Akihiro Bessho

The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells

A simplified method for cryopreservation at −80°C of peripheral blood stem cells (PBSC) has been increasingly used for autologous PBSC transplantation in Japan. Although this method, using 6% hydroxyethyl starch (HES) and 5% dimethyl sulfoxide (DMSO) as a cryoprotectant without rate-controlled freezing, has several advantages over the conventional method using 10% DMSO with rate-controlled freezing, little is known about effects of long-term cryopreservation for years and thawing process on hematopoietic progenitors. We examined the recovery rates of BFU-E and CFU-GM in sample tubes cryopreserved by the simplified method under various conditions as follows: (1) long-term storage for 1–5 years; (2) DMSO exposure for 1 h after rapid thawing; and (3) thawing at a lower temperature other than 37°C. In our study, we found that the recovery rates of BFU-E and CFU-GM were not affected by the length of cryopreservation period; they remained at more than 70% on average for 16–61 months. In our hands, a 1-h exposure to DMSO after rapid thawing was not toxic for hematopoietic progenitors. Furthermore, there was no significant difference in the recovery rates of BFU-E and CFU-GM between thawing at 37°C and 20°C. These observations indicate that PBSC cryopreserved for at least 5 years by the simplified method can be used clinically without losing hematopoietic activity, and suggest that hematopoietic activity of the thawed PBSC may be unaffected when PBSC are infused slowly within 60 min or even when PBSC are thawed gradually at room temperature.

A Phase II Trial of Erlotinib Monotherapy in Pretreated Patients with Advanced Non-small Cell Lung Cancer Who Do Not Possess Active EGFR Mutations: Okayama Lung Cancer Study Group Trial 0705

Backgrounds: Efficacy of gefitinib therapy strongly depends on epidermal growth factor receptor (EGFR)-mutation status in Asian patients with non-small cell lung cancer. Recently, the survival advantage of erlotinib, another tyrosine kinase inhibitor, was not affected by EGFR mutation status in a phase III trial, indicating that patients with EGFR-wild-type (EGFR-wt) tumors might also benefit from this tyrosine kinase inhibitor. The aim of this trial was to evaluate the efficacy and toxicity of erlotinib in Japanese patients with EGFR-wt tumors. Methods: The primary end point was an objective response. Patients with EGFR-wt tumors previously receiving one to three chemotherapy regimens were enrolled in this trial. The mutation status was assessed using the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method. Erlotinib was administered (150 mg/d) until disease progression or unacceptable toxicities occurred. Results: Thirty patients were enrolled between January and December 2008. Objective response was observed in one patient (3.3%), and the disease became stable in 18 patients (60.0%). Skin rash was the most common side effect. Grades 3–4 adverse events included pulmonary embolism, keratitis, and anemia. Two other patients developed interstitial lung disease (grades 1 and 2). Nevertheless, all these events were reversible, resulting in no treatment-related deaths. With a median follow-up time of 10.7 months, the median survival time and median progression-free survival times were 9.2 and 2.1 months, respectively. Conclusion: This is the first prospective biomarker study showing that erlotinib therapy for pretreated patients with EGFR-wt tumors seems to have a modest activity with no irreversible toxicity.

Phase I/II study of docetaxel and cisplatin with concurrent thoracic radiation therapy for locally advanced non-small-cell lung cancer.

Recent studies have suggested the superiority of concomitant over sequential administration of chemotherapy and radiotherapy. Docetaxel and cisplatin have demonstrated efficacy in advanced non-small-cell lung cancer (NSCLC). This study evaluated the safety, toxicity, and antitumour activity of docetaxel/cisplatin with concurrent thoracic radiotherapy for patients with locally advanced NSCLC. Patients with locally advanced NSCLC (stage IIIA or IIIB), good performance status, age ⩽75 years, and adequate organ function were eligible. Both docetaxel and cisplatin were given on days 1, 8, 29, and 36. Doses of docetaxel/cisplatin (mg m−2) in the phase I study portion were escalated as follows: 20/30, 25/30, 30/30, 30/35, 30/40, 35/40, 40/40, and 45/40. Beginning on day 1 of chemotherapy, thoracic radiotherapy was given at a total dose of 60 Gy with 2 Gy per fraction over 6 weeks. In the phase I portion, the maximum tolerated doses (MTD) among 33 patients were docetaxel 45 mg m−2 and cisplatin 40 mg m−2. The major dose-limiting toxicity (DLT) was radiation oesophagitis. The recommended doses (RDs) for the phase II study were docetaxel 40 mg m−2 and cisplatin 40 mg m−2. A total of 42 patients were entered in the phase II portion. Common toxicities were leukopenia, granulocytopenia, anaemia, and radiation oesophagitis, with frequencies of grade ⩾3 toxicities of 71, 60, 24, and 19%, respectively. Toxicity was significant, but manageable according to the dose and schedule modifications. Dose intensities of docetaxel and cisplatin were 86 and 87%, respectively. Radiotherapy was completed without a delay in 67% of 42 patients. The overall response rate was 79% (95% confidence interval (CI), 66–91%). The median survival time was 23.4+ months with an overall survival rate of 76% at 1 year and 54% at 2 years. In conclusion, chemotherapy with cisplatin plus docetaxel given on days 1, 8, 29, and 36 and concurrent thoracic radiotherapy is efficacious and tolerated in patients with locally advanced NSCLC and should be evaluated in a phase III study.

