Radioimmunotherapy of human colon carcinoma by 131I-labelled monoclonal anti-CEA antibodies in a nude mouse model.

A mixture of 3 MAbs directed against 3 different CEA epitopes was radiolabelled with 131I and used for the treatment of a human colon carcinoma transplanted s.c. into nude mice. Intact MAbs and F(ab')2 fragments were mixed because it had been shown by autoradiography that these 2 antibody forms can penetrate into different areas of the tumor nodule. Ten days after transplantation of colon tumor T380 a single dose of 600 microCi of 131I MAbs was injected i.v. The tumor grafts were well established (as evidenced by exponential growth in untreated mice) and their size continued to increase up to 6 days after radiolabelled antibody injection. Tumor shrinking was then observed lasting for 4-12 weeks. In a control group injected with 600 microCi of 131I coupled to irrelevant monoclonal IgG, tumor growth was delayed, but no regression was observed. Tumors of mice injected with the corresponding amount of unlabelled antibodies grew like those of untreated mice. Based on measurements of the effective whole-body half-life of injected 131I, the mean radiation dose received by the animals was calculated to be 382 rads for the antibody group and 478 rads for the normal IgG controls. The genetically immunodeficient animals exhibited no increase in mortality, and only limited bone-marrow toxicity was observed. Direct measurement of radioactivity in mice dissected 1, 3 and 7 days after 131I-MAb injection showed that 25, 7.2 and 2.2% of injected dose were recovered per gram of tumor, the mean radiation dose delivered to the tumor being thus more than 5,000 rads. These experiments show that therapeutic doses of radioactivity can be selectively directed to human colon carcinoma by i.v. injection of 131I-labelled anti-CEA MAbs.

Utilisation en tomoscintigraphie d'anticorps monoclonaux radio-marqués pour la détection chez l'homme des cancers digestifs et des cancers médullaires de la thyroïde

Two 131-Iodine radiolabelled monoclonal antibodies were used to perform tomoscintigraphy in 42 patients: 11 patients bearing medullary thyroid cancers and 19 patients bearing gastrointestinal cancers received an antibody directed against carcino-embryonic antigen; 12 patients bearing gastro-intestinal cancers received an antibody directed against a non circulating antigen expressed by human colorectal cancers cell lines. Tomoscintigraphy is particularly useful for analysing the complex biodistribution of radiolabelled antibodies and the low contrast images encountered in immunoscintigraphy; the problems related to the true positive rate and to the clinical specificity of the method are discussed.

Iodine-131-labeled MAb F(ab')2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice.

During one week, beginning 18 days after transplantation, nude mice bearing human colon carcinoma ranging from 115 to 943 mm3 (mean 335 mm3) were treated by repeated intravenous injections of either iodine-131-(131I) labeled intact antibodies or 131I-labeled corresponding F(ab')2 fragments of a pool of four monoclonal antibodies (MAbs) directed against distinct epitopes of carcinoembryonic antigen (CEA). Complete tumor remission was observed in 8 of 10 mice after therapy with F(ab')2 and 6 of the animals survived 10 mo in good health. In contrast, after treatment with intact MAbs, tumors relapsed in 7 of 8 mice after remission periods of 1 to 3.5 mo despite the fact that body weight loss and depression of peripheral white blood cells, symptoms of radiation toxicity, and the calculated radiation doses for liver, spleen, bone, and blood were increased or equal in these animals as compared to mice treated with F(ab')2.

Mesenteric fat-control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

MACROPHAGES FROM CROHN'S DISEASE PATIENTS EXHIBIT DEFICIENT REPAIR FUNCTIONS

AbstractBackground: Mucosal healing is becoming a major goal in the treatment of Crohn's disease. It has been previously reported that myeloid cells induce mucosal healing in a mouse model of acute colitis. The aim in this study is to investigate the pro-repair function of myeloid cells in healthy donors (HD) and Crohn's disease patients (CD).Methods: Peripheral blood mononuclear cells (PBMC) from HD and CD patients were isolated from blood samples and tested either directly or after differentiation ex-vivo into macrophages (Μφ). Intestinal macrophages (IMACs) were isolated from the bowel mucosa of patients undergoing intestinal surgical resections. Through an in vitro wound healing assay the repairing ability of these various human myeloid cells and the mechanisms responsible of wound healing were evaluated.Results: PBMC and myeloid CD14+ cells from HD and CD were not able to repair at any tested cell concentration. Μφ from HD and ulcerative colitis (UC) patients were able to induce wound healing and this capacity was partially mediated by Hepatocyte Growth Factor (HGF). Remarkably, CD Μφ were unable to promote wound healing and produced lower levels of HGF as compared to Μφ from HD or UC patients. In particular, Μφ from CD in active phase (ACD) exhibited the weakest repair function, but this defect was rescued if rh- GM-CSF was added during the differentiation of PBMCs. Interestingly, IMACs from HD promoted wound healing and produced HGF.Conclusion: We demonstrated that CD Μφ, unlike HD or UC Μφ, were defective in promoting wound healing, in particular if coming from an ACD. This deficient pro-repair function was related to a lower production of HGF. IMACs from HD colonic mucosa induced wound healing, confirming the results obtained with Μφ. Our results are in keeping with the current theory of CD as an innate immunodeficiency. In this context, Μφ may be responsible for the mucosal repair defects observed in CD patients and for the subsequent chronic activation of the adaptive immune response.

