Remote delivery of rAAV-GFP to the rat brainstem through the recurrent laryngeal nerve.

OBJECTIVE: To demonstrate that a recombinant adeno-associated viral vector (rAAV) carrying the gene for green fluorescent protein (GFP) could be delivered to the rat brainstem by remote injection into the recurrent laryngeal nerve. STUDY DESIGN/METHODS: rAAV-GFP is a serotype 2 adeno-associated vector containing the cDNA of GFP and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under the control of the CAG promoter (obtained from Matthew During, PhD, Thomas Jefferson Medical College). Five microliters or 10 microL of 1.4 x 109 particles/microL of rAAV-GFP were injected into the right recurrent laryngeal nerve of adult Sprague-Dawley rats. Rats were killed and perfused at 3 (n = 3) and 11 weeks (n = 3). Brainstems were removed and cryosectioned. Fluorescent in-situ hybridization (FISH) was performed on cryosections from animals killed at 3 weeks using a cDNA probe for woodchuck polyribosomal enzyme within the rAAV vector. In a third group (n = 2), Fluoro-Gold (Fluorochrome, Inc., Denver, CO) was injected into the right thyroarytenoid muscle for comparison of neuronal uptake distribution. These rats were killed and perfused at 3 weeks. RESULTS: The presence of GFP was noted in neurons throughout the medulla of all rat brainstems after unilateral rAAV-GFP injection at both 3 and 11 weeks. In contrast to the Fluoro-Gold, GFP was noted bilaterally and outside of the nucleus ambiguus. FISH confirmed the presence of virus within neurons expressing GFP at 3 weeks. CONCLUSIONS: Remote delivery of rAAV-GFP to the rat brainstem is possible through injection into the recurrent laryngeal nerve. This has important therapeutic implications for the future treatment of recurrent laryngeal nerve injury and neurodegenerative diseases.

Initial experience with high-dose radioimmunotherapy of metastatic medullary thyroid cancer using 131I-MN-14 F(ab)2 anti-carcinoembryonic antigen MAb and AHSCR.

UNLABELLED: This phase I study was initiated to determine the toxicity and therapeutic potential of high-dose 131I-MN-14 F(ab)2 anti-carcinoembryonic antigen monoclonal antibody (MAb) combined with autologous hematopoietic stem cell rescue (AHSCR) in patients with rapidly progressing metastatic medullary thyroid cancer. METHODS: Twelve patients were entered into the study. Dose escalation was based on prescribed radiation doses to critical organs (i.e., kidney, lung, and liver). Starting doses were 900 cGy to the kidney and no more than 1200 cGy to the lung and liver, with dose increments of 300 cGy until the maximum tolerable dose is determined. Tumor targeting was assessed by external scintigraphy, toxicity was assessed according to the common toxicity criteria of the National Cancer Institute, and therapy responses were assessed by CT, serum carcinoembryonic antigen, and calcitonin. RESULTS: One patient received 9.95 GBq 131I-MN-14 F(ab)2, for an initial dose of 656 cGy to critical organs, 8 received 900 cGy (8.69-17.98 GBq), and 3 received 1200 cGy (15.17-17.69 GBq). The MAb scans of all patients showed positive findings. Autologous hematopoietic stem cells were given to all patients 1-2 wk after therapy, when the total body radiation exposure was less than 5.2 x 10(-7) C/kg/h. Dose-limiting toxicity, defined as grade 3 or 4 nonhematologic toxicity, was not seen in the patient who received the 656-cGy dose, and only 1 of the 8 patients treated at the 900-cGy dose level had grade 3 toxicity, which was gastrointestinal and reversible. No dose-limiting toxicity was seen in the 3 patients treated at the 1200-cGy dose level. Except for the instance of grade 3 gastrointestinal toxicity, nonhematologic toxicity was relatively mild, with only grade 1 or 2 toxicity observed in 9 patients. No renal toxicity was seen. Of the 12 patients, 1 had partial remission for 1 y, another had a minor response for 3 mo, and 10 had stabilization of disease lasting between 1 and 16 months. CONCLUSION: The results show the safety of administering high myeloablative doses of 131I-MN-14 F(ab)2 with AHSCR in patients with metastatic medullary thyroid cancer. The antitumor responses in patients with aggressive, rapidly progressing disease are encouraging and warrant further research to optimize the effectiveness of this new treatment.

