Cancer Biotherapy and Radiopharmaceuticals

The cytocidal potency of a molecule can be augmented by conjugating a radionuclide for molecular targeted radionuclide therapy (MTRT) for cancer. Radioimmunotherapy (RIT) should be incorporated into the management of patients with B-cell non-Hodgkin's lymphoma (NHL) soon after the patients have proven incurable. Better drugs, strategies, and combinations with other drugs seem certain to make RIT integral to the management of patients with NHL and likely to lead to a cure of the currently incurable NHL. These improved drugs, strategies, and combinations thereof also offer opportunities for RIT to become part of the management of solid malignancies, including epithelial cancers. Smaller radionuclide carriers, such as those used for pretargeted strategies, provide dose intensification. The potential of pretargeted RIT to improve patient outcomes is striking.

Objective: The 4.2-day half-life of ¹²⁴I favors its use for positron emission tomography (PET) of monoclonal antibodies (mAbs). However, high positron energy and β⁺ -associated cascade γ rays pose image resolution and background noise problems for ¹²⁴I. This study evaluated quantitative PET of an ¹²⁴I mAb in tumor-bearing mice. Methods: An R4 microPET™ (Siemens/CTIMI, Knoxville, TN) was used with standard energy and coincidence timing windows (350–750 keV and 6 ns, respectively), delayed random coincidence subtraction, iterative image reconstruction, and no attenuation or scatter correction. Image resolution, contrast, and response linearity were compared for ¹²⁴I and ¹⁸F, using phantoms. Nude mice bearing human colon tumors (LS-174T) were injected intravenously with a chimeric ¹²⁴I anti-CEA mAb (cT84.66) and imaged serially 1 hour to 7 days postinjection. Venous blood was sampled to validate image-derived blood curves. Mice were sacrificed after the final scan, and the biodistribution of ¹²⁴I was measured by direct tissue assay. Images were converted to units of kBq/g for each tissue of interest by comparing the final scans with the direct assays. Results: Measured resolution (FWHM) 0–16 mm from the scanner axis was 2.3–2.7 mm for ¹²⁴I versus 1.9–2.0 mm for ¹⁸F. Due to true coincidence events between annihilation photons and cascade γ rays, background was greater for ¹²⁴I than ¹⁸F, but the signal-to-background ratio was still more than 20, and ¹²⁴I image intensities varied linearly with activity concentration. Tissue-based calibration worked well (i.e., PET blood curves agreed with direct measurements within 12% at all time points), while calibration, based on a cylindrical phantom approximating the mouse body, yielded tumor quantitation that was 46%–66% low, compared with direct assay. Conclusions: Images of quantitative accuracy sufficient for biodistribution measurements can be obtained from tumor-bearing mice by using ¹²⁴I anti-CEA mAbs with standard microPET acquisition and processing techniques, provided the calibration is based on the direct assay of excised tissue samples.

The use of single-chain variable fragment (scFv) constructs has been investigated in cancer radioimmunotherapy (RIT) and radioimmunodetection, as these molecules permit rapid tumor penetration and clearance from the serum relative to whole IgG. Multimerization of scFv constructs has demonstrated improvements in functional affinity (i.e., avidity) and maximal tumor uptake. In this paper, we report the first biodistribution and pharmacokinetics studies of a noncovalent, direct-linked scFv (V_L-0-V_H) trimeric/tetrameric “multimer” of the anti-Lewis Y monoclonal antibody, hu3S193. The in vitro binding and in vivo biodistribution of the hu3S193 multimer was characterized alongside the hu3S193 F(ab′)₂ following radiolabeling with the Indium-111 (¹¹¹In) radioisotope. Immunoreactivities of the radiolabeled multimer and F(ab′)₂ were 73% and 53.2%, and binding affinities (K_a) were 1.58 × 10⁷ M⁻¹ and 4.31 × 10⁶ M ⁻¹ for the multimer and F(ab′)₂, respectively. Maximal tumor uptake in Le^y-positive MCF-7 breast cancer xenografted BALB/c nude mice was 12.6 ± 2.5 percent injected dose/per gram (%ID/g) at 6 hours postinjection for the multimer and 15.7 ± 2.1 %ID/g at 24 hours postinjection for the F(ab′)₂. However, limited in vitro stability and high renal localization of radiolabeled constructs were observed, which, despite the observed tumor targeting of the hu3S193 multimer, most likely preclude its use in RIT and imaging modalities.

