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Cancer Therapy: Clinical |
Authors' Affiliations: 1 Institut de Recherche en Cancérologie de Montpellier (IRCM); 2 Institut National de la Sante et de la Recherche Medicale U896; 3 Université Montpellier1; 4 CRLC Val d'Aurelle-Paul Lamarque, Montpellier, France; 5 Schering-CIS Biointernational, Gif-sur-Yvette, France; 6 Département de Biochimie, Université de Lausanne, Epalinges, Switzerland; and 7 Department of Radiation Oncology, Maisonneuve-Rosemont Hospital, Montreal, Canada
Requests for reprints: André Pèlegrin, Institut de Recherche en Cancérologie de Montpellier, CRLC Val d'Aurelle-Paul Lamarque, 34298 Montpellier Cedex 5, France. Phone: 33-4-67-61-30-32; Fax: 33-4-67-61-37-87; E-mail: Andre.Pelegrin{at}valdorel.fnclcc.fr.
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Abstract |
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Patients and Methods: Twenty-two patients planned for surgery of one to four LM received a preoperative diagnostic dose of a 131I-F(ab')2–labeled anti-carcinoembryonic antigen monoclonal antibody F6 (8-10 mCi/5 mg). 131I-F(ab')2 uptake was analyzed using direct radioactivity counting, and tumor-to-normal liver ratios were recorded. Ten patients with tumor-to-normal liver ratios of >5 and three others were treated with a therapeutic injection [180-200 mCi 131I/50 mg F(ab')2] 30 to 64 days after surgery.
Results: Median 131I-F(ab')2 immunoreactivity in patient serum remained at 91% of initial values for up to 96 hours after injection. The main and dose-limiting-toxicity was hematologic, with 92% and 85% grades 3 to 4 neutropenia and thrombocytopenia, respectively. Complete spontaneous recovery occurred in all patients. No human anti-mouse antibody response was observed after the diagnosis dose; however, 10 of the 13 treated patients developed human anti-mouse antibody 
3 months later. Two treated patients presented extrahepatic metastases at the time of RIT (one bone and one abdominal node) and two relapsed within 3 months of RIT (one in the lung and the other in the liver). Two patients are still alive, and one of these is disease-free at 93 months after resection. At a median follow-up of 127 months, the median disease-free survival is 12 months and the median overall survival is 50 months.
Conclusion: RIT is feasible in an adjuvant setting after complete resection of LM from colorectal cancer and should be considered for future trials, possibly in combination with chemotherapy, because of the generally poor prognosis of these patients.
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Patients and Methods |
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Clinical grade F6 F(ab')2 was obtained from Schering-CIS Biointernational. It was purified from culture supernatant by protein A chromatography. F(ab')2 was obtained by pepsin digestion and purified by ion exchange chromatography. The cell banks and the purified mAb and F(ab')2 tested negative for viruses and Mycoplasma contamination.
Radioiodination of F6 F(ab')2 fragments. Radiolabeling was done by Schering-CIS Biointernational. The diagnostic dose consisted of 5 mg F6 F(ab')2 labeled with 8 to 10 mCi of 131I (1 mCi = 37 MBq) in sterile PBS containing 1.6% human serum albumin. The therapeutic dose consisted of 50 mg F6 F(ab')2 labeled with 200 mCi of 131I in the same buffer. Each therapeutic dose was stored in two vials of 9 mL to reduce radiolysis of the antibody. The radiochemical purity, as determined by radio-TLC, was >90%. The endotoxin (Limulus assay) was lower than 175 endotoxin units/dose. The immunoreactivity of 131I-F(ab')2 was determined as described by Buchegger et al. (19) using a direct binding assay to CEA coupled to CNBr-Sepharose (Pharmacia) and ranged from 44% to 85% (median, 71%).
