Yohei Funakoshi

Inhibition of the mTOR/S6K signal is necessary to enhance fluorouracil-induced apoptosis in gastric cancer cells with HER2 amplification.

The purpose of this study was to explore the effect of trastuzumab in enhancing the activity of chemotherapeutic agents and the molecular basis of this effect. Two gastric cancer cell types with HER2 amplification, one sensitive (NCI‑N87) and one insensitive (MKN-7) to trastuzumab, were tested for the effects of trastuzumab on cell growth and cell signaling using MTS assay and western blotting, respectively. Interaction between trastuzumab and chemotherapeutic agents (fluorouracil, doxorubicin, cisplatin and paclitaxel) was evaluated by the combination index (CI). Fluorouracil-induced apoptosis was evaluated using western blot for poly (ADP-ribose) polymerase (PARP). Trastuzumab decreased phosphorylation of S6K, showed synergistic effect with fluorouracil or doxorubicin, and increased fluorouracil-induced apoptosis in NCI-N87 cells, but not in MKN-7 cells. While the mTOR inhibitor everolimus enhanced fluorouracil-induced apoptosis in both HER2-amplified cell lines, this was not the case in the gastric cancer cell lines without HER2 amplification. Consistently, while the EGFR/HER2 inhibitor Cl-387,785 inhibited cell growth of MKN-7, this growth inhibition did not accompany decrease in phosphorylation of S6K, and the compound did not enhance fluorouracil-induced apoptosis. In summary, inhibition of the mTOR/S6K signal may be a key molecular event in enhancing fluorouracil-induced apoptosis specifically in gastric cancer cells with HER2 amplification. mTOR inhibitors may therefore be attractive alternative drugs in gastric cancers with HER2 amplification regardless of their sensitivity to trastuzumab.

Excessive MET signaling causes acquired resistance and addiction to MET inhibitors in the MKN45 gastric cancer cell line

SummaryThe clinical efficacy of MET tyrosine kinase inhibitors (MET-TKIs) is hindered by the emergence of acquired resistance, presenting an obstacle to drug discovery. To clarify the mechanisms underlying acquired resistance to MET-TKIs, we established resistance models by continuous exposure of the MET-amplified gastric cancer cell line MKN45 to MET-TKIs, PHA665752 (MKN45-PR) and GSK1363089 (MKN45-GR). Baseline expression and phosphorylation of MET were elevated in MKN45-PR and MKN45-GR compared to MKN45 cells, and higher concentrations of MET-TKIs were required to inhibit MET phosphorylation compared to parental cells. Alterations in MET previously associated with resistance to MET-TKIs were observed in resistant cells, including elevated MET copy number, observed in both resistant lines compared to MKN45 cells, and the Y1230H mutation, detected in MKN45-PR cells. Notably, the growth of resistant lines was lower in the absence of MET-TKIs, suggesting “addiction” to inhibitors. While MKN45-PR cells exhibited a higher S-phase fraction in the absence of PHA665752, bromodeoxyuridine (BrdU) uptake was identical. Baseline phosphorylation of ATR, Chk1 and p53 and p21waf1/Cip1 expression was higher in MKN45-PR compared to MKN45 cells, and levels were reduced to those observed in untreated MKN45 cells following PHA665752 treatment. Furthermore, targeted knockdown of MET enhanced the growth of MKN45-PR cells. These findings suggest that alterations in MET leading to acquired MET-TKI resistance, may cause excessive MET signaling, subsequent replication stress and DNA damage response, and intra-S-phase arrest in the absence of MET-TKIs. Thus, partial MET inhibition is necessary for resistant cells to proliferate, a phenomenon we refer to as MET-TKI “addiction”.