Importance of timing of radioimmunotherapy after granulocyte colony-stimulating factor administration for peripheral blood stem cell harvest.

Nine radioimmunotherapy (RAIT)-naive patients with medullary thyroid cancer received high doses of 131I-MN-14 F(ab)2 anti-carcinoembryonic antigen monoclonal antibody (232-486 mCi), five in combination with bone marrow harvest, without prior granulocyte colony stimulating factor (G-CSF) injections (group 1) and the other four using peripheral blood stem cell harvest (PBSCH) preceded by G-CSF administration of 10 microg/kg per day for 5 days for stem cell mobilization, 6-8 days before RAIT (group 2). The amounts of radioactivity (mCi) given in both groups were similar (312 +/- 93 versus 424 +/- 65; P = NS). The percent platelet loss at nadir, duration of grade 4 thrombocytopenia, and time to complete recovery (TTCR, measured from the day of treatment), were 83 +/- 17%, 2.5 +/- 0.7 days, and 45 +/- 8 days in group 1, respectively, compared with 88 +/- 6%, 3.0 +/- 2.6 days, and 50 +/- 24 days in group 2 (P = NS), respectively. In contrast, the percent WBC loss at nadir, duration of grade 4 leukopenia, and TTCR of WBCs were 72 +/- 12%, 0.0 +/- 0.0 day, and 42 +/- 12 days in group 1, respectively, compared with 93 +/- 3%, 8.0 +/- 3.6 days, and 263 +/- 136 days in group 2, respectively (P < 0.02, 0.03, and 0.05 for differences of percent loss, duration of nadir, and TTCR, respectively). The difference in WBC toxicity after RAIT with bone marrow harvest and PBSCH is thought to be due to the administration of G-CSF for stem cell mobilization within 1 week before RAIT, which may sensitize the "endogenous" granulocyte precursors to subsequent RAIT. Preclinical data of RAIT in mice showed that the time of G-CSF administration before RAIT is critical: increased WBC toxicity was seen in mice given RAIT 3 or 7 days after a 5-day course of G-CSF (81 and 57% WBC loss, respectively) compared with those given no G-CSF or G-CSF 10 or 14 days before RAIT (45-50%) WBC loss). In conclusion, our data indicate that the timing of RAIT after the administration of G-CSF for PBSCH may influence WBC toxicity and recovery after this treatment and may have important implications on the design of high-dose RAIT trials combined with PBSCH.

Peripheral clear cell cholangiocarcinoma: a rare histologic variant.

We present the case of a 50-year-old diabetic male who underwent open cholecystectomy for acute gangrenous cholecystitis. At the time of exploration, a 1.5-cm mass was found peripherally in the right lobe of his liver, and an incisional biopsy was performed. Microscopic examination revealed a distinct overgrowth of clear cells in an acinar pattern, with tumor cells emerging directly from bile ducts. The tumor cells were periodic acid-Schiff reactive and diastase resistant, indicating the presence of mucin. No bile canaliculi were demonstrated by immunostaining with carcinoembryonic antigen. CT scans of the chest and abdomen were otherwise normal. Based on these microscopic, immunohistochemical, and clinical data, a diagnosis of clear cell cholangiocarcinoma was established. The patient later underwent reexploration and generous hepatic wedge resection. He did well postoperatively and is free of disease after 12 months.