Interstitial lung disease in Japanese patients with non-small cell lung cancer receiving gefitinib : An analysis of risk factors and treatment outcomes in okayama lung cancer study group

ABSTRACTRisk factors for the development of interstitial lung disease in patients with non-small cell lung cancer receiving gefitinib and the prognostic factors after interstitial lung disease development have not been established. The aim of this study was to retrospectively identify and evaluate these possible factors. PATIENTS AND METHODSWe reviewed the clinical records and radiographs of 365 consecutive patients with non-small cell lung cancer who received gefitinib in West Japan between 2000 and 2003. RESULTSIn total, 330 patients were eligible for interstitial lung disease evaluation, and 15 patients (4.5%) were finally confirmed to have developed interstitial lung disease by blinded expert review. Multivariate analysis revealed that preexisting pulmonary fibrosis, poor performance status, and prior thoracic irradiation were independent risk factors for interstitial lung disease, with odds ratios of 21.0 (95% confidence interval, 5.12–86.3, P < 0.0001), 9.70 (2.27–41.4, P= 0.001), and 4.33 (1.27–14.8, P= 0.019), respectively. Among the 15 patients who developed interstitial lung disease, eight have died of the condition. Short interval from the initiation of gefitinib treatment to the onset of interstitial lung disease, acute interstitial pneumonia pattern, and the presence of pre-existing pulmonary fibrosis were associated with poor prognosis. DISCUSSIONOur results suggest the importance of patient selection for gefitinib treatment based on interstitial lung disease risk factors in the Japanese population identified.

Parathyroid hormone‐related protein measured at the time of first visit is an indicator of bone metastases and survival in lung carcinoma patients with hypercalcemia

Parathyroid hormone‐related protein (PTH‐rP) is a major cause of tumor‐induced hypercalcemia (TIH) and frequently is found to be elevated in serum of patients with TIH. In the current study, the authors examined the usefulness of PTH‐rP measurement at the time of first presentation in the follow‐up of lung carcinoma patients with TIH.

Non–Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases

Crizotinib is the standard of care for advanced non-small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult.

Comparison of the Incidence and Pattern of Interstitial Lung Disease During Erlotinib and Gefitinib Treatment in Japanese Patients with Non-small Cell Lung Cancer: The Okayama Lung Cancer Study Group Experience

Background: Data comparing the incidence and pattern of interstitial lung disease (ILD) in non-small cell lung cancer patients receiving treatment with gefitinib versus erlotinib, both of which are epidermal growth factor receptor tyrosine kinase inhibitors, are scarce. We investigated the incidence of ILD in Japanese patients treated with gefitinib or erlotinib. Methods: We reviewed the clinical records of 209 patients treated with erlotinib in 2008 (cohort A) and 330 treated with gefitinib between 2000 and 2003 (cohort B). Toxicity within the first month of treatment was investigated. Results: The patients in cohort A had fewer known risk factors for ILD (e.g., poor performance status and prior pulmonary fibrosis). ILD was detected in two patients (1.0%) from cohort A and eight patients (2.4%) from cohort B during the first month of treatment. The events were graded as follows: one patient each in grades 1 and 2 (cohort A), and one, one, and six patients in grades 3, 4, and 5, respectively (cohort B). Multivariate analysis revealed that poor performance status and prior pulmonary fibrosis were significantly correlated with the occurrence of ILD, but the type of epidermal growth factor receptor tyrosine kinase inhibitor administered was not. Conclusion: There was a somewhat lower incidence of ILD with erlotinib therapy than with gefitinib therapy, despite no statistically significant difference. Patient selection based on awareness by Japanese physicians of the risk factors for ILD, rather than the type of agent, may explain the difference in ILD incidence between the two treatments.

