inhibitor. The expression of CD16 was significantly increased at 1 and 5 µM of the ADAM17 inhibitor (p=0.046, 0.028, respectively) during the culturing period. Inhibition of ADAM10 reduced the expression of CD16 on NK cells. The cytotoxic activity of the ADAM17 inhibitor treated NK cells against MCF-7 (p=0.039) and BT-474 (p=0.027) cells was significantly elevated. The ADCC activity of NK cells treated with 5 µM of ADAM17 inhibitor was significantly increased against SKBR-3 and BT-474 (p=0.027). Inhibition of ADAM17 increased the production of IFN-γ in expanded NK cells. CONCLUSION: The inhibition of ADAM17 enhanced the purity of expanded NK cells and the ADCC activity of these cells against trastuzumab treated breast cancer cell lines.

3-Dimensional micropillar drug screening identifies FGFR2-IIIC overexpression as a potential target in metastatic giant cell tumor.

We established two patient derived tumor cells (PDCs) from right and left pulmonary metastatic lesions respectively of a patient with giant cell tumor. At that time, patient-derived tumor cells from right and left surgical specimens were collected and cultured. High-throughput screening (HTS) for 24 drugs was conducted with a micropillar/microwell chip platform using giant cell tumor PDCs. Using 6 doses per drug in 6 replicates for giant cell tumor PDCs, the dose response curves and corresponding IC50 values were calculated from the scanned images using the S+ Chip Analyzer. A sensitive response was more significantly achieved for AZD4547 (FGFR2 inhibitor) in giant cell tumor PDCs originated from the right pulmonary nodule under the micropillar/microwell chip platform using 3D culture. This sensitivity was consistent with the target expression patterns of giant cell tumor PDCs (FGFR2-IIIC mRNA expression in giant cell tumor PDCs originated from the right pulmonary nodule was increased significantly as compared to those originated from left). However, in a conventional 2D cultured MTT assay, there was no difference for IC50 values of AZD4547 between giant cell tumor PDCs originated from right and left pulmonary nodules. An HTS platform based on 3D culture on micropillar/microwell chips and PDC models could be applied as a useful preclinical tool to evaluate the intrapatient tumor/response heterogeneity. This platform based on 3D culture might reflect far better the relation between the tumor-biology and the matched targeted agent as compared to a conventional 2D cultured MTT assay.

Acquired resistance to LY2874455 in FGFR2-amplified gastric cancer through an emergence of novel FGFR2-ACSL5 fusion.

BACKGROUND: Fibroblast growth factor 2 (FGFR2) amplification, occurring in ~2-9% of gastric cancers (GC), is associated with poor overall survival. RESULTS: RNA sequencing identified a novel FGFR2-ACSL5 fusion in the resistant tumor that was absent from the matched pre-treatment tumor. The FGFR2-amplified PDC line was sensitive to FGFR inhibitors whereas the PDC line with concomitant FGFR2 amplification and FGFR2-ACSL5 fusion exhibited resistance. Additionally, the FGFR2-amplified GC PDC line, which was initially sensitive to FGFR2 inhibitors, subsequently also developed resistance. MATERIALS AND METHODS: We identified an FGFR2-amplified patient with GC, who demonstrated a dramatic and long-term response to LY2874455, a pan-FGFR inhibitor, but eventually developed an acquired LY2874455 resistance. Following resistance development, an endoscopic biopsy was performed for transcriptome sequencing and patient-derived tumor cell line (PDC) establishment to elucidate the underlying molecular alterations. CONCLUSIONS: FGFR inhibitors may function against FGFR2-amplified GC, and a novel FGFR2-ACSL5 fusion identified by transcriptomic characterization may underlie clinically acquired resistance. IMPLICATIONS FOR PRACTICE: Poor treatment response represents a substantial concern in patients with gastric cancer carrying multiple FGFR2 gene copies. Here, we show the utility of a general FGFR inhibitor for initial response prior to treatment resistance and report the first characterization of a potential resistance mechanism involving an FGFR2-ACSL5 fusion protein.