Prediction of Glomerular Filtration Rate in Cancer Patients by an Equation for Japanese Estimated Glomerular Filtration Rate

BACKGROUND Assessment of renal function is important for safe cancer chemotherapy, and eligibility criteria for clinical trials often include creatinine clearance. However, creatinine clearance overestimates glomerular filtration rate, and various new formulae have been proposed to estimate glomerular filtration rate. Because these were developed mostly in patients with chronic kidney disease, we evaluated their validity in cancer patients without kidney disease. METHODS Glomerular filtration rate was measured by inulin clearance in 45 Japanese cancer patients, and compared with creatinine clearance measured by 24-h urine collection as well as that estimated by the Cockcroft-Gault formula, Japanese estimated glomerular filtration rate developed in chronic kidney disease patients, the Modification of Diet in Renal Disease study equation and the Chronic Kidney Disease Epidemiology Collaboration equation. The Modification of Diet in Renal Disease study and Chronic Kidney Disease Epidemiology Collaboration equations were adjusted for the Japanese population by multiplying by 0.808 and 0.813, respectively. RESULTS The mean inulin clearance was 79.2 ± 18.7 ml/min/1.73 m(2). Bias values to estimate glomerular filtration rate for Japanese estimated glomerular filtration rate, the Cockcroft-Gault formula, creatinine clearance measured by 24-h urine collection, the 0.808 × Modification of Diet in Renal Disease study equation and the 0.813 × Chronic Kidney Disease Epidemiology Collaboration equation were 0.94, 9.75, 29.67, 5.26 and -0.92 ml/min/1.73 m(2), respectively. Precision (root-mean square error) was 14.7, 22.4, 39.8, 16.0 and 14.1 ml/min, respectively. Of the scatter plots of inulin clearance versus each estimation formula, the Japanese estimated glomerular filtration rate correlated most accurately with actual measured inulin clearance. CONCLUSION The Japanese estimated glomerular filtration rate and the 0.813 × Chronic Kidney Disease Epidemiology Collaboration equation estimated glomerular filtration rate with lower bias and higher precision than the other formulae. We therefore propose Japanese estimated glomerular filtration rate for the estimation of glomerular filtration rate in Japanese cancer patients.

Left atrial extension of metastatic lung tumor via pulmonary vein: report on the first case of Ewing sarcoma

Extension of metastatic lung tumors into the left atrium via pulmonary veins is rare. Here, we report the first case of Ewing sarcoma exhibiting such extension. A 31-year-old man with pulmonary metastasis from Ewing sarcoma presented with a mass in the left lung, extending to the left atrium through the left inferior pulmonary vein. As the patient was considered to be at risk of tumor embolism, the mass was excised surgically.

Randomized trial of standard pain control with or without gabapentin for pain related to radiation-induced mucositis in head and neck cancer

OBJECTIVE Radiation-induced mucositis (RIM) in chemoradiotherapy (CRT) for head and neck cancer (HNC) causes severe pain and worsens CRT compliance, QOL and outcome. Following retrospective reports, we conducted a randomized trial of the safety and efficacy of gabapentin for RIM-associated pain during CRT. METHODS HNC patients (pts) receiving CRT were randomized to standard pain control (SPC) with acetaminophen and opioids, or SPC plus gabapentin (SPC+G). Gabapentin was maintained at 900mg/day for 4 weeks after CRT. Primary endpoint was maximum visual analogue scale (VAS) score during CRT, and secondary endpoints were total opioid dose, changes in QOL (EORTC QLQ-C30 and QLQ-HN 35) from baseline to 4 weeks after CRT, and adverse events. RESULTS Twenty-two eligible Stage III or IV pts were randomly assigned to SPC or SPC+G (n=11 each). Twelve were treated in a locally advanced setting and 10 in a postoperative setting. Median maximum VAS scores, median total dose of opioids at maximum VAS and total dose of opioids at 4 weeks after CRT tended to be higher in the SPC+G arm (47 in SPC vs. 74 in SPC+G, p=0.517; 215mg vs. 745.3mg, p=0.880; and 1260mg vs. 1537.5mg, p=0.9438, respectively), without significance. QOL analysis showed significantly worse scores in the SPC+G arm for weight gain (p=0.005). Adverse events related to gabapentin were manageable. CONCLUSIONS This pilot study is the first prospective randomized trial of gabapentin for RIM-related pain. Gabapentin had no apparent beneficial effect. Further research into agents for RIM-related pain is warranted.

