Hirotoshi Dosaka-Akita

B7-H1 Expression on Non-Small Cell Lung Cancer Cells and Its Relationship with Tumor-Infiltrating Lymphocytes and Their PD-1 Expression

Purpose: B7-H1/PD-L1 (B7-H1) and B7-DC/PD-L2 (B7-DC) are ligands for the receptor PD-1, which is known to negatively regulate T-cell activation. In the present study, we investigated the expression of B7-H1 and B7-DC in tumor specimens of non-small cell lung cancer and their relationships with clinicopathological variables and postoperative survival. Furthermore, we examined the correlation between B7-H1 expression on tumor cells and the number of tumor-infiltrating lymphocytes (TILs) or PD-1 expression on TILs. Experimental Design: The expression of B7-H1 and B7-DC in 52 surgically resected specimens of non-small cell lung cancer was evaluated immunohistochemically. Results: Expression of B7-H1 and B7-DC was focally observed in all non-small cell lung cancer tumor specimens. No relationship was found between the expression of B7-H1 or B7-DC and clinicopathological variables or postoperative survival. However, in the same sections evaluated, significantly fewer TILs were identified in B7-H1-positive tumor regions than in B7-H1-negative tumor regions in a subset of five patients (P = 0.01). Moreover, the percentage of TILs expressing PD-1 was significantly lower in B7-H1-positive tumor regions than in B7-H1-negative tumor regions (P = 0.02). Conclusions: The expression of B7-H1 on tumor cells in local areas reciprocally correlated with the number of TILs, and this may contribute to negative regulation in antitumor immune responses in non-small cell lung cancer.

Feasibility of insertion/implantation of 2.0-mm-diameter gold internal fiducial markers for precise setup and real-time tumor tracking in radiotherapy.

PURPOSE To examine the feasibility and reliability of insertion of internal fiducial markers into various organs for precise setup and real-time tumor tracking in radiotherapy (RT). MATERIALS AND METHODS Equipment and techniques for the insertion of 2.0-mm-diameter gold markers into or near the tumor were developed for spinal/paraspinal lesions, prostate tumors, and liver and lung tumors. Three markers were used to adjust the center of the mass of the target volume to the planned position in spinal/paraspinal lesions and prostate tumors (the three-marker method). The feasibility of the marker insertion and the stability of the position of markers were tested using stopping rules in the clinical protocol (i.e., the procedure was abandoned if 2 of 3 or 3 of 6 patients experienced marker dropping or migration). After the evaluation of the feasibility, the stability of the marker positions was monitored in those patients who entered the dose-escalation study. RESULTS Each of the following was shown to be feasible: bronchoscopic insertion for the peripheral lung; image-guided transcutaneous insertion for the liver; cystoscopic and image-guided percutaneous insertion for the prostate; and surgical implantation for spinal/paraspinal lesions. Transcutaneous insertion of markers for spinal/paraspinal lesions and bronchoscopic insertion for central lung lesions were abandoned. Overall, marker implantation was successful and was used for real-time tumor tracking in RT in 90 (90%) of 100 lesions. No serious complications related to the marker insertion were noted for any of the 100 lesions. Using three markers surgically implanted into the vertebral bone, the mean +/- standard deviation in distance among the three markers was within 0.2 +/- 0.6 mm (range -1.4 to 0.8) through the treatment period of 30 days. The distance between the three markers gradually decreased during RT in five of six prostate cancers, consistent with a mean rate of volume regression of 9.3% (range 0.015-13%) in 10 days. CONCLUSIONS Internal 2.0-mm-diameter gold markers can be safely inserted into various organs for real-time tumor tracking in RT using the prescribed equipment and techniques. The three-marker method has been shown to be a useful technique for precise setup for spinal/paraspinal lesions and prostate tumors.

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1

The mechanisms by which tumor microenvironments modulate nucleic acid–mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.

A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations

Retrospective analysis has shown that activating mutations in exons 18–21 of the epidermal growth factor receptor (EGFR) gene are a predictor of response to gefitinib. We conducted a phase II trial to evaluate the efficacy and safety of gefitinib as first-line therapy for advanced non-small cell lung cancer (NSCLC) with EGFR mutations. Patients with stage IIIB or IV chemotherapy-naïve NSCLC with EGFR mutation were treated with 250 mg gefitinib daily. For mutational analysis, DNA was extracted from paraffin-embedded tissues and EGFR mutations were analysed by direct sequence of PCR products. Twenty (24%) of the 82 patients analysed had EGFR mutations (deletions in or near E746-A750, n=16; L858R, n=4). Sixteen patients were enrolled and treated with gefitinib. Twelve patients had objective response and response rate was 75% (95% CI, 48–93%). After a median follow-up of 12.7 months (range, 3.1–16.8 months), 10 patients demonstrated disease progression, with median progression-free survival of 8.9 months (95% CI, 6.7–11.1 months). The median overall survival time has not yet been reached. Most of the toxicities were mild. This study showed that gefitinib is very active and well tolerated as first-line therapy for advanced NSCLC with EGFR mutations.

