Quantitative Evaluation of Hormesis in Breast Cancer Using Histoculture Drug Response Assay.

PURPOSE: Hormesis is a phenomenon of growth stimulation at low doses and inhibition at higher doses. In cancer treatment, little is known about how hormesis affects cancer cell proliferation. We evaluated the hormetic dose-response relationship of paclitaxel using surgically resected breast cancer specimens on the basis of histoculture drug response assay (HDRA). METHODS: We used surgically resected fresh tumor specimens from 22 patients with breast cancer: 17 invasive ductal, 3 mucinous, and 2 other "special-type" cancers. All patients were female, ranging in age between 40 and 86 (median 60) years. Small pieces of viable cancer tissue were placed on collagen gel and cultured for 7 days with paclitaxel. Inhibition rates of paclitaxel at several concentrations were measured and fitted to a sigmoid dose-response curve. RESULTS: Hormesis was observed in 9 of the 22 cases; ED50 of cytotoxic effect was significantly higher (P = .0036) in hormesis (H) group (44.6 ± 4.2 µg/mL) than in nonhormesis (N) group (26.7 ± 3.5 µg/mL). CONCLUSION: We evaluated hormesis in breast cancer tissue using HDRA for the first time although previously confirmed in cultured cells. Hormesis seems to occur in patients undergoing treatment with anticancer agents, especially in a metastatic setting. Meanwhile, tumor growth may be stimulated in patients who are resistant to paclitaxel.

[Application of Bevacizumab beyond Progressive Disease in Patients with Lung Cancer Recurrence after Surgery].

BACKGROUND: The benefits of continuing bevacizumab (BEV) beyond progressive disease (PD) in patients with non-small cell lung cancer (NSCLC) remain unclear. We present our experience of continuing chemotherapy with BEV in patients with recurrent NSCLC after surgery. PATIENTS: From January 2010 to December 2016, chemotherapy with BEV was continued beyond PD in 20 patients. These patients included 10 men and 10 women, and their mean age at surgery was 71±10 years. Recurrence was observed at 630±460 days after surgery. RESULTS: The average number of protocols with BEV was 3±1 (1-6). The presented side effects were acceptable. Eight patients died of cancer. The 5-year survival rates after surgery, recurrence, and initiation of BEV were 78.8%, 50.1%, and 34.3%, respectively, and the median survival times were 2,465, 2,017, and 1,120 days, respectively. CONCLUSION: The majority of patients with operable NSCLC had a good performance status. We could detect recurrence early on, before the symptoms appeared, by regular examination. Therefore, these patients had an advantage in that more chemotherapeutic regimens could be administered to them and their prognosis could be improved by the continuation of BEV beyond over PD.

A Theoretical Model for the Hormetic Dose-response Curve for Anticancer Agents.

In the present article, we quantitatively evaluated the dose-response relationship of hormetic reactions of anticancer agents in vitro. Serial dilutions of gemcitabine, cisplatin, 5-fluorouracil, vinorelbine, and paclitaxel were administered to the A549 non-small-cell lung cancer cell line. The bi-phasic sigmoidal curve with hormetic and cytotoxic effects is given by the formula y=(a-b/(1+exp(c(*)log(x)-d)))/(1+exp(e(*)log(x)-f)), that was used to perform a non-linear least square regression. The dose-responses of the five anticancer agents were fitted to this equation. Gemcitabine and 5-fluorouracil, which had the lowest ED50 for their hormetic reaction, had the most pronounced promotive effects out of the five anticancer agents tested. The hormetic reaction progressed exponentially with culturing time. Our theoretical model will be useful in predicting how hormetic reactions affect patients with malignant tumors.

Class III Beta-tubulin Expression in Non-small Cell Lung Cancer: A Predictive Factor for Paclitaxel Response.