Interleukin-24 (IL-24) has been shown to be a tumor-suppressor gene and the protein product found to be constitutively expressed by melanocytes, nerve cells, and some primary melanomas. The potential effect of adenovirus (AdV)-mediated IL-24 gene therapy was explored on human pancreatic carcinoma by using a pancreatic carcinoma cell line, patu8988. A recombinant adenovirus, AdVGFP/IL-24, expressing the marker, green fluorescent protein (GFP), and the tumor-suppressor gene, IL-24, was constructed. AdVGFP/IL-24 treatment of pancreatic carcinoma cells in vitro significantly induced pancreatic carcinoma cell cytotoxicity and apoptosis, compared with AdVGFP without IL-24 expresssion. In nude mice bearing patu8988 tumors, intratumoral injections of AdVGFP/IL-24 significantly inhibited pancreatic carcinoma growth. In addition, the molecular mechanism of tumor suppression was elucidated by downregulating the expression of vascular endothelial growth factor, CD34, and Bcl-2, as well as inhibiting tumor angiogenesis. Therefore, AdVGFP/IL-24 has the potential to serve as a novel tool for pancreatic carcinoma gene therapy.

Affibody molecules are a novel class of targeting proteins, demonstrating promising results in the molecular imaging of tumor markers. The aim of this study was to investigate the cellular processing of Affibody molecules bound to human epidermal growth-factor-receptor type 2 (HER2). Cellular processing of the synthetic Affibody molecule, DOTA-Z_{HER2:342-pep2} (K_D = 65 _pM) labeled with indium-111, was studied both during continuous and interrupted incubation with HER2-expressing cell lines (SKOV-3, SKBR-3, and BT474). The internalized and membrane bound fractions of Affibody molecule were discriminated by treatment with 4 M of urea solution in 0.2 M of glycine buffer (pH 2.0). Incubation media collected after an interrupted incubation was analyzed for the presence of radiocatabolites. Continuous incubation of tumor cells with ¹¹¹In-DOTA-Z_{HER2:342-pep2} led to the saturation of HER2 and slow internalization. Sixty (60)- to 80% of the radioactivity remained cell associated 24 hours after interrupted incubation. The rate of Affibody molecule internalization was the same after interrupted incubation, as in the continuous incubation experiments. Internalization of ¹¹¹ In-DOTA-Z_{HER2:342-pep2} was relatively slow. A high level of cellular retention of the tracer was provided by strong binding to cell-surface receptors. These data suggest that good tumor targeting with anti-HER Affibody molecules may be obtained by using short-lived, nonresidualizing labels.

We reviewed data from 160 consecutive patients (89 M/71 F; 53.5 [range, 9–88] years) who had under-gone lymphoscintigraphy and sentinel lymph node biopsy (SNB) in our hospital for histologically proven cutaneous malignant melanoma (CMM) (located on the upper limb: 33; lower limb: 57; trunk: 44; and head and neck: 26 patients), with a Breslow index > 1 mm and without clinical or radiologic evidence of metastatic spread. Colloidal ^99mTc-rhenium sulfide (36–76 MBq) was injected intradermally in the four quadrants around the tumorectomy scar, followed by dynamic acquisition and static imaging. SN(s) were identified in 157 patients (overall identification rate, 98%). Fast (< 20 minutes), intermediate (20–30 minutes), or slow (> 30 minutes) lymphatic drainage was observed, respectively, in 122 (78%), 24 (15%), or 11 (7%) cases. Overall malignancy rate was 15%, respectively found in 19 (16%), 2 (8%), and 2 (18 %) patients with fast, intermediate, or slow drainage. No statistical difference between SN-positivity rates of patients with fast (19/122 = 16%) versus intermediate or slow drainage (4/35 = 11.4%) was observed (p = 0.69). Therefore, lymphoscintigraphic SN appearance time in CMM patients is unable both to predict SN metastasis and spare them from undergoing SN excision.

Promyelocytic leukemia zinc finger (PLZF) is a transcriptional repressor and tumor suppressor inhibiting melanoma cell growth in vitro and in vivo in animal models. In this study, we analyzed the impact of in vivo primary tumor gene expression of PLZF on the long-term survival of malignant melanoma patients. PLZF expression was assessed by using DNA microarray and real-time polymerase chain reaction analysis of 41 primary malignant melanomas from patients with a defined histology and a close to 20-year clinical follow-up, of 29 melanoma metastases, and of 6 different melanoma cell lines. Kaplan-Meier survival analyses, log-rank statistics and Cox regression analysis were employed to identify the impact of PLZF expression on long-term survival. We detected PLZF expression in 92% of primary melanoma tumors in vivo but not in melanoma cell lines in vitro. By univariate analysis, we identified: (1) PLZF mRNA expression ≤ 10,000 mRNA copies/μg total tumor RNA, (2) Breslow tumor thickness >4 mm, and (3) American Joint Committee on Cancer stages IIC, IIIB, IIIC, and IV as statistically significant pretreatment risk factors. We defined a continuous prognostic index (i.e., risk score) for primary melanoma patients based on the regression coefficient of PLZF mRNA expression. Applying a cutpoint to the prognostic index at − 1.65, patients were assigned to one of two risk groups: low-risk patients (n = 28) with a median overall survival of 79 months (5-year survival of 61%) and high-risk patients (n = 13) with a median overall survival of 32 months (5-year survival of 23%) (p < 0.05). This is the first time that PLZF mRNA expression has been linked to a prognostic model for primary malignant melanoma patients to derive prognostic groups for clinical purposes (e.g., improved melanoma immunotherapies).