Tolerance of organs at risk in small-volume, hypofractionated, image-guided radiotherapy for primary and metastatic lung cancers

PURPOSE To determine the organ at risk and the maximum tolerated dose (MTD) of radiation that could be delivered to lung cancer using small-volume, image-guided radiotherapy (IGRT) using hypofractionated, coplanar, and noncoplanar multiple fields. MATERIALS AND METHODS Patients with measurable lung cancer (except small-cell lung cancer) 6 cm or less in diameter for whom surgery was not indicated were eligible for this study. Internal target volume was determined using averaged CT under normal breathing, and for patients with large respiratory motion, using two additional CT scans with breath-holding at the expiratory and inspiratory phases in the same table position. Patients were localized at the isocenter after three-dimensional treatment planning. Their setup was corrected by comparing two linacographies that were orthogonal at the isocenter with corresponding digitally reconstructed images. Megavoltage X-rays using noncoplanar multiple static ports or arcs were used to cover the parenchymal tumor mass. Prophylactic nodal irradiation was not performed. The radiation dose was started at 60 Gy in 8 fractions over 2 weeks (60 Gy/8 Fr/2 weeks) for peripheral lesions 3.0 cm or less, and at 48 Gy/8 Fr/2 weeks at the isocenter for central lesions or tumors more than 3.0 cm at their greatest dimension. RESULTS Fifty-seven lesions in 45 patients were treated. Tumor size ranged from 0.6 to 6.0 cm, with a median of 2.6 cm. Using the starting dose, 1 patient with a central lesion died of a radiation-induced ulcer in the esophagus after receiving 48 Gy/8 Fr at isocenter. Although the contour of esophagus received 80% or less of the prescribed dose in the planning, recontouring of esophagus in retrospective review revealed that 1 cc of esophagus might have received 42.5 Gy, with the maximum dose of 50.5 Gy. One patient with a peripheral lesion experienced Grade 2 pain at the internal chest wall or visceral pleura after receiving 54 Gy/8 Fr. No adverse respiratory reaction was noted in the symptoms or respiratory function tests. The 3-year local control rate was 80.4% +/- 7.1% (a standard error) with a median follow-up period of 17 months for survivors. Because of the Grade 5 toxicity, we have halted this Phase I/II study and are planning to rearrange the protocol setting accordingly. The 3-year local control rate was 69.6 +/- 10.6% for patients who received 48 Gy and 100% for patients who received 60 Gy (p = 0.0442). CONCLUSIONS Small-volume IGRT using 60 Gy in eight fractions is highly effective for the local control of lung tumors, but MTD has not been determined in this study. The organs at risk are extrapleural organs such as the esophagus and internal chest wall/visceral pleura rather than the pulmonary parenchyma in the present protocol setting. Consideration of the uncertainty in the contouring of normal structures is critically important, as is uncertainty in setup of patients and internal organ in the high-dose hypofractionated IGRT.

Tumor-associated macrophages regulate tumorigenicity and anticancer drug responses of cancer stem/initiating cells

Recent evidence has unveiled the critical role of tumor cells with stem cell activities in tumorigenicity and drug resistance, but how tumor microenvironments regulate cancer stem/initiating cells (CSCs) remains unknown. We clarified the role of tumor-associated macrophages (TAMs) and their downstream factor milk-fat globule-epidermal growth factor-VIII (MFG-E8) in the regulation of CSC activities. Bone marrow chimeric systems and adoptive cell transfers elucidated the importance of MFG-E8 from TAMs in conferring to CSCs with the ability to promote tumorigenicity and anticancer drug resistance. MFG-E8 mainly activates signal transducer and activator of transcription-3 (Stat3) and Sonic Hedgehog pathways in CSCs and further amplifies their anticancer drug resistance in cooperation with IL-6. Thus, the pharmacological targeting of key factors derived from tumor-associated inflammation provides a unique strategy to eradicate therapy-resistant tumors by manipulating CSC activities.

CD4+ T cells in cancer stroma, not CD8+ T cells in cancer cell nests, are associated with favorable prognosis in human non‐small cell lung cancers