Test of four colon cancer risk-scores in formalin fixed paraffin embedded microarray gene expression data.

BACKGROUND: Prognosis prediction for resected primary colon cancer is based on the T-stage Node Metastasis (TNM) staging system. We investigated if four well-documented gene expression risk scores can improve patient stratification. METHODS: Microarray-based versions of risk-scores were applied to a large independent cohort of 688 stage II/III tumors from the PETACC-3 trial. Prognostic value for relapse-free survival (RFS), survival after relapse (SAR), and overall survival (OS) was assessed by regression analysis. To assess improvement over a reference, prognostic model was assessed with the area under curve (AUC) of receiver operating characteristic (ROC) curves. All statistical tests were two-sided, except the AUC increase. RESULTS: All four risk scores (RSs) showed a statistically significant association (single-test, P < .0167) with OS or RFS in univariate models, but with HRs below 1.38 per interquartile range. Three scores were predictors of shorter RFS, one of shorter SAR. Each RS could only marginally improve an RFS or OS model with the known factors T-stage, N-stage, and microsatellite instability (MSI) status (AUC gains < 0.025 units). The pairwise interscore discordance was never high (maximal Spearman correlation = 0.563) A combined score showed a trend to higher prognostic value and higher AUC increase for OS (HR = 1.74, 95% confidence interval [CI] = 1.44 to 2.10, P < .001, AUC from 0.6918 to 0.7321) and RFS (HR = 1.56, 95% CI = 1.33 to 1.84, P < .001, AUC from 0.6723 to 0.6945) than any single score. CONCLUSIONS: The four tested gene expression-based risk scores provide prognostic information but contribute only marginally to improving models based on established risk factors. A combination of the risk scores might provide more robust information. Predictors of RFS and SAR might need to be different.

Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids.

The binding and penetration of two 125I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) and their F(ab')2 and Fab fragments were measured in multicellular spheroids of poorly (HT29) and moderately well differentiated (Co112) human colon adenocarcinomas which express different amounts of CEA. Spheroids cultured in vitro model tumor microenvironments where poor vascular supply may modulate antigen expression and accessibility. The two MAb studied, 202 and 35, were shown previously to react with different CEA epitopes and to have high affinities of 1.2 and 5.8 X 10(9) M-1, respectively. MAb 202 has also been shown to cross-react with antigens present on human granulocytes and normal epithelial cells from human lung and pancreas. Specific binding of intact MAb and fragments of both antibodies was demonstrated for both types of human colon carcinoma spheroids compared to mouse colon carcinoma (CL26) and mammary tumor (EMT6/Ro) spheroids. Total binding of MAb and fragments was greater (1.5- to 2.5-fold) after 4 h compared to 1 h of exposure; the amount of binding compared to control IgG1 was 5- to 30-fold greater after 1-h incubation and 15 to 200 times greater after 4 h. This binding was stable as demonstrated by short and long wash experiments at 37 degrees and 4 degrees C. The binding of F(ab')2 and Fab fragments of the anti-CEA MAb 35 to spheroids of human colon Co112 was almost 2-fold greater than that of the intact MAb. However, for MAb 202, the binding of intact MAb and F(ab')2 was greater than that of Fab fragments. In addition the binding of both intact and F(ab')2 fragments of MAb 202 was greater than that obtained with MAb 35. Specific binding of both antibodies to HT29 spheroids, which express less CEA, was decreased for MAb and fragments of both 202 and 35. Autoradiography and immunoperoxidase experiments were performed to determine the penetration of MAb and fragments after incubation with intact spheroids. Comparisons were made with labeled MAb directly applied to frozen sections of spheroids. F(ab')2 and Fab fragments of both antibodies were bound at the surface of intact spheroids and penetrated to eight to ten cells, but the intact MAb were localized mainly at the spheroid surface and the outer one to three cell layers. There was much less binding at the surfaces of HT29 compared to Co112 spheroids. An enzyme immunoassay using MAb 35 and 202 demonstrated that Co112 spheroids produced about 8-fold more CEA/mg of cell protein than did monolayer cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice.

