Antibodies and Engineered Antibody Fragments against M13 Filamentous Phage to Facilitate Phage-Display-Based Molecular Breeding.

Antibodies are essential for characterizing various analytes. "Molecular-breeding" approaches enable rapid generation of antibody mutants with desirable antigen-binding abilities. Typically, prototype antibodies are converted to single-chain Fv fragments (scFvs), and random mutations are genetically introduced to construct molecular libraries with a vast diversity. Improved species therein are then isolated via phage display genotype-phenotype-connecting systems to separate them from a large excess of nonspecific scFvs. During these experiments, counting of phage particles is routinely performed. However, current methods depend on the time-consuming overnight cultivation of phage-infected bacteria on agar plates to estimate phage numbers as plaque-forming units (pfu) or colony-forming units, the results of which fluctuate considerably. Immunochemical systems capturing phage particles should be a more convenient and robust alternative. We therefore generated monoclonal antibodies against M13 filamentous phage, which is commonly used for phage display, by employing hybridoma technology. Combinatorial use of two such antibodies (Ab-M13#53 and #71; both specific to the major coat protein pVIII) enabled development of a sandwich enzyme-linked immunosorbent assay (ELISA) that could measure ca. 107-1010 phage pfu/mL. To construct a more convenient system, Ab-M13#71 was converted to the scFv form and further fused with an alkaline phosphatase variant. Using this fusion protein, the sandwich ELISA enabled rapid (within 90 min) and reliable phage counting without reducing the sensitivity, and the results were reasonably consistent with those of infection-based methods. The present anti-phage antibodies and scFvs might also enable visualization of individual phage particles by combining them with sensitive fluorescent staining.

The biochemical recurrence-free rate in patients who underwent prostate low-dose-rate brachytherapy, using two different definitions.

BACKGROUND: To assess the biochemical recurrence (BCR)-free rate in patients who underwent prostate low-dose-rate brachytherapy (LDR-brachytherapy), using two different definitions (Phoenix definition and PSA ≥ 0.2 ng/mL). METHODS: Two hundreds and three patients who were clinically diagnosed with localized prostate cancer (cT1c-2cN0M0) and underwent LDR-brachytherapy between July 2004 and September 2008 were enrolled. The median follow-up period was 72 months. We evaluated the BCR-free rate using the Phoenix definition and the PSA cut-off value of 0.2 ng/mL, as in the definition for radical prostatectomy. To evaluate an independent variable that can predict BCR, Cox's proportional hazard regression analysis was carried out. RESULTS: The BCR-free rate in patients using the Phoenix definition was acceptable (5-year: 92.8%). The 5- year BCR-free rate using the strict definition (PSA ≥ 0.2 ng/mL) was 74.1%. Cox's proportional hazard regression analysis showed that a higher biological effective dose (BED) of ≥180 Gy2 was the only independent variable that could predict BCR (HR: 0.570, 95% C.I.: 0.327-0.994, p = 0.048). Patients with a higher BED (≥180 Gy2) had a significantly higher BCR-free rate than those with a lower BED (<180 Gy2) (5-year BCR-free rate: 80.5% vs. 67.4%). CONCLUSIONS: A higher BED ≥180 Gy2 promises a favorable BCR-free rate, even if the strict definition is adopted.

Efficacy of FDG-PET for defining gross tumor volume of head and neck cancer.

We analyzed the data for 53 patients with histologically proven primary squamous cell carcinoma of the head and neck treated with radiotherapy between February 2006 and August 2009. All patients underwent contrast-enhanced (CE)-CT and (18)F-fluorodeoxyglucose (FDG)-PET before radiation therapy planning (RTP) to define the gross tumor volume (GTV). The PET-based GTV (PET-GTV) for RTP was defined using both CE-CT images and FDG-PET images. The CE-CT tumor volume corresponding to a FDG-PET image was regarded as the PET-GTV. The CE-CT-based GTV (CT-GTV) for RTP was defined using CE-CT images alone. Additionally, CT-GTV delineation and PET-GTV delineation were performed by four radiation oncologists independently in 19 cases. All four oncologists did both methods. Of these, PET-GTV delineation was successfully performed in all 19 cases, but CT-GTV delineation was not performed in 4 cases. In the other 15 cases, the mean CT-GTV was larger than the PET-GTV in 10 cases, and the standard deviation of the CT-GTV was larger than that of the PET-GTV in 10 cases. Sensitivity of PET-GTV for identifying the primary tumor was 96%, but that of CT-GTV was 81% (P < 0.01). In patients with oropharyngeal cancer and tongue cancer, the sensitivity of CT-GTV was 63% and 71%, respectively. When both the primary lesions and the lymph nodes were evaluated for RTP, PET-GTV differed from CT-GTV in 19 cases (36%). These results suggested that FDG-PET is effective for defining GTV in RTP for squamous cell carcinoma of the head and neck, and PET-GTV evaluated by both CE-CT and FDG-PET images is preferable to CT-GTV by CE-CT alone.

Changes of tumor size and tumor contrast enhancement during radiotherapy for non-small-cell lung cancer may be suggestive of treatment response.

We evaluated sequential dynamic contrast-enhanced CT (DCE-CT) scans to assess the possibility of early prediction of treatment responses by quantifying the tumor size reduction and the change in tumor enhancement during and after a course of radiotherapy (RT). Thirty-nine patients with non-small-cell lung cancer were treated with RT for initial treatment. DCE-CT scan was performed within one week before the beginning of treatment, after 17 or 18 fractions (34 or 36 Gy), and 1 week and 1 month after the end of RT. The correlation between the relative decrease in tumor diameter and that in the attenuation value was evaluated. Nineteen patients were evaluated in this study. The median tumor size was 39.5 mm at the start of treatment, 30.8 mm at 34-36 Gy, and 16.1 mm 1 month after the end of RT. The relative decrease in tumor diameter at 34-36 Gy well correlated with that 1 month after treatment (r = 0.85, r: Pearson's correlation coefficient, p < 0.001). Relative change in the attenuation value at the rim of the tumor at 34-36 Gy did not significantly correlate with the change in tumor diameter 1 month after the completion of RT, but in the center of the tumor, the change of the attenuation value in the delayed phase correlated with the change in tumor diameter. The decrease of tumor diameter during RT may be predictive of treatment response. The relative change of tumor enhancement in the center of the tumor in the delayed phase correlated with tumor shrinkage 1 month after the completion of RT.

[Application of PET in radiation oncology].

Positron emission tomography (PET), especially F-18 fluorodeoxyglucose (FDG)-PET, has been recently used to verify the target volume in radiation treatment planning (RTP) for malignancies. The utility of FDG-PET/CT in defining gross tumor volume (GTV) has been shown in many studies, and the target delineation by a fixed threshold of the maximum standardized uptake value (40-50%) is suggested to be useful in RTP for lung cancer, head and neck cancer, etc. But, the spatial resolution, sensitivity, and specificity of PET are not always enough to define the difference between the GTV and the clinical target volume(CTV). Furthermore, FDG-PET is frequently used in the clinical staging before the treatment, and is also applied to the response evaluation after the treatment. This review focuses on the developing applications of PET in radiation oncology.