AIM: In order to clarify whether class III beta-tubulin (TUBB3) is a predictive marker for paclitaxel (PTX) chemotherapy, chemosensitivity was examined using an in vitro drug sensitivity assay. PATIENTS AND METHODS: Twelve specimens from non-small cell lung cancer (NSCLC) patients were obtained for dose-response curve analysis and measurement of the half-maximal effective dose (ED50) of PTX using the histoculture drug response assay (HDRA). Forty-one specimens were evaluated using the HDRA and the inhibition ratio (IR) at a concentration of 25 µg/ml PTX (IR25) was measured. TUBB3 expression was evaluated by H-score in immunohistochemical staining. RESULTS: The ED50 of PTX was 24.5 ± 8.06 µg/ml. The median H-score was significantly higher (p=0.0076) in the high effective dose (HE)-group (ED50 >25 µg/ml) than in the low effective (LE)-group (ED50 ≤ 25 µg/ml). The mean IR25 was 53.8 ± 26.6%. The median H-score for the high-inhibition ratio (HI)-group (IR25 >50%) was significantly higher (p=0.0337) than the low-inhibition ratio (LI)-group (IR25 ≤ 50%). CONCLUSION: High TUBB3 expression in NSCLC appeared to correlate with lower PTX sensitivity.

Fast Fourier transform analysis of pulmonary nodules on computed tomography images from patients with lung cancer.

PURPOSE: The outlines of primary lung cancers are more complicated than those of metastatic lung tumors on computed tomography (CT) images. This feature is useful for clarifying the diagnosis of pulmonary nodules before surgery. We applied fast Fourier transform (FFT) analysis for quantification of complexity of tumor outline. METHODS: Sequential cases of 72 primary lung cancers (Group PL) and 54 metastatic lung tumors (Group MT) were included. The outline of each tumor on chest CT images was described using polar coordinates, and converted to rectangular coordinates, yielding wave data of the tumor outline. The FFT was then used to analyze the wave data. The complexity index (Cxi) was defined as the sum of the amplitude of all harmonics over a fundamental frequency. RESULTS: The Cxi was higher (P <0.0001) for group PL (10.3 ± 6.7 mm) than for group MT (3.2 ± 2.4 mm), and it was correlated with tumor diameter in both groups. The cut-off equation "Cxi = 0.127 DT + 2.23" provided the highest diagnostic accuracy for distinguishing Group PL from Group MT such as a sensitivity of 95.8%, a specificity of 81.5%, and an accuracy of 89.7%. CONCLUSION: FFT analysis appears useful for quantification of complexity of the tumor outline.

Primary clear-cell sarcoma in the mediastinum.

We report a case of primary clear-cell sarcoma (CCS) in the mediastinum. In October 2011, a 63-year-old man was admitted to our hospital for surgical resection. The tumor was completely excised by video-assisted thoracoscopic surgery. The tumor was well encapsulated and did not invade the pleura. Histological examination led to a final diagnosis of primary CCS in the mediastinum. The patient remains alive without evidence of recurrence at 15 months after surgery.

Intrapleural administration of gelatin-embedded, sustained-release basic fibroblast growth factor for the regeneration of emphysematous lungs in rats.

BACKGROUND: Intra-airway and intra-arterial administration of gelatin-embedded, sustained-release basic fibroblast growth factor has stimulated regeneration of emphysematous lungs in animal experiments, but these routes of administration may also cause harm. This study investigated the effectiveness of intrapleural administration of gelatin-embedded, sustained-release basic fibroblast growth factor. This animal experiment preceded our clinical trial of intrapleural administration of sustained-release basic fibroblast growth factor in patients with chronic obstructive pulmonary disease accompanied by pneumothorax. METHODS: Pulmonary emphysema was induced in Sprague-Dawley rats using porcine elastase. Gelatin-embedded, sustained-release basic fibroblast growth factor was administered via the left pleural cavity. The rats were divided into a group that received gelatin-embedded, sustained-release basic fibroblast growth factor (FGF(+) group, n = 6), and a group that did not (FGF(-)group, n = 6). Animals were sacrificed after 14 days, and the results were evaluated by histologic examination. RESULTS: In the FGF(+) group, the mean linear intercept value of the alveolar septa was significantly shorter on the treated side than on the untreated side (65.1 ± 7.0 vs 114.4 ± 7.5 µm; P = .0005). There was no significant difference in the mean linear intercept value between the treated and untreated sides in the FGF(-) group. CONCLUSIONS: Intrapleural administration of sustained-release basic fibroblast growth factor induced lung regeneration in rats with elastase-induced pulmonary emphysema.