Four monoclonal antibodies against carcinoembryonic antigen (CEA) have been selected from 32 hybrids that produce antibodies against this antigen, by the criteria of high affinity for CEA and low cross-reactivity with granulocyte glycoprotein(s). The specificity of tumor localization in vivo of the four MAb, and their F(ab')2 and Fab fragments was compared in nude mice bearing grafts of a serially transplanted, CEA-producing, human colon carcinoma. The distribution of radiolabeled MAb and their fragments after intravenous injection was analyzed by direct measurement of radioactivity in tumor and normal organs, as well as by whole-body scanning and by autoradiography of tumor sections. Paired labeling experiments, in which 131I-labeled antibody or fragments and 125I-labeled control IgG are injected simultaneously, were undertaken to determine the relative tumor uptakes of each labeled protein. The tumor antibody uptake divided by that of control IgG defines the specificity index of localization. Tumor antibody uptakes (as compared with the whole mouse), ranging between 7 and 15, and specificity indices ranging between 3.4 and 6.8, were obtained with the four intact MAb at day 4-5 after injection. With F(ab')2 fragments of the four MAb, at day 3, the tumor antibody uptakes ranged between 12 and 24 and the specificity indices between 5.3 and 8.2. With the Fab fragments prepared from the two most promising MAb, the antibody uptakes reached values of 34 and 82 at day 2-3 and the specificity indices were as high as 12 and 19. The scanning results paralleled those obtained by direct measurement of radioactivity. With intact MAb, tumor grafts of 0.5-1 g gave very contrasted positive scans 3 d after injection. Using MAb fragments, tumors of smaller size were detectable earlier. The best results were obtained with Fab fragments of MAb 35, which gave clear detections of tumors weighing only 0.1 g as early as 48 h after injection. Autoradiographs of tumor sections from mice injected with 125I-labeled MAb demonstrated that the radioactivity was localized in the tumor tissues and not in the stromal connective tissue of mouse origin. The highest radioactivity concentration was localized in areas known to contain CEA such as the pseudolumen of glands and the apical side of carcinoma cells. The penetration of radioactivity in the central part of tumor nodules and the pseudolumen appeared to be increased with the use of MAb fragments.

The "reversed" treatment approach for synchronous liver metastases and colorectal cancer. Feasability and first results

Background: The « reversed treatment» approach inverts the treatment sequence of¦advanced synchronous colorectal liver metastases - i.e. the liver metastasis is¦treated first, followed by resection of the primary tumor. Chemotherapy is performed¦before and after liver surgery. We recently started to use a reversed treatment¦approach in selected patients. The aim of this study is to critically assess this new¦treatment modality.¦Methods: Nine patients (7 male, 2 female, mean age 62 years) benefited from this¦new treatment between November 2008 and May 2010. The data were collected¦retrospectively.¦Results: All patients responded to the neoadjuvant chemotherapy. The median¦number of liver metastases was 6 (range 1 - 22). The median size of the largest liver¦metastases was 4.3 cm (range 2.6 - 13 cm). Three patients had portal vein¦embolization prior to liver surgery. Two patients could not complete the treatment.¦One had to undergo emergency surgery for occluding colonic tumor. The second one¦showed liver recurrence before starting the adjuvant chemotherapy. The seven¦patients who completed the treatment are still alive after a median time of 27 months¦(range 17 - 37 months). Seven of them had recurrence (1 rectal, 6 liver). The median¦disease-free survival was 9 months (range 0 - 17 months).¦Conclusion: Based on our preliminary experiences, the reversed strategy shows¦encouraging results for the treatment of advanced synchronous colorectal liver¦metastases in well selected patients. The treatment was generally well tolerated and¦long term survival seems to be prolonged.

Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments.

Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.

Anti-idiotypic monoclonal antibody AB3, reacting with the primary antigen (CEA), can localize in human colon-carcinoma xenografts as efficiently as AB1.