Patient selection. Patients were eligible if complete resection (R0) of LM from CRC was deemed possible after assessment by an experienced committee of surgeons, medical and radiation oncologists, and nuclear physicians. Other eligibility criteria included absence of chemotherapy in the 6 wk before the injection, a serum creatinine concentration of <120 µmol/mL, bilirubinemia of <30 µmol/mL, prothrombine time at >60%, a platelet count of >100,000/mm3, and a granulocyte count of >2,000/mm3. Patients with severe cardiac, pulmonary, or infectious disease, clinical evidence of central nervous system tumor involvement, history of prior administration of mouse-derived antibodies, protein constructs or human anti-mouse antibody (HAMA) serum reactivity were excluded, as were those with a performance status of >2 according to the CTC v. 2.0 classification (20). Patients with prior pelvic radiation therapy could be included. Postoperative chemotherapy was not permitted in the absence of recurrence. The clinical protocol and consent form were approved by our ethical committee, and written informed consent was obtained from all patients before study entry.
Dose and administration of 131I F6 F(ab')2. Three days before antibody administration and continuing for 2 wk, patients received a daily iodine solution (150 mg/d) to prevent uptake of the 131I by the thyroid gland. A first diagnostic dose (8-10 mCi 131I/5 mg) was given as an i.v. bolus 5 to 7 d before the surgery. Patients were eligible to receive a second, therapeutic injection (200 mCi 131I/50 mg) 4 to 8 wk after surgery if the tumor-to-normal liver (TTL) uptake ratio of 131I F6 F(ab')2 was superior to 5, based on analysis of tissues obtained during surgery. This ratio corresponds to the lower limit proposed by Welt et al. for the screening of patients potentially eligible for RIT (21). The therapeutic dose was given in the form of an i.v. 250 mL isotonic sodium chloride solution in a 60-min perfusion. The radionuclide doses were not adjusted for body weight or surface area because we believe that individualized calculation of doses should be based on antibody biodistribution and pharmacokinetics rather than body weight or surface area. In the future, we will adjust doses based on data from this trial. Patients were hospitalized in a protected nuclear medicine ward allowing for safe radioactive urine collection and adequate radiation-isolation. Strict radioprotection regulations were reviewed with nursing and auxiliary staff. Visits were forbidden during the entire week. Patients were discharged five to seven days after treatment, depending on the results of dose-rate measurements. They were given recommendations on safety precautions to follow for the next week with the aim of reducing radiation exposure to family members.
Scintigraphic and metabolic monitoring. The day before surgery (4-6 d after the 131I F6 F(ab')2 diagnostic dose), 0.08 mCi/kg 99mTc-sodium phytate (Phytacis, CisBio International) was given i.v. to image normal liver and a whole body scan and single-photon emission computed tomography (SPECT) were carried out with a Sopha Medical NXT 
camera and a high-energy, parallel-hole, general purpose collimator. Scintigraphic data were collected through a double isotope acquisition, each with a 20% symmetrical window around 140 and 364 keV, respectively. SPECT was implemented with a 360° elliptic rotation, 64 projections, and 30 s/projection, whereas slice reconstruction was completed by a Weiner-filtered backprojection technique.
After the therapeutic dose, complete blood cell counts were done at least twice weekly until week 8 or until the neutrophil and platelet counts returned to normal. Blood chemistry tests and a coagulation profile were done before discharge from the hospital and within 4 to 8 wk after the therapeutic dose.
Blood samples were collected at the end of the therapeutic injection (t0), at 1, 2, 4, and 8 h postinjection, and then once daily until day 5. These samples were used to determine the circulating half-lives (
and β) using the biexponential model and the immunoreactivity of the injected F(ab')2 fragments, as described by Buchegger (19).
Blood samples were also collected from weeks 3 to 15 to detect the formation of HAMAs using an ELISA assay, which provided a quantitative result concerning the antiisotype human anti-mouse IgG response (HAMA-ELISA, Medac Diagnostika).
CEA values were determined before surgery, before the RIT treatment, every month for the first 3 mo, and then every 3 mo.