Response of meningeal carcinomatosis from breast cancer to capecitabine monotherapy: a case report.

A 62-year-old woman with breast cancer received neoadjuvant chemotherapy followed by breast-conserving surgery and sentinel node biopsy. During adjuvant endocrine therapy with aromatase inhibitor, she developed multiple bone metastases. Thereafter, she received tamoxifen and zoledronate therapy. In May 2011, she developed a tongue deviation and was diagnosed as having meningeal carcinomatosis. The tongue deviation disappeared 3 weeks after taking capecitabine (2,400 mg/day). Magnetic resonance imaging of the brain showed regression of meningeal carcinomatosis. Levels of tumor markers CEA and CA15-3 changed from 96.0 IU/ml and 3.5 ng/ml to 47.0 IU/ml and 1.5 ng/ml, respectively. Progression-free survival with capecitabine monotherapy was 5 months.

Preoperative serum pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen level predicts postoperative distant metastasis in patients with non-small-cell lung cancer.

OBJECTIVES: To examine the relationship between preoperative serum pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (I-CTP) levels and postoperative distant metastasis in patients with non-small-cell lung cancer (NSCLC). METHODS: We retrospectively reviewed 143 patients in whom preoperative serum I-CTP level was measured from January 2006 to March 2011, including 91 males and 52 females with an average age of 70.1 ± 8.2 years. Histological subtypes included adenocarcinoma (n = 95), squamous cell carcinoma (n = 34) and other (n = 14). Preoperative serum carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA) levels were also measured. Patients with abnormal renal function or preoperative bone fractures were excluded. RESULTS: The mean preoperative serum I-CTP level was 4.1 ± 1.6 ng/ml, and the preoperative serum I-CTP level was elevated (>4.5 ng/ml) in 29 patients. Distant metastasis was detected in 21 patients during the 39 ± 18 (range 1-79) months of follow-up. The rate of distant metastasis was significantly higher in patients with elevated preoperative serum I-CTP levels than those with normal preoperative I-CTP levels (≤4.5 ng/ml) (P < 0.0001). The 5-year recurrence-free survival rate was lower in patients with elevated preoperative serum I-CTP levels than those with normal preoperative I-CTP levels (41.8 vs 92.9%; P < 0.0001). CONCLUSIONS: An elevated preoperative serum I-CTP level predicts postoperative distant metastasis in patients with NSCLC.

Surgical treatment for metachronous second primary lung cancer after radical resection of primary lung cancer.

PURPOSE: We retrospectively reviewed our experience of surgical resection for second primary lung cancer (SPLC) in our institute. And to clarify whether periodic follow-up after resection of first primary lung cancer (FPLC) is associated with earlier detection of SPLC. METHODS: From January 2003 to March 2011, a total of 386 patients underwent surgical resection for primary lung cancer in our institute. Of these patients, 21 (5.4%) with SPLC were observed during follow-up after surgery. Radiation therapy was selected instead of surgical resection in 7 patients to preserve respiratory function. The other14 patients are reviewed in this paper. RESULTS: Histological types were different between FPLC and SPLC in only one patient(FPLC: adenosquamous carcinoma, SPLC: squamous cell carcinoma). The average SPLC tumor size (18±8 mm) was smaller (P = 0.07) than the average FPLC tumor size (26±14 mm). Recurrence was not observed in these patients.The follow-up period after resection of SPLC was 31±30 (5-94) months. During followup, 2 patients died of de novo malignancies, and the other 12 patients were alive without recurrence. CONCLUSION: Systematic and periodic long-term follow-up after FPLC probably resulted in earlier detection of SPLC and yielded this good prognosis.