BALB/c mice were immunized with anti-idiotypic monoclonal (MAb) antibody (anti-Id or Ab2) directed against an AB1 MAb anti-carcinoembryonic (CEA) in order to obtain AB3 MAbs (anti-anti-Id). AB3 MAbs were shown to recognise the primary antigen (CEA) and one of them was tested extensively in vitro and in vivo. This AB3 MAb was shown to bind specifically to CEA on frozen sections of a human colon carcinoma by immunoperoxidase. Scatchard plot analyses showed that the affinity of this AB3 was of the same order of magnitude as the AB1. In vivo experiments, in nude mice bearing CEA-producing human colon-carcinoma xenografts showed that up to 30% of the intravenously injected dose of 125I-labelled AB3 were localized per gram of tumour tissue. Furthermore, calculation of the ratios of AB3 concentration in the tumour over those in normal organs such as lung, liver, kidney, spleen and bone gave relatively high values similar to results obtained with AB1. All together our results show that AB3 can localize as efficiently and specifically in the tumour as AB1, despite the fact that the mice from which it was derived were immunized with a mouse MAb (AB2) and had never been exposed to CEA.

Nodal staging in colorectal cancer: should distant lymph nodes be recovered in surgical specimens?

To assess the value of sampling lymph nodes located far sidelong colorectal cancer specimens, we analyzed retrospectively surgical specimens from 345 colorectal cancer patients. The mesocolic and perirectal fat was divided into 2 fractions: close to (<5 cm) and distant from (>5 cm) the tumor. Tumors were located in the cecum (n = 61), ascending colon (n = 29), transverse colon (n = 31), descending colon (n = 27), sigmoid colon (n = 108), and rectum (n = 89). The median number of lymph nodes sampled was 17 in both fractions (range, 4-66), 12 (range, 0-46) in the close fraction, and 3 (range, 0-33) in the distant fraction. There were 169 pN0, 104 pN1, and 72 pN2 cases. The pN staging was accurate in all cases except 10 based on the close fraction alone; of these, 6 were upstaged from pN0 to pN1 and 4 from pN1 to pN2 when the distant fraction was considered. Among pN1-upstaged cases, 5 were rectal (3/5 with neoadjuvant radiotherapy) and 1 colonic. In the colon, we found that lymph node location is more important than lymph node number because metastatic lymph nodes were present mostly in the peritumoral area. This suggests that lymph nodes should be initially recovered from the pericolic fat close to the tumor. If there are less than 4 positive lymph nodes and less than 12 lymph nodes examined in total, additional lymph nodes should be retrieved from the distal fraction for potential upstaging. In the rectum, systematic sampling of close and distant lymph nodes is mandatory because in rare cases, metastases are detected only in distant lymph nodes, particularly in patients who have undergone neoadjuvant radiotherapy.

Isolation and characterization of carcinoembryonic antigen (CEA) extracted from normal human colon mucosa.

Carcinoembryonic antigen (CEA) was identified in perchloric acid (PCA)_extract from normal colon mucosa by 2 immunological criteria: a line of identity in double diffusion and a parallel inhibition curve in radioimmunoassay (RIA), both with reference colon carcinoma-CEA (CEA-Tu). The average concentration of CEA in normal colon mucosa (CEA-No) was 35 times lower than in primary large bowel carcinomas and 230 times lower than in metastatic colon or rectum carcinomas. CEA-No was purified from PCA extracts of normal colon mucosa by Sephadex G-200 filtration and immunoadsorbent columns. Purified CEA-No had quatitatively the same inhibition activity in RIA as the British Standard CEA coded 73/601. Purified CEA-No was labelled with 125I. The percentage of binding of labelled CEA-No to a specific goat anti-CEA-Tu antiserum was similar to that of CEA-Tu. Labelled CEA-No could be used as radioactive tracer in RIA as well as labelled CEA-Tu. The physico-chemical properties of purified CEA-Tu as demonstrated by Sepharose 6 B filtration, SDS Polyacrylamide gel analysis and cesium chloride density gradient, were found to be almost identical to those of reference CEA-Tu. Preliminary results showed that CEA-No and CEA-Tu contained the same types of carbohydrates in similar proportions. A rabbit antiserum against CEA-No was obtained which demonstrated the same specificity as conventional anti-CEA-Tu antisera.

Imaging and therapy with monoclonal antibodies in non-hematopoietic tumors.

Although radiolabelled monoclonal antibodies are useful in tumor imaging, in our opinion their most important role is in the evaluation of the capacity of newly developed monoclonal antibodies to localize in tumors specifically. Intravenous injections of monoclonal antibody fragments, labelled with beta-emitting radionuclides, can completely eradicate large human colon carcinoma xenografts in nude mice whereas this is not achieved by unconjugated monoclonal antibodies. New strategies are being developed to make radioimmunotherapy applicable to carcinoma patients.