Loss of Tuberous Sclerosis Complex 2 (TSC2) as a Predictive Biomarker of Response to mTOR Inhibitor Treatment in Patients with Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death globally. Mechanistic target of rapamycin (mTOR) is frequently up-regulated in HCC and plays an important role in HCC tumorigenesis. Tumors with loss of tuberous sclerosis complex 2 (TSC2), a negative regulator of mTOR signaling, tend to respond well to mTOR inhibitors. We analyzed TSC2 expression status in Korean patients with HCC and evaluated the correlation between TSC2 loss and response to the mTOR inhibitor, everolimus. METHODS: We retrospectively assessed 36 patients with advanced HCC who had received sorafenib at a single center in Korea between 2008 and 2014, and for whom tumor specimens were available for TSC2 immunohistochemical analysis (IHC). Three patient-derived tumor cell lines (PDCs) were analyzed by western blotting to determine TSC2 expression and drug sensitivity to mTOR. RESULTS: Twelve of 36 patients (33.3%) showed low to undetectable levels of TSC2 expression. No significant differences were observed in progression-free survival (PFS) or overall survival with sorafenib treatment based on TSC2 expression status. Two patients were treated with everolimus after sorafenib failure; one patient, with moderate TSC2 expression, experienced stable disease with a PFS of 5.8 months; the other, with high TSC2 expression, experienced rapid progression. PDC models demonstrated that the TSC2-low HCC PDC line was significantly more sensitive to everolimus than the TSC2-high HCC PDC lines. CONCLUSION: Loss of TSC2 may predict improved response to everolimus in HCC patients, but further studies are needed to confirm the predictive role of TSC2 expression for everolimus treatment.

Comparison of FcRγ-Deficient and CD57+ Natural Killer Cells Between Cord Blood and Adult Blood in the Cytomegalovirus-Endemic Korean Population.

BACKGROUND: FcRγ-deficient natural killer (NK) cells (g(-)NK cells) have been associated with cytomegalovirus (CMV) infection. However, the frequency of g(-)NK cells in a CMV-endemic area (i.e., Korea) has not yet been studied. We examined the frequency of g(-)NK cells and expression of CD57 on NK cells in cord blood (CB) and adult blood (AB). METHODS: Of the 24 AB samples collected, 95.8% (23/24) were CMV IgG(+)/IgM(-), while 100% of the 13 healthy CB samples were CMV IgG(+)/IgM(-). We performed whole-blood flow cytometry assays to analyze intracellular FcRγ and CD3ζ expression of CD3(-)/CD56(dim) NK cells from 13 CB and 24 AB samples, and surface CD57 expression on CD3(-)/CD56(dim)/CD16(+) NK cells from 13 CB and 19 AB samples. RESULTS: All CMV seropositive AB samples contained g(-)NK cells (23/23), and the median proportion of g(-)NK cells in the CD3(-)/CD56(dim) NK cell pool was 35.0% (range: 11-77%). CD57(+) NK cells in the CD3(-)/CD56(dim)/CD16(+) NK cell population were detected in all 19 AB samples tested, but not in any CB samples. CONCLUSIONS: Our data suggest that g(-)NK cells and CD57(+) NK cells are present at a very high frequency in CMV-seropositive AB, but rare in CMV-naïve CB.

Interleukin-21 induces proliferation and modulates receptor expression and effector function in canine natural killer cells.

Interleukin (IL)-21 is an important modulator of natural killer (NK) cell function. However, little is known about IL-21 function in canine NK cells because the phenotype of these cells remains undefined. In this study, we selectively expanded non-B and non-T large granular NK lymphocytes (CD3(-)CD21(-)CD5(-)CD4(-)TCRαß(-)TCRγδ(-)) ex vivo from the peripheral blood mononuclear cells (PBMCs) of healthy dogs using a combination of IL-2, IL-15, and IL-21 in the presence of 100 Gy-irradiated K562 cells. We investigated the effects of varying the duration and timing of IL-21 treatment on stimulation of proliferation, expression of NK-related receptors, anti-tumor activity and production of interferon (IFN)-γ. The expanded NK cells in each treatment group became enlarged and highly granular after 21 days in culture. NK cells proliferated rapidly in response to activation by IL-21 for 3 weeks, and IL-21 was able to induce changes in the mRNA expression of NK cell-related receptors and enhance the effector function of NK cells in perforin- and granzyme-B-dependent manners. The duration, frequency and timing of IL-21 stimulation during culture affected the rate of proliferation, patterns of receptor expression, cytokine production, and anti-tumor activity. The optimal conditions for maximizing the IL-21-induced proliferation and effector function of NK cells in the presence of IL-2 and IL-15 were seen in cells treated with IL-21 for the first 7 days of culture but without any further IL-21 stimulation other than an additional 2-day treatment prior to harvesting on day 21. The results of this study suggest that synergistic interactions of IL-21 with IL-2 and IL-15 play an important role in the proliferation, receptor expression, and effector function of canine NK cells.