[The importance of p53 and ras mutation as predictive markers for adjuvant chemotherapy in non-small-cell lung cancer].

OBJECTIVE: A previous large, randomized, control trial(JBR.10)revealed that adjuvant chemotherapy with cisplatin(CDDP) and vinorelbine(VNR)was effective for non-small-cell lung cancer(NSCLC). It was also reported that adjuvant chemotherapy was not effective for p53-negative or K-ras mutation-positive patients. To clarify whether p53 and ras statuses are true predictive markers for chemotherapy with cisplatin, chemosensitivity was examined using an in vitro drug sensitivity assay. MATERIALS AND METHODS: Surgically resected fresh tumor specimens obtained from 27 patients at our institute with NSCLC were used for this study. Histoculture Drug Response Assay(HDRA)was applied to evaluate the chemosensitivity of CDDP and VNR in these specimens. p53 expression was evaluated by immunohistochemistry, and K-ras mutation by direct sequencing. RESULT: Four of the 27 patients were positive for K-ras mutation. The inhibition rate of CDDP was 54±5% for K-ras mutation positive-patients, and 50±13% for negative-patients. There was no significant difference between these two groups. p53 overexpression was observed in 14 patients, but not in 13 patients. The inhibition rate of CDDP was 50±12% for p53- overexpressed patients, and 50±12% for patients not overexpressed. The inhibition rate of VNR was 36±17% for p53- overexpressed patients, and 33±14%for patients not overexpressed. As for 8 adenocarcinoma patients, the inhibition rate of CDDP of p53-overexpressed patients(59±8%)was significantly(p=0. 018)higher than that of patients not overexpressed (45±9%). p53 overexpression may be a predictive marker for chemotherapy using CDDP in lung adenocarcinoma. CONCLUSION: p53 overexpression may be a possible predictive marker for adjuvant chemotherapy using CDDP in NSCLC.

Epidermal growth factor receptor mutations are associated with docetaxel sensitivity in lung cancer.

INTRODUCTION: A recent large randomized controlled trial revealed that patients with lung cancer with epidermal growth factor receptor (EGFR) mutations had better prognoses when treated with the EGFR-tyrosine kinase inhibitor, gefitinib, than with cytotoxic chemotherapeutic agents. Lung cancer with EGFR mutations is highly sensitive to EGFR-tyrosine kinase inhibitors. The previous trial implied that EGFR mutations might be predictive of the response to cytotoxic chemotherapy. METHODS: Forty-six tumor tissue specimens (32 adenocarcinomas and 14 nonadenocarcinomas) were obtained from patients with lung cancer who underwent surgical resection. EGFR mutations were detected using polymerase chain reaction-invader assay. A histoculture drug response assay was used as an in vitro drug sensitivity test. The inhibition rates of cisplatin, docetaxel (DOC), vinorelbine, and gemcitabine were measured. RESULTS: Sensitizing EGFR mutations were detected in samples from 14 patients, all with adenocarcinomas. The inhibition rate of cisplatin in tumors with EGFR mutations (group M) was 34.8 ± 15.5%, which was significantly lower (p = 0.0153) than in wild-type tumors (group W; 46.6 ± 14.0%). The inhibition rate of DOC in group M (18.8 ± 13.4%) was also significantly lower (p = 0.0051) than in group W (35.4 ± 19.1%). There were no significant differences in inhibition rates of gemcitabine and vinorelbine between groups M and W. Inhibition rates of DOC were significantly lower in group M (p = 0.0256) than in group W (32.6 ± 18.4) in samples from patients with adenocarcinoma. CONCLUSION: The histoculture drug response assay indicated that lung cancers with EGFR mutations were less sensitive to DOC than EGFR wild-type tumors.

In vitro evaluation of the effect of cardiac surgery on cancer cell proliferation.