Validity of new methods to evaluate renal function in cancer patients treated with cisplatin

PurposeCreatinine clearance (Ccr) is used as a marker of renal function in cancer chemotherapy, but it is not correlated with glomerular filtration rate (GFR) after high-dose cisplatin treatment. In addition to Ccr, measured using 24-h urine collection (24-h Ccr) or Cockcroft–Gault formula (CGF), the Chronic Kidney Disease Epidemiology Collaboration (CKD–EPI) equation and the Japanese GFR estimation equation (the Japanese equation) have been recently developed to estimate GFR for predicting renal function. However, these equations remain to be evaluated, particularly in cancer patients treated with cisplatin. Therefore, we investigated the validity of these equations for predicting the GFR in cancer patients treated with cisplatin.MethodsGFR was measured by inulin clearance (Cin) in 50 cancer patients and compared with GFR estimated by the CKD–EPI equation, the Japanese equation, and Ccr estimated by CGF or measured by 24-h Ccr before the first and third cisplatin-containing chemotherapy cycles (considered pretreatment and posttreatment, respectively).ResultsBefore treatment, the CKD–EPI and the Japanese equations estimated GFR with higher accuracy than Ccr. Posttreatment bias values for GFR estimation using the CKD–EPI and the Japanese equations were lower than those for Ccr. The CKD–EPI and the Japanese equations were also more precise than Ccr. However, for patients with low renal function, these equations still overestimated Cin.ConclusionThe CKD–EPI and the Japanese equations estimated GFR with lower bias and higher precision than Ccr pre- and postcisplatin treatment. This study is registered at UMIN: 000002167.

Expression of the novel NUP98/PSIP1 fusion transcripts in myelodysplastic syndrome with t(9;11)(p22;p15).

Objectives:  The t(9;11)(p22;p15) is a very rare but recurrent translocation in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) blast crisis. The translocation results in a fusion gene between NUP98 at 11p15 and PSIP1 encoding two transcriptional coactivators, p52 and p75, at 9p22. Here, we describe the first case of myelodysplastic syndrome (MDS) with t(9;11)(p22;p15).

Mediastinal Germ Cell Tumor Exhibiting a Discrepancy between Tumor Markers and Imaging: A Case Study

We report a mediastinal germ cell tumor (GCT) that exhibited a discrepancy between the time course of serum human chorionic gonadotropin (hCG) levels and clinical consequences. An otherwise healthy man, aged 34 years, was diagnosed with a nonseminomatous GCT, most likely embryonal carcinoma (EC), based on a mediastinal tumor biopsy. Standard chemotherapy resulted in an optimal decrease in serum hCG levels. However, multiple lesions in the liver continued to enlarge, which led to his death. Autopsy revealed few viable tumor cells in the liver, with the great majority of the tumor cells appearing to have undergone necrosis, suggesting that they responded to the chemotherapy. The residual tumor cells in the mediastinum and the liver were similar to syncytiotrophoblast cells, suggesting a choriocarcinoma (CC). On immunohistochemical analysis, the mediastinal tumor cells in the diagnostic biopsy specimen expressed both CD30 and hCG, whereas residual mediastinal and hepatic tumor cells in the autopsy specimen after chemotherapy also expressed hCG, but not CD30. These findings suggested that the patient suffered from a primary mixed GCT consisting of an EC and a CC. Both pre- and postchemotherapy tumors strongly expressed matrix metalloproteinase-2, supporting the aggressive and invasive features of the tumor phenotype. We speculate that the extremely invasive tumor destroyed normal liver structure, whereas chemotherapy and central necrosis reduced the number of viable cells themselves, causing a discordant decrease in serum hCG levels.