Surgical procedures. Of the 13 patients receiving a therapeutic dose of RIT, seven underwent a major hepatectomy, three underwent a minor hepatectomy, and the remaining three patients underwent wedge resections. Major hepatectomy was defined as resection of more than three segments according to the Couinaud classification. Minor hepatectomy was defined as resection of one to three liver segments. Intraoperative ultrasound was systematically used for complete hepatic evaluation. Postoperative variables of hepatocyte damage and recovery, including serum transaminase, bilirubin levels, and prothrombin time (expressed as a percentage of controls), were measured on postoperative days 1, 2, 5, and 7. Histologic margin to the nearest metastasis was characterized as negative (
1 cm), close (<1 cm), or positive.
Patient follow-up. All patients underwent a computed tomography of the chest, abdomen, and pelvis before therapy. Follow-up consisted of clinical evaluation with routine blood chemistry, tumor marker dosage (CEA), HAMA production, computed tomography or X-rays of the chest, and computed tomography or ultrasound of the abdomen every 3 mo during the first 2 y, and then every 6 mo for the following 3 y. Clinical and radiologic response were assessed until relapse or death.
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Results |
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All surgical resections of LM were done with curative intent at a single institution. Complete resection (R0) was obtained for all 22 patients. Intraoperative ultrasound did not detect LM other than those found by the preoperative staging procedures described above. No postoperative bleeding or hepatocellular failure was observed.
For one patient (patient 6), the suspected LM on preoperative imaging in segment IV was not palpable intraoperatively after neoadjuvant chemotherapy. However, a hyperdense image on intraoperative ultrasound was noted and an immunoscintigraphy of segment IV allowed the visualization of a metastatic nodule (0.7-cm diameter). The patient underwent a metastatectomy of segment VII and a resection of segment IV. This nodule showed an uptake of 34.5 x 10–3%ID/g and a metastasis-to-normal liver uptake ratio of 68 (Fig. 1 ).
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Pharmacokinetic analysis. Extensive analysis of the radiolabeled F(ab')2 serum clearance rates was done for 12 of the 13 treated patients. This was done using radioactivity counting of serum samples collected until day 5, as previously described. The calculated circulating and β half-lives were found to range from 30 minutes to 6 hours 37 minutes and from 23 hours 9 minutes to 40 hours 53 minutes, respectively. The median mean residence time was 51 hours 42 minutes, with extreme values of 37 hours 45 minutes for patient 2 and 65 hours 9 minutes for patient 1. Table 3
shows the kinetic variables and HAMA responses for each of the 13 treated patients.
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HAMA response. All 22 patients were tested negative for HAMA before the study, and they all remained negative after the diagnostic injection. Of the 13 RIT-treated patients, 12 were analyzed further for HAMA response. Ten developed HAMA, and two patients remained negative until testing was stopped at 15 weeks after RIT (Table 3).
Toxicity. One patient experienced an allergic reaction to the diagnostic dose. Symptomatic clinical toxicity was mild to moderate (grade 1 or grade 2) and consisted mostly of fatigue and anorexia, occurring in 61.5% and 23%, respectively. Unexplained fever was observed in one patient without concurrent neutropenia. Hematologic toxicity was significant (Table 4 ), but there were no toxicity-related deaths. Cytopenic nadirs usually occurred between weeks 4 and 6, with recovery by weeks 6 to 9. No febrile neutropenia was observed, and no patient required granulocyte colony-stimulating factor. The grade 4 thrombocytopenias lasted from 9 to 33 days. No patient experienced symptomatic bleeding, and only one patient (patient 6) required platelet transfusions.
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Patient follow-up. At the time of writing, with 127 months of median follow-up, 2 of the 13 treated patients are still alive. Median DFS was 12 months, and median overall survival was 50 months.
Patient 13 has no evidence of disease at 93 months, and his CEA levels remain within normal limits. Patient 8 presented liver recurrences that were resected in 1999 and 2001 (one of which was treated with neoadjuvant chemotherapy). At 127 months of follow-up, he has nonresectable LM but is responding well to chemotherapy. Six other patients developed further LM and were not candidates for curative treatment. No patient underwent retreatment with RIT.