Phase I/II trial of (131)I-MN-14F(ab)2 anti-carcinoembryonic antigen monoclonal antibody in the treatment of patients with metastatic medullary thyroid carcinoma.

BACKGROUND: Monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) have been recognized as targeting agents for medullary thyroid carcinoma (MTC). This Phase I/II study was initiated to determine the safety, maximum tolerated dose (MTD), and therapeutic potential of (131)I-MN-14 F(ab)2 anti-CEA MAb for patients with metastatic MTC. METHODS: Fifteen patients were enrolled in this study. Dose escalation was based on estimates of radiation dose to the bone marrow, and the radioactive dose given was determined by a pretherapy diagnostic study in which 8 mCi (0.6-20 mg) of (131)I-MN-14 F(ab)2 was administered 1 week prior to therapy. RESULTS: Three patients received an initial dose of 140 centigray (cGy) to bone marrow, 11 received 180 cGy, and 1 received 220 cGy. Myelosuppression was the only significant treatment-related dose-limiting toxicity (DLT), and the MTD appeared to be 180 cGy to the bone marrow. Human antimouse antibodies (HAMA) developed in 8 patients 2-6 weeks after therapy. Seven patients had a median of 55% reduction of tumor markers. One patient showed a dramatic improvement in the mass effect on the airways caused by 3 tumor lesions in the neck, with a 45% reduction of overall tumor burden. The disease has continued to be radiologically stable in 11 of 12 assessable patients for periods ranging from 3+ to 26+ months. CONCLUSIONS: Therapy with (131)I-MN-14 F(ab)2 is well tolerated and shows evidence of biochemical and radiologic antitumor activity. HAMA development suggests that humanized MAbs will be required in trials with repeated dose schedules. Further dose escalation, alone or in combination with other therapy modalities, is indicated for future trials, preferably with humanized anti-CEA MAbs.

99Tcm-LL1: a potential new bone marrow imaging agent.

LL1, a monoclonal antibody (MAb) to HLA Class-II-like antigen (li determinant) on the surface of B-lymphocytes, monocytes and histiocytes, was evaluated as an agent for bone marrow imaging. Six patients with diverse diseases (non-Hodgkin's lymphoma, n = 2; multiple myeloma, n = 1; polycythaemia vera, n = 1; lung cancer, n = 1; breast cancer, n = 1) were given low protein doses (< 1 mg) of 99Tcm (30 mCi) labelled Fab' of LL1. 99Tcm-sulphur colloid (SC) imaging was performed in three patients for comparison. Both planar and single photon emission tomographic images were acquired using Sopha gamma cameras. As early as 2 h post-MAb injection, excellent bone marrow images were achieved in all patients, demonstrating both normal or hyperproliferative marrow, as well as 'cold' bone marrow abnormalities such as radiation defects or cancer metastases. Similar to SC, relatively high uptake of LL1 was found in the liver and spleen. However, the bone marrow-to-liver and -spleen uptake ratios were approximately 19-fold higher (0.75 vs 0.04) and 6-fold higher (1.23 vs 0.22), respectively, with LL1 than with SC. The higher bone marrow uptake allowed clearly superior visualization of the thoracic spine when compared to SC. The mean T1/2 of blood and whole-body clearance were 0.4 and 66 h, respectively. The highest radiation absorbed doses (in cGy mCi-1) were observed in the spleen (0.47 +/- 0.24), kidneys (0.25 +/- 0.09) and liver (0.14 +/- 0.04). The bone marrow dose was only 0.05 +/- 0.02 cGy mCi-1. These results indicate that bone marrow imaging with 99Tcm-LL1 is feasible, and that LL1 may be a suitable alternative to SC because of better visualization of the lower thoracic spine. Potential applications include the improved detection of bone marrow metastases of solid tumours and the assessment of haematological disorders.

Regression of advanced refractory ovarian cancer treated with iodine-131-labeled anti-CEA monoclonal antibody.