Abstract 319: Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer

While it is becoming recognized that screening of oncology drugs on a platform using two-dimensionally (2D)-cultured cell lines is unable to precisely select clinically active drugs, three-dimensional (3D)-culture systems are emerging and show potential for better simulating the in vivo tumor microenvironment. The aim of this study was to reveal differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We first evaluated differences between 2D- and 3D-cultured breast cancer cell lines by assessing drug sensitivity, oxygen status, and expression of Ki-67 and caspases. Three cell lines (BT-549, BT-474, and T-47D) developed dense multi-cellular spheroids (MCSs) in 3D-culture, and tended to show greater resistance to paclitaxel and doxorubicin than in 2D-culture. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB-231) developed only loose MCSs in 3D-culture, and showed drug sensitivities similar to those in 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved PARP expression in 2D- than in 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected cells from paclitaxel-induced apoptosis. Hypoxia, as measured with phosphorescent LOX-1, was observed only in dense 3D-MCSs. BT-549 had considerably fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0 dormant sub-population may be responsible for its drug resistance in 3D-culture. In addition, BT-474 had a lower level of caspase-3 in 3D- than in 2D-culture, suggesting that the 3D-environment was anti-apoptotic. We next examined whether these findings were reproduced in primary cells utilizing a breast cancer patient-derived xenograft (PDX). The fresh disaggregated PDX tissue developed dense MCSs in 3D-culture. We compared staining for Ki-67, and caspase-3 and -8 in 2D- and 3D-cultured primary cells obtained from the PDX, and the original patient tumor (Ki-67 only): 2D-cultured cells showed much greater proportions of Ki-67-positive and caspase-3-positive cells than the others. 3D-cultured cells tended to show greater resistance to paclitaxel and doxorubicin compared to the others. Residual spheroids after exposure to paclitaxel did not grow after removal of the drug but majority of cells in the spheroids appeared alive based on their excluding trypan blue dye. In the residual spheroids, greater population of cells showed phosphorylated histone H2AX expression than those in the spheroids unexposed to paclitaxel, suggesting that chemotherapy-induced senescence associated with drug resistance. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important characteristics of tumor grown in vivo, namely hypoxia, dormancy, anti-apoptotic features, and their resulting drug resistance. 3D primary culture will be a model for the study of chemotherapy-induced senescence. Citation Format: Yoshinori Imamura, Toru Mukohara, Yohei Shimono, Yohei Funakoshi, Naoko Chayahara, Masanori Toyoda, Naomi Kiyota, Shintaro Takao, Seishi Kono, Tetsuya Nakatsura, Hironobu Minami. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. [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 319. doi:10.1158/1538-7445.AM2015-319

Hypothyroidism in patients with colorectal carcinoma treated with fluoropyrimidines

Targeted therapy with tyrosine kinase inhibitors, including vascular endothelial growth factor receptors, has been demonstrated to induce hypothyroidism and thyroid dysfunction. Cancer patients with thyroid dysfunction may be underdiagnosed and undertreated. Thyroid function in colorectal cancer patients receiving fluoropyrimidine-based chemotherapy with or without bevacizumab was evaluated at baseline and monthly. In the present study, 3 of 27 (11.1%) patients who received fluoropyrimidine-based chemotherapy developed a thyroid-stimulating hormone (TSH) level >10 µU/ml, and 13 (48.1%) developed an elevation above the upper limit of the normal range. No difference in TSH elevation was noted between the bevacizumab and chemotherapy-alone group (50 vs. 45%; P=1.00, respectively). Three (11.1%) patients developed a TSH level >10 µU/ml and 2 with hypothyroidism were treated with thyroid hormone replacement therapy. We demonstrated that bevacizumab does not affect thyroid function but fluoropyrimidines may induce thyroid dysfunction in patients with colorectal cancer. Further investigation is required to clarify the mechanism of fluoropyrimidine-induced thyroid dysfunction.