OBJECTIVE: Operative stress of cardiovascular surgery can alter the blood levels of various physiologically active substances (e.g., cytokines, growth factors), and thus potentially affect cancer cell proliferation. How the combination of changes in blood levels of these substances affects cancer cells has not been adequately addressed. We investigated the stimulatory capacity on cancer cells of serum from patients after cardiovascular surgery, using a novel in vitro assay method. METHODS: The subjects were 22 patients undergoing cardiovascular surgery, consisting of 11 off-pump and 11 on-pump procedures. Blood was sampled from each subject immediately before surgery, immediately after surgery, and after transfer to the intensive care unit. Human lung cancer cells were exposed to the serum of each blood sample from each patient, and an MTT assay was conducted to evaluate cell proliferation. RESULTS: Serum samples of all patients showed an inhibitory effect for lung cancer cell proliferation. This inhibitory effect was lower in postoperative serum compared with serum samples before surgery. As a result, lung cancer cell proliferation was better with postoperative serum samples than preoperative serum samples. The proliferation rate after surgery, when it was compared with preoperative serum, was significantly higher in patients with on-pump procedures than in patients with off-pump procedures. CONCLUSION: The results of this study suggest that the operative stress of cardiovascular surgery induces changes in serum to make it less inhibitory for the cancer cell proliferation. This phenomenon is greater in patients with extracorporeal circulation.

Is class III beta-tubulin a true predictive marker of sensitivity to vinorelbine in non-small cell lung cancer? Chemosensitivity data evidence.

AIM: To clarify whether class III ß-tubulin (TUBB3) is a true predictive marker for chemotherapy with vinorelbine, chemosensitivity was examined using an in vitro drug sensitivity assay. PATIENTS AND METHODS: Initially, 9 specimens were obtained to analyze the dose-response curve and to measure the median effective dose 50 (ED(50)) in the histoculture drug response assay (HDRA). Subsequently, 68 surgically resected non-small cell lung cancer (NSCLC) specimens were applied to the HDRA and H-scores were calculated by immunohistochemical staining. RESULTS: The mean (±SD) slope factor, ED(50) and maximal response was 8.7±5.4, 39.0±17.9 µg/ml and 85.5±5.1% respectively. The mean inhibition rate was 26.4±16.2% and the mean H-score was 1.09±1.07. The inhibition rate was significantly correlated with TUBB3 expression (r=0.27, p=0.03), and was significantly higher in TUBB3-positive specimens than in TUBB3-negative specimens (p=0.003). CONCLUSION: Tumors with high TUBB3 levels exhibited greater chemosensitivity to vinorelbine than tumors with low TUBB3 levels. This finding provides support for the results of the JBR.10 trial.

Mediastinal germ cell tumor with acute myeloid leukemia and growing teratoma syndrome.

A 39-year-old man was referred to hospital with a giant mediastinal mass, thrombocytopenia and high levels of serum tumor markers. Flow cytometry of bone marrow and peripheral blood samples led to the diagnosis of a mediastinal germ cell tumor associated with hematologic neoplasia. He was treated with combination chemotherapy for a germ cell tumor and acute myeloid leukemia. After chemotherapy, the tumor was enlarged, although serum tumor marker levels had decreased. After induction therapy, the tumor was surgically resected. This syndrome is rare, and more cases need to be studied to enable effective treatment.

[Feasibility of adjuvant docetaxel plus cyclophosphamide therapy for breast cancer].

PURPOSE: To evaluate the feasibility of adjuvant docetaxel plus cyclophosphamide(TC)therapy for breast cancer. PATIENTS AND METHODS: A total of 16 patients with intermediate risk-breast cancer were enrolled. TC therapy consisted of four courses of docetaxel 75 mg/m² plus cyclophosphamide 600 mg/m² intravenous administration over three weeks. Every infusion was premedicated with intravenous administration of granisetron 3 mg plus dexamethasone 16 mg, followed by dexamethasone 8 mg p. o. on days 2 and 3. RESULTS: Due to the allergic reaction, one patient discontinued TC therapy. Fifteen (94%) of the 16 patients completed the scheduled TC therapy. Feasibility was 93.7%. Grade 3/4 toxicity was limited to leucopenia, neutropenia, and febrile neutropenia. No non-hematological serious adverse events were observed. CONCLUSION: Adjuvant TC therapy is a feasible option for breast cancer.