Long-term survival, which extends beyond 5 years, is a desired outcome for colorectal cancer patients. In the present study, we retrospectively compared the 10-year overall survival between the control group and the polysaccharide kureha (PSK) group and analyzed the factors influencing the prognosis. The control group was treated exclusively with oral fluoropyrimidines, whereas the PSK group was treated with fluoropyrimidines, given in conjunction with PSK for 24 months. The 10-year survival rates for the PSK group (81.9%) were significantly superior to those of the control group (50.6%). In Dukes' C cases, the 10-year overall survival rates for the PSK group were also significantly higher than those of the control group. In cases with lymphatic invasion graded ly2–ly3 or venous invasion graded v2–v3, the 10-year overall survival rates were 80.6% in the PSK group, which were significantly superior, compared to 25.9% in the control group. Analysis by Cox's proportional hazard model showed a significant difference between the control and PSK groups. These results indicate that postoperative adjuvant immunochemotherapy with PSK greatly improves prognosis at 10 years. On the basis of these results, we recommend postoperative adjuvant immunochemotherapy combined with PSK for patients with Dukes' C and in cases with ly2–ly3 or v2–v3 invasion.

Breast carcinomas show a trend toward bone metastasis that is prevalent worldwide. Celecoxib (CX) and minocycline hydrochloride (MH) have both been widely used in treating breast cancer; however, their combined effects on the osseous metastasis of breast cancer have not yet been studied. In the present study, breast cancer cells were injected into the back of the femoral bone of nude mice, and CX and MH were intraperitoneally administered every other day at doses of 30 and 40 mg/kg/day, respectively, for 30 days. Tumor weights and volumes were significantly lower and the tumor inhibition rate was significantly higher in the CX + MH group than those of the control and CX or MH alone groups (p < 0.05). The cell density in the tumor tissue was significantly decreased and apoptotic and necrotic cell death was significantly increased in the CX + MH group, as compared with those of the control and CX or MH alone groups. Microvessel density and expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 in the tumor tissues of the CX + MH group were significantly lower than those of the CX, MH, and control groups. The serum alkaline phosphatase level of the CX + MH group was significantly lower than those of the other groups (p < 0.01). These results suggest that a combined use of CX and MH has better inhibitory effects on the osseous metastasis of breast cancer, as compared to CX or MH alone. They exerted their combined effects by increasing tumor-cell death and decreasing the tumor expression of MMP-9 and VEGF systems.

Objective: The fermented wheat germ extract (FWGE) nutraceutical (Avemar™), manufactured under “good manufacturing practice” conditions and, fulfilling the self-affirmed “generally recognized as safe” status in the United States, has been approved as a “dietary food for special medical purposes for cancer patients” in Europe. In this paper, we report the adjuvant use of this nutraceutical in the treatment of high-risk skin melanoma patients. Methods: In a randomized, pilot, phase II clinical trial, the efficacy of dacarbazine (DTIC)-based adjuvant chemotherapy on survival parameters of melanoma patients was compared to that of the same treatment supplemented with a 1-year long administration of FWGE. Results: At the end of an additional 7-year-long follow-up period, log-rank analyses (Kaplan-Meier estimates) showed significant differences in both progression-free (PFS) and overall survival (OS) in favor of the FWGE group. Mean PFS: 55.8 months (FWGE group) versus 29.9 months (control group), p = 0.0137. Mean OS: 66.2 months (FWGE group) versus 44.7 months (control group), p = 0.0298. Conclusions: The inclusion of Avemar into the adjuvant protocols of high-risk skin melanoma patients is highly recommended.

Imaging can take advantage of developments in “omics” approaches and go from routine individual biomarkers to multiple-scale biomarker profiles. Imaging structural, functional, metabolic, cellular, and molecular changes will be made possible by multimodality hybrid techniques, such as positron emission tomography–magnetic resonance imaging. Imaging should predict treatment response, look at stratification for specific treatment modalities, and look at the “omic” characterization of an individual patient or a specific tumor. This should lead to the development of “personalized” medicine. In cancer radiotherapy, patient responses should be accurately predicted. In specific cases, proton and hadrontherapy will be further enhanced by the irradiation dose delivered to the tumors. For disseminated or metastatic disease, targeted radionuclide therapy is an effective addition to the arsenal against cancer. The clinical efficacy of radiolabeled antibodies has been clearly demonstrated in lymphoma as well as that of radiolabeled peptides derived from somatostatin in the treatment of neuroendocrine tumors. Preliminary studies now show interesting results in solid tumors, too. Even if the number of objective clinical responses based on tumor shrinkage is small, targeted radionuclide therapy increases progression-free survival or overall survival in some specific cases where tumor burden is small. Avenues for further improvement are multiple and include combination with other therapeutic modalities, development of new approaches (e.g., small molecules, pretargeting, and antibody alternatives). Using alpha-emitting radionuclides is another possibility for specific diseases, such as leukemias, multiple myeloma, or brain tumor remnants.