Advanced chemotherapy-resistant ovarian cancer has a poor prognosis, thus requiring new therapeutic modalities. A complete clinical remission, using two cycles of 131I-labeled murine MN-14 anti-CEA monoclonal antibody (MAb), given intravenously, is reported in a patient with advanced ovarian cancer refractory to paclitaxel (Taxol) therapy. The patient first received radioimmunotherapy with approximately 74 mCi 131I-MN-14 IgG, followed 4 mo later by a similar dose of radiolabeled MAb. A partial remission was seen by CT 1 mo after the first radioimmunotherapy, and a further objective response was documented after the second radioimmunotherapy. CT scans performed 6 and 11 mo after the second radioimmunotherapy showed stable and minimal residual changes. However, a whole-body PET scan with [18F]fluorodeoxyglucose (FDG-PET) was negative in these regions. The CA-125 also decreased to only 13 U/ml, compared to the baseline value of 7700 U/ml. Based on CT, FDG-PET, serum CA-125 and physical exam, the patient was in complete clinical remission for 8 mo when the CA-125 levels rose. CT also showed a new suspicious lesion, presumably a peritoneal implant. No toxicity was seen after the first injection, and only Grade 1 thrombocytopenia and Grade 2 leukopenia developed after the second injection, both reversing within 6 wk. This is a report of a complete clinical remission with radiolabeled anti-CEA antibodies in a patient with chemotherapy-refractive metastatic ovarian cancer.

Prospects of radioimmunotherapy in epithelial ovarian cancer: results with iodine-131-labeled murine and humanized MN-14 anti-carcinoembryonic antigen monoclonal antibodies.

OBJECTIVES: Epithelial ovarian cancer (EOC) is known to produce carcinoembryonic antigen (CEA), and the plasma CEA level is frequently elevated in patients with advanced stage and bulk of tumor. This study reports the results of a phase I therapy trial with intravenously administered 131I-MN-14 anti-CEA monoclonal antibody (MAb) in patients with EOC. METHODS: Fourteen patients with advanced refractory EOC were given escalating intravenous doses (two received 30 mCi/m2, six 40 mCi/m2, and six 50 mCi/m2) of 131I-MN-14 IgG. All patients received a diagnostic study with 8 mCi (0.6 mg) of 131I-MN-14 IgG 1 week prior to their therapy infusion. Tumor targeting was assessed by external scintigraphy, toxicity according to the Radiation Therapy Oncology Group criteria, and therapy responses by CT and serum CA-125. RESULTS: The MAb scan was positive in all 14 treated patients. Myelosuppression was the only observed treatment-related toxicity. Dose-limiting toxicity, defined as grade 4 leukopenia or thrombocytopenia of any duration, or grade 3 leukopenia or thrombocytopenia of > 2 weeks, was not seen at the 30 or 40 mCi/m2 dose levels. However, 2 of 6 patients treated at 50 mCi/m2 had a grade 4 thrombocytopenia or a grade 3 thrombocytopenia lasting 18 days. Of the 14 patients, 1 with diffuse peritoneal implants of < or = 2 cm had a complete clinical remission by CT and CA-125 for 8 months, following an initial partial remission for 10 months, both at the 40 mCi/m2 dose level. Another patient had a mixed response for 1 month. CONCLUSION: MN-14 anti-CEA MAb is a suitable agent for tumor targeting and may have a therapeutic potential in patients with chemotherapy-refractive EOC, especially those with minimal disease.

Radioimmunotherapy of medullary thyroid cancer with iodine-131-labeled anti-CEA antibodies.