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Discussion |
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The natural radiosensitivity of lymphomas make them ideal tumors for this treatment when given as a single agent and may explain in part why similar success has not been obtained with solid, bulky malignancies, notably in CRC (13–15). Other explanations may include the more heterogeneous antigen expression and density of solid tumors or factors that limit agent delivery, such as location in solid organs or on peritoneal surfaces, heterogeneous tumor vascularity, or slow diffusion of antibodies to tumoral tissues (22, 23). Furthermore, it has been shown in solid tumors that mAb uptake and the resulting radiation doses are inversely correlated with tumor size (13, 18). Uptake is also higher in the periphery of the tumor than at its core (18). Hence, microscopic disease is probably more suited for RIT, and clinical benefit might be observed only in the presence of smaller tumor burden. Behr et al. tested this hypothesis in 19 metastatic CRC patients with small volume disease. All lesions were smaller than 3 cm, and patients received 60 mCi/m2 of 131I-hMN14, a humanized anti-CEA antibody (11). Partial response rate of 16% were reported, and the overall response rate was 58%. In the same study, nine other patients were treated postoperatively after surgical resection of LM and seven of these remained disease-free for up to 36 months. Transient myelosuppression was the only toxicity observed.
The present study is a phase II trial of RIT, in which the feasibility and toxicity of high-dose 131I F6 F(ab')2 after resection of LM were determined. One of the major limitations in RIT of clinical tumors is the small percentage of injected mAb that is selectively delivered to the tumor (19). In nude mice xenografted with human colon cancer, we showed that 131I-labeled F(ab')2 fragments are more efficient and less toxic than their intact anti-CEA counterpart (18). These results can be explained by the fact that F(ab')2 fragments give higher tumor-to-normal tissue ratios and have a shorter half-life than intact mAbs. In the present study, as in our initial phase I study (18), most TTL uptake ratios ranged from 2 to 30. Treatment was well tolerated, and similar to other studies of RIT, the main toxicity was significant but transient myelosupresssion. Ten of 13 patients experienced grade 3 or grade 4 neutropenia, and 11 of 13 patients had grade 3 or grade 4 thrombopenia. However, no patient experienced spontaneous bleeding, none required granulocyte colony-stimulating factor, and only one patient required platelet transfusions.
We cannot draw conclusions as to the efficacy of this treatment, because this was not taken into account during the design of our phase II study. However, median overall survival (50 months) and DFS (12 months) are provided for information and seem to be comparable with historical controls despite the small number of patients, which included two patients with metastases at the time of treatment.
Although much effort has been put into investigating perioperative treatment to improve the outcome of these patients, there is no standard approach in this setting. Studies have included hepatic arterial infusion with or without systemic chemotherapy (24–29) and systemic 5-fluorouracil/leucovorin–based chemotherapy alone (6). Some of these treatments have shown enhancement of DFS, but an overall survival benefit over surgery alone remains unclear. To date, none of these regimens has established itself as standard, and toxicities from these treatments are nonnegligible, especially with regard to hepatic catheter complications with hepatic arterial infusion. Moreover, there are no published randomized studies using active chemotherapy agents, such as oxaliplatin, irinotecan, or bevacizumab, that have shown superiority in stage III and stage IV patients with measurable disease, although a phase II trial of postoperative irinotecan has been shown to be tolerable after major liver resection (30). Preliminary results of the EORTC 40983 trial, which randomizes patients with potentially resectable liver-only metastases to surgery alone or 3 months of preoperative and 3 months of postoperative FOLFOX4 chemotherapy, were presented at the American Society of Clinical Oncology 2007 annual meeting. They found an 8.1% increase in DFS at 3 years, which was the primary end-point (hazard ratio, 0.77; P = 0.041). The ongoing NSABP C-09 trial is evaluating the role of systemic capecitabine and oxaliplatin alone or alternating with hepatic arterial infusion chemotherapy in these patients.