[Histoculture drug response assay guided adjuvant chemotherapy in patients with ERCC1-positive non-small cell lung cancer].

PURPOSE: A previous large randomized control study(IALT)revealed that cisplatin(CDDP)-based adjuvant chemotherapy was not effective for patients with ERCC1-positive non-small cell lung cancer(NSCLC). We evaluated the chemosensitivity of surgically resected specimens of NSCLC using in vitro chemosensitivity test and searched for promising adjuvant chemotherapy protocols in the ERCC1-positive subgroup of NSCLC. PATIENTS AND METHODS: Chemosensitivities of 10 anticancer agents including cisplatin were evaluated by histoculture drug response assay(HDRA) using 28 surgically resected NSCLC specimens. ERCC 1 status was evaluated by immunohistochemistry. RESULTS: ERCC1 was positive in 22 and negative in 6 specimens. All ERCC1-negative specimens were sensitive for CDDP in HDRA, and all CDDP-resistant specimens in HDRA showed positive ERCC1 staining. ERCC1 status was significantly correlated with CDDP sensitivity(p=0.01). HDRA showed average 3(0-6)sensitive anticancer agents except for CDDP even in ERCC1-positive specimens. CONCLUSION: HDRA may provide effective non-platinum adjuvant chemotherapy protocols for patients with ERCC1-positive, i.e. CDDP resistant, NSCLC.

Histoculture drug response assay for gefitinib in non-small-cell lung cancer.

OBJECTIVE: There are many predictive factors for gefitinib sensitivity, including epidermal growth factor receptor (EGFR) gene mutation, EGFR copy number, and k-ras mutation. To investigate all of them is too expensive. We evaluated the chemosensitivity for gefitinib in non-small-cell lung cancer (NSCLC) using a histoculture drug response assay (HDRA). METHODS: Surgically resected fresh tumor specimens from 22 patients with NSCLC were used. There were 13 male and 9 female patients, ranging in age from 49 to 84 (average 70) years old. Sixteen patients (73%) were smokers. Sixteen adenocarcinomas, four squamous cell carcinomas, and two other histological types were included. Small pieces of viable cancer tissue were placed on the collagen gel and then cultured for 7 days in the presence of gefitinib. RESULTS: The HDRA was successful in all specimens. A dose-response relation was observed between inhibition rates and gefitinib concentration (p = 0.016). The inhibition rate at 20 microg/ml (IR(20)) in adenocarcinoma without smoking (39.2% +/- 35.1%, n = 6) was higher than that with smoking (2.2% +/- 5.0%, n = 10, P = 0.001) and that of nonadenocarcinoma (16.9% +/- 23.6%, n = 6, P = 0.09). Gene mutation analysis was performed in two of three adenocarcinomas without smoking, which showed especially high IR(20) values, and sensitizing mutations were observed in these specimens. A cutoff inhibition rate of approximately 40%-50% appeared to be suitable for a concentration of 20 microg/ml. CONCLUSION: HDRA appears to be applicable for evaluating sensitivity to gefitinib in NSCLC. It provides a convenient method for predicting the response to gefitinib in patients with NSCLC whose fresh tumor specimens are available.

[Safety and efficacy of weekly paclitaxel followed by FEC100 as primary systemic therapy for breast cancer].

The efficacy and safety of primary systemic therapy with weekly paclitaxel (wPTX; 80 mg/m2) followed by FEC100 (cyclophosphamide 500 mg/m2, epirubicin 100 mg/m2, 5-fluorouracil 500 mg/m2)were investigated in 52 patients with stage I-III A locally advanced breast cancer. Clinical response was complete in 30 patients (58%) and partial in 19 patients (37%). Pathological complete response was found in 23 primary lesions and 17 sentinel and/or sampling lymph nodes. No patients developed progressive disease and major adverse events except for febrile neutropenia in ten patients. These results showed that primary systemic therapy with wPTX followed by FEC100 is a feasible therapeutic option for breast cancer.