Phase II Trial of Gefitinib in Combination with Bevacizumab as First-Line Therapy for Advanced Non–Small Cell Lung Cancer with Activating EGFR Gene Mutations: The Okayama Lung Cancer Study Group Trial 1001

Purpose: Whether bevacizumab enhances the effect of the epidermal growth factor receptor (EGFR) inhibitor gefitinib on EGFR mutant non–small cell lung cancers (NSCLCs) remains unknown. We conducted a phase II trial to investigate the efficacy and safety of gefitinib when combined with bevacizumab as first-line therapy in patients with advanced NSCLC harboring EGFR gene mutations. Methods: In this trial, 42 patients with a performance status of 0 to 2 received gefitinib (250 mg/d) and bevacizumab (15 mg/kg, every 3 weeks). The primary end point of this study was the 1-year progression-free survival (PFS) rate. We assumed that a 1-year PFS rate of 55% would indicate potential usefulness and that a 1-year PFS rate of 40% would constitute the lower limit of interest. Results: Forty-two patients were enrolled in the study with a median age of 73 (range 42–86) years. Activating EGFR gene mutations included exon 19 deletion (57%) and L858R point mutations in exon 21 (38%). The objective response rate was 73.8% and included two complete responses. The 1-year PFS rate and median PFS time were 56.7% (95% confidence interval [CI] 39.9–70.5) and 14.4 months (95% CI 10.1–19.2), respectively. The median PFS differed significantly between EGFR exon 19 deletion and the L858R point mutation (18.0 versus 9.4 months, respectively; p = 0.006). The median overall survival had not yet been reached. Severe adverse events included grade 3 skin rash (15%), hypertension (17%), aspartate transaminase/alanine aminotransferase elevation (17%), proteinuria (7%), intracranial hemorrhage (2%), and grade 4 perforation of the digestive tract (2%). There were no treatment-related deaths. Conclusion: Gefitinib in combination with bevacizumab as first-line therapy seems to be a favorable and well-tolerated treatment for patients with advanced NSCLC with activating EGFR gene mutations, especially those with EGFR exon 19 deletion mutations, although the primary end point was not met because the lower limit of the CI was less than 40%.

Detection of the T790M mutation of EGFR in plasma of advanced non–small cell lung cancer patients with acquired resistance to tyrosine kinase inhibitors (West Japan oncology group 8014LTR study)

Introduction Next-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to overcome resistance to earlier generations of such drugs mediated by a secondary T790M mutation of EGFR, but the performance of a second tumor biopsy to assess T790M mutation status can be problematic. Methods We developed and evaluated liquid biopsy assays for detection of TKI-sensitizing and T790M mutations of EGFR by droplet digital PCR (ddPCR) in EGFR mutation–positive non–small cell lung cancer (NSCLC) patients with acquired EGFR-TKI resistance. Results A total of 260 patients was enrolled between November 2014 and March 2015 at 29 centers for this West Japan Oncology Group (WJOG 8014LTR) study. Plasma specimens from all subjects as well as tumor tissue or malignant pleural effusion or ascites fluid from 41 patients were collected after the development of EGFR-TKI resistance. All plasma samples were genotyped successfully and the results were reported to physicians within 14 days. TKI-sensitizing and T790M mutations were detected in plasma of 120 (46.2%) and 75 (28.8%) patients, respectively. T790M was detected in 56.7% of patients with plasma positive for TKI-sensitizing mutations. For the 41 patients with paired samples obtained after acquisition of EGFR-TKI resistance, the concordance for mutation detection by ddPCR in plasma compared with tumor tissue or malignant fluid specimens was 78.0% for TKI-sensitizing mutations and 65.9% for T790M. Conclusions Noninvasive genotyping by ddPCR with cell-free DNA extracted from plasma is a promising approach to the detection of gene mutations during targeted treatment.

Predictive value of circulating immature cell counts in peripheral blood for timing of peripheral blood progenitor cell collection after G–CSF plus chemotherapy-induced mobilization

BACKGROUND: Enumeration of CD34+ cells in peripheral blood (PB) before apheresis predicts the number of CD34+ cells collected, although flow cytometric techniques used are complex and expensive. In an attempt to determine the optimal timing for peripheral blood progenitor cell (PBPC) collection, the usefulness of circulating immature cell (CIC) counts in PB was evaluated.