Effect of exposure to interleukin-21 at various time points on human natural killer cell culture.

BACKGROUND AIMS: Interleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture. METHODS: IL-2/IL-15-activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry. RESULTS: Compared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the "first week" group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the "first week" group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of "first week" NK cells that received IL-21 treatment for an additional 2 days compared with the "first week" NK cells (P < 0.05). CONCLUSIONS: These data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells.

Immunological characterization of glioblastoma cells for immunotherapy.

The aim of this study was the immunological characterization of glioblastoma cells. Glioblastoma cell lines were cultured in serum and serum-free neurobasal (NBE) medium conditions. These cell lines were characterized by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), western blot and natural killer (NK) cell-cytotoxicity assays. A previously described NK cell expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) was used. RT-PCR and western blots for the expression of tumor-associated antigens (TAAs), were carried out in 32 glioblastoma and seven normal brain tissues. U87 and U343 tumor cell lines showed increased expression for major histocompatibility complex (MHC)-I and -II molecules. No significant differences in the levels of CD133, MHC class I/II, MHC class I-related chain A (MICA), MICB, UL16 binding protein 1-3 (ULBP 1-3) expression in these cell lines and in NK cell cytotoxicity were observed between serum and NBE conditions. Regardless of culture conditions, U87 and U343 cell lines were sensitive to expanded NK cells, with median cytotoxicities at 4:1 effector/target ratio of 43.2% and 46.5%, respectively. In RT-PCR, U343 and U87 showed the expression of most TAAs at a high ratio compared with U251. Western blots demonstrated positive expression for BIRC5, CD99 and ERBB2 in U251, U87 and U343 cell lines and tissues. These highly-expressed TAAs such as BIRC5, CD99 and ERBB2 in glioblastoma tissue could be the targets for immunotherapy. U87 and U343 cell lines could be useful for studying the efficacy of immunotherapy related to various TAAs and NK cell immunotherapy.

Ex vivo expansion of natural killer cells using cryopreserved irradiated feeder cells.

Currently, feeder cells are γ-irradiated immediately before use for the ex vivo expansion of natural killer (NK) cells from human peripheral blood. Storing irradiated feeder cells by cryopreserving them in multiple vials would be more convenient than irradiating cells each time they are needed. We compared NK cell expansion using cryopreserved-irradiated feeder cells (cryopreserved group) and freshly-irradiated feeder cells (fresh group). To expand NK cells, peripheral blood mononuclear cells were isolated and co-cultured with-100 Gy-irradiated K562 leukemia cells that had been modified to express 4-1BB ligand and membrane-bound (mb) interleukin (IL)-15 (K562-mb15-41BBL cells) for three weeks in the presence of IL-2 and IL-15. Fresh and cryopreserved K562-mb15-41BBL feeder cells expressed similar levels of 4-1BB ligand, whereas membrane-bound IL-15 expression was lower in the cryopreserved cells than in the fresh cells. The NK cell expansion rate did not differ between the two groups (980-fold vs. 1058-fold, respectively), although the mean NK cell purity was higher in the fresh-group than in the cryopreserved-group at day 14 (94.1% vs. 92.5%, respectively) and day 21 (97.1% vs. 95.4%, respectively). The NK cells from the two feeder cell groups did not differ in cytotoxicity against various malignant cell lines at effector-to-target ratios of 4:1, 2:1, and 1:1, or in the expression pattern of NK cell receptors [cluster of differentiation (CD)-16, natural killer group-2, member D (NKG2D), CD69, NKp30, NKp44, NKp46, and CD158b] and level of interferon-γ secretion. Our results demonstrate that cryopreserved irradiated feeder cells can be used for the ex vivo expansion of human NK cells and provide a convenient improvement on current methods.