According to clinical dosimetric data obtained in a previous study, the maximum delivered dose to LM can be estimated as being 10 Gy/100 mCi of injected 131I-F6 F(ab')2 (18). This is insufficient to eradicate macroscopic metastases (which require at least 50-70 Gy) or even to eradicate microscopic disease. One strategy to improve the results of RIT in CRC would be repeated administration of antitumor radiolabeled mAb to increase the cumulative dose. This could be done using humanized antibody to avoid HAMA formation in response to repeated injection of murine antibody. A limitation of this approach is the need to wait at least 2 or 3 months to allow full bone marrow recovery before delivering a second therapeutic dose. However, this opens the possibility of tumor cell repopulation during this relatively long waiting period. Another option would be to combine RIT with chemotherapy, which could also act as a radiosensitizer, but cumulative hematologic toxicity is of concern. Wong et al. conducted a phase I dose escalation study of 90Y–anti-CEA chimeric T84.66 RIT in combination with continuous infusion of 5-fluorouracil in heavily pretreated patients with chemotherapy-refractory metastatic CRC (31). Thirteen patients received one cycle, and eight patients received two cycles. The maximal tolerated doses of both agents in combination were comparable with maximal tolerated dose levels of each agent alone. Hematologic toxicity was dose-limiting and reversible with 52% grades 3 to 4 platelet toxicity and 29% grade 3 WBC toxicity. RIT did not seem to enhance the nonhematologic toxicities usually associated with 5-fluorouracil. They concluded that RIT, in combination with chemotherapy, is feasible. At this time, it is not possible to compare adjuvant RIT with adjuvant chemotherapy in this particular setting, but a further trial incorporating RIT combined with chemotherapy seems indicated.
There is only one other published trial delivering RIT to metastatic CRC patients in the postoperative setting (12). In this phase II trial, Liersch et al. treated 23 patients who underwent hepatectomies for LM of CRC with 40 to 60 mCi/m2 of 131I-labetuzumab, a humanized mAb against CEA. Recently updated data with 91 months median follow-up show a median overall survival of 58 months, a 5-year survival rate of 42%, and a median DFS of 18 months (32). Although retrospective comparisons must always be interpreted with caution, this seems encouraging compared with the median survival of 28 to 40 months of historical controls (3) and of 31 months for a group of 19 contemporaneous patients with similar prognostic scores at their center. Of note, 13 of these 19 patients used for comparison had received adjuvant chemotherapy, whereas this was not permitted in the RIT-treated patients. Similar to our study, the only significant toxicity was transient myelosuppression with a 52% rate of grades 3 to 4 cytopenias. These effects seemed to be independent of prior chemotherapy.
In our study, a single-shot treatment protocol was used. Behr et al. tested retreatment with the same dose (60 mCi/m2) in five patients with metastatic lesions having previously responded to RIT with 131I-hMN14, after a delay of 8 to 16 months. Two patients experienced partial responses and another experienced disease stabilization. Of importance, there was no evidence of increased toxicity, with no hematologic toxity greater than grade 3 (11). Liersch et al. also retreated two patients for recurrent disease 5 and 31 months after the first treatment and reported no cumulative toxicity (12). Retreatment with RIT is feasible and deserves further study.
Regarding HAMA response, 10 of the 12 patients with longer follow-up eventually tested positive after the therapeutic injection. Future research should be geared toward humanized anti-CEA antibodies, such as 131I-labetuzumab and 131I-hMN14. A fully human anti-CEA IgG2
has been developed at our center and is a promising candidate for RIT in intact form, as a F(ab')2, or as a bispecific antibody (33). Other promising avenues include use of affinity enhancement systems with pretargeting bispecific mAb (34).
In conclusion, this phase II study shows that RIT, after complete resection of LM in CRC, is both feasible and well tolerated. These findings support those of others and suggest that RIT, either alone with surgery or in a multimodality setting including chemotherapy, should be evaluated in a randomized trial.
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Disclosure of Potential Conflicts of Interest |
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: M. Ychou and D. Azria contributed equally to this work.
Received 10/22/07; revised 12/20/07; accepted 1/31/08.
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