We investigated intratumoral tumor‐infiltrating lymphocytes (TILs), including CD4+ and CD8+ T cells, in non‐small cell lung cancers (NSCLCs) and their relationships with clinicopathological variables and post‐operative survival. Tumor specimens from 178 NSCLCs were consecutively obtained by surgery at the Hokkaido University Medical Hospital between 1976 and 1994. CD8+ T cells, CD4+ T cells and Ki‐67/CD8+ T cells were visualized immunohistochemically, and counted within cancer cell nests and in cancer stroma. CD8+ T cells and CD4+ T cells were observed at higher frequencies within cancer cell nests in moderately and poorly differentiated tumors compared with well differentiated tumors (P<0.01), and in tumors with high Ki‐67 expression compared with low Ki‐67 expression (P<0.01), that showed severe cellular atypia and a higher growth rate. Patients with higher numbers of CD8+ T cells within cancer cell nests showed significantly shorter survival times compared to those with lower numbers of CD8+ T cells within cancer cell nests (5‐year survival rates, 47% and 60%, respectively; P=0.03). Moreover, patients with higher labeling index of Ki‐67/CD8+ T cells showed significantly shorter survival than those with lower labeling index of Ki‐67/CD8+ T cells within cancer cell nests (5‐year survival rates, 41% and 69%, respectively; P=0.02), and the labeling index of Ki‐67/CD8+ T cells within cancer cell nests was found to be a significant and independent unfavorable prognostic factor by multivariate analysis (P=0.01). On the other hand, higher numbers of CD4+ T cells in cancer stroma, but not within cancer cell nests, were correlated with longer survival times in patients with NSCLC (5‐year survival rates, 64% and 43%, respectively; P=0.04). CD4+ T cells in cancer stroma might reflect immune responses against cancer cells, while CD8+ T cells do not appear to work as effectors in tumor tissues of NSCLC. Moreover, the higher labeling index of Ki‐67/CD8+ T cells within cancer cell nests is a strong indicator of unfavorable clinical outcome.

Real-time tumor-tracking radiation therapy for lung carcinoma by the aid of insertion of a gold marker using bronchofiberscopy.

The authors developed fluoroscopic real‐time tumor‐tracking radiation therapy (RTRT) by insertion of a gold marker using bronchofiberscopy to reduce uncertainties in organ motion and set‐up error in external radiotherapy for moving tumors. The purpose of the current study was to evaluate RTRTs feasibility in lung carcinoma treatment.

Small-volume image-guided radiotherapy using hypofractionated, coplanar, and noncoplanar multiple fields for patients with inoperable Stage I nonsmall cell lung carcinomas

Occasionally, medically compromised and/or elderly patients with nonsmall cell lung carcinomas (NSCLCs) cannot be treated surgically. We investigated small‐volume hypofractionated image‐guided radiotherapy (IGRT) without the need for breath control in patients with inoperable Stage I NSCLCs.

Mediastinal Lymph Node Staging by FDG-PET in Patients with Non-Small Cell Lung Cancer: Analysis of False-Positive FDG-PET Findings

Background: Accurate staging of mediastinal and hilar lymph nodes is a critical factor determining operability in patients with non-small cell lung cancer (NSCLC). Positron emission tomography with 2-[18F] fluoro-2-deoxy-D-glucose as a tracer (FDG-PET) has recently been reported to be more effective in detecting tumor involvement in mediastinal and hilar lymph nodes than computed tomography (CT). Objective: In this study, we analyzed the accuracy of FDG-PET in mediastinal and hilar lymph node staging in patients with NSCLC and the factors associated with false-positive or false-negative FDG-PET findings in mediastinal and hilar lymph node staging. Methods: Fifty-four patients with NSCLC who underwent preoperative analysis including chest CT and whole-body FDG-PET were evaluated retrospectively. Using FDG-PET, lesions were considered to be positive if a definite, localized area of higher uptake, excluding physiologic uptake, than in surrounding normal tissue was present. On CT findings, lymph nodes were considered to be positive if they were >10 mm in short-axis diameter, except subcarinal lymph nodes (#7), which were considered to be positive if they were >15 mm in short-axis diameter. All patients underwent surgical resection of primary tumors and mediastinal and hilar lymph nodes between 1999 and 2001 in our institute. Resected lymph nodes were histologically examined for the existence of tumor cells. Results: A total of 306 lymph nodes were resected and used for analysis. The sensitivity, specificity, positive predictive value and negative predictive value of FDG-PET were 73, 98, 70 and 98%, while those of CT were 55, 96, 55 and 96%, respectively. When pre-operative nodal staging was compared with post-operative histopathological staging, 44 patients (81%) were correctly staged, 7 (13%) were overstaged and 3 (6%) were understaged by FDG-PET, while 39 patients (72%) were correctly staged, 8 (15%) were overstaged and 7 (13%) were understaged by CT. All 7 overstaged patients by FDG-PET had other pulmonary complications, including interstitial pneumonitis (n = 2), previous pulmonary tuberculosis (n = 3), silicosis (n = 1) and emphysema (n = 1), although they were not in the active stage. In 3 understaged patients by FDG-PET, lymph nodes were also undetectable by CT. Conclusion: FDG-PET is superior to CT in mediastinal and hilar lymph node staging of patients with NSCLC. However, care should be taken in lymph node staging for patients who have other pulmonary complications, including interstitial pneumonitis, previous pulmonary tuberculosis and silicosis.