UNLABELLED: This study evaluates the pharmacokinetics, dosimetry, toxicity and therapeutic potential of radiolabeled NP-4 and MN-14 anti-CEA antibodies in medullary thyroid cancer (MTC). METHODS: Eighteen patients with advanced MTC entered exploratory clinical studies with therapeutic doses of 131I-labeled NP-4 and MN-14 murine monoclonal antibodies (MAbs) reactive with carcinoembryonic antigen (CEA). Doses administered ranged from 46 mCi for 131I-MN-14 lgG to 195 mCi for 131I-MN-14 F(ab)2 in patients negative for human anti-mouse antibodies (HAMA). RESULTS: The radioconjugate blood half-life (T1/2) for the whole lgG was 42.5+/-5.0 hr compared to 18.8+/- 4.1 hr for the bivalent fragments. Tumor doses of 17.5+/-11.0 and 11.4+/-6.3 cGy/mCi were estimated for 131I-MN-14 lgG and F(ab)2, respectively. Tumor/red marrow dose ratios exceeded 3:1 for most lesions. Red marrow doses of up to 350 cGy generally could be delivered with < grade 4 toxicity. Seven of 14 evaluable patients showed evidence of anti-tumor effects lasting up to 26 months, based on physical exam, tumor markers or computed tomography. CONCLUSION: This study demonstrates that anti-CEA MAbs may be suitable for radioimmunotherapy of metastatic or recurrent MTC.

Improved detection of medullary thyroid cancer with radiolabeled antibodies to carcinoembryonic antigen.

PURPOSE: This investigation was undertaken to assess the targeting of established and occult medullary thyroid cancer (MTC) with radiolabeled monoclonal antibodies (MAbs) reactive with carcinoembryonic antigen (CEA). PATIENTS AND METHODS: Twenty-six assessable patients with known (n = 17) or occult (n = 9) MTC were studied with radiolabeled anti-CEA MAbs. Scintigraphic images were collected to determine targeting of tumor lesions. RESULTS: The targeting results of technetium 99m (99mTc)-,iodine 123 (123I)-, and iodine 131 (131I)-labeled anti-CEA antibodies (all directed against the same epitope of CEA) indicated that all these reagents were capable of detecting established and occult MTC. The sensitivity for detection of known sites of disease ranged from 76% to 100% for the various anti-CEA MAbs used, when compared with computed tomography (CT), magnetic resonance imaging (MRI), bone scan, or other imaging modalities. Moreover, the antibody scan was positive in seven of nine patients with occult disease (patients with negative conventional imaging studies, but who had elevated calcitonin and/or CEA levels). Three of seven patients underwent surgery and the disease was confirmed by histopathology in all three. CONCLUSION: Anti-CEA MAbs are excellent agents for imaging recurrent, residual, or metastatic MTC. The high lesion sensitivity in patients with known lesions, combined with the ability to detect disease, may make these agents ideal for staging patients, monitoring disease pretherapy or posttherapy, and especially for evaluating patients with recurrent or persistent hypercalcitonemia or CEA elevations after primary surgery. Analogous to radioiodine in the evaluation of patients with differentiated thyroid cancer, radiolabeled anti-CEA MAbs may achieve a similar role in diagnosing and monitoring patients with MTC.

Treatment of non-Hodgkin's lymphoma with radiolabeled murine, chimeric, or humanized LL2, an anti-CD22 monoclonal antibody.

LL2 is a murine IgG2a anti-CD22 monoclonal antibody found to react with virtually all non-Hodgkin's lymphomas (NHLs). Twenty-one patients with chemotherapy-resistant NHL received nonmyeloablative doses of 131I-labeled LL2 IgG and F(ab')2 ranging from 15 to 343 mCi given in cycles of 15-50 mCi, for up to seven treatment cycles. The cumulative protein dose ranged from 1.1 mg IgG to 157 mg F(ab')2. Seventeen patients were assessable for treatment response, and antitumor effects were seen in five (one complete remission, two partial remissions, and two minor or mixed responses). In addition, one complete response was seen in a patient who received only "diagnostic" doses of 131I-LL2 IgG. Thus, a total of six patients had responses according to the defined response criteria. Three additional patients have been treated with potentially myeloablative doses of 131I-LL2 IgG at a starting dose level of 90 mCi/m2 (100 mg). Two patients were evaluable, and both had partial remissions lasting 8 and 3 months, respectively. Chimeric and complementarity-determining region-grafted LL2 have been developed. Initial clinical studies have shown that these agents have targeting properties similar to the murine LL2 and, therefore, may be suitable alternatives to murine LL2 in the treatment of NHL. LL2 is a promising agent for the treatment of lymphoma, particularly when the maximum tolerated dose is given either with or without autologous bone marrow transplantation.

Targeting and initial radioimmunotherapy of medullary thyroid carcinoma with 131I-labeled monoclonal antibodies to carcinoembryonic antigen.

The targeting potential of 131I-labeled NP-4 and MN-14 anti-CEA (carcinoembryonic antigen) monoclonal antibodies (MAbs) was assessed in 19 patients with metastatic medullary thyroid cancer (MTC). Seventeen of these patients also entered pilot radioimmunotherapy studies with nonmyeloablative doses of 131I-anti-CEA MAbs. Tumor targeting was possible in all 19 patients, with an overall lesion sensitivity of 91%. Tumor dosimetry with 131I-MN-14 IgG or F(ab)2 was very favorable, with tumor doses of 14.3 +/- 8.3 cGy/mCi and tumor:red marrow dose ratios exceeding 3:1 for most lesions. Limited antitumor effects lasting up to 26+ months, based on physical exam, tumor markers, computed tomography, or a followup MAb scan, were seen in 5 of 11 assessable patients given relatively low doses of 131I-labeled anti-CEA MAbs. We conclude that anti-CEA MAbs are excellent agents for targeting metastatic MTC. The high tumor uptake of the 131I-anti-CEA antibodies and evidence of tumor response in some patients suggest that radioimmunotherapy with radioiodinated anti-CEA MAbs may be an effective treatment for MTC, particularly when the maximum tolerated dose is given alone or in combination with autologous red marrow or peripheral stem cell support.

Home-based intravenous therapy for oncology patients.

It is desirable to keep cancer patients at home whenever possible to maximize their physical comfort and mental well-being. However, physicians should be aware of all the serious limitations in terms of patient selection and avoidance of the abuses of overutilization of the system.

Further characterization of the circulating cell in chronic lymphocytic leukemia.

Peripheral lymphocytes from normal individuals and from patients with chronic lymphocytic leukemia (CLL) were cultured in vitro for 1-7 days. The growth response to phytohemagglutinin (PHA) was quantitated by the incorporation of tritiated uridine into RNA nucleotide during a 2-hr pulse with the radioisotope. While the maximum response in PHA-stimulated normal cultures appeared at 2-3 days, CLL cultures required 5-7 days to develop their maximal response, which was 50%-60% of the normal magnitude. Dilution of the number of normally reactive lymphocytes by culturing them with totally unreactive, mitomycin-treated cells produced a normal 72-hr maximal response, no matter what proportion of unreactive cells was included in the PHA-stimulated cultures. In addition, the response of peripheral lymphocytes from patients with myeloblastic leukemia, where large numbers of unreactive myeloblasts diluted the normal small lymphocytes, a depressed reaction occurred at the anticipated 2-3 days. Nylon fiber-adherent lymphocytes consisting of 85% immunoglobulin (Ig)-bearing cells responded minimally to PHA, but showed no evidence of a delay. When isolated from CLL patients, both fiber-adherent cells (Ig-bearing) as well as non-fiber-adherent (sheep erythrocyterosetting) cells responded to PHA in a delayed fashion. Similarly, a case of CLL, in which 93.5% of the circulating lymphocytes bore sheep red blood cell receptors, showed its peak response to PHA at 7 days. Therefore, using surface marker criteria considered characteristic of normal T cells and B cells, the delayed response to PHA on the part of CLL lymphocytes was independent of thymic or nonthymic origin.