Extensive surgical history prior to cytoreductive surgery and hyperthermic intraperitoneal chemotherapy is associated with poor survival outcomes in patients with peritoneal mucinous carcinomatosis of appendiceal origin.

BACKGROUND: Patients with PMCA commonly undergo surgery before CRS/HIPEC. We evaluated the role of extensive surgical treatment before CRS/HIPEC in terms of overall survival (OS). METHODS: 105 patients with PMCA who underwent a CRS/HIPEC procedure were identified from a prospective database. Patients were divided into two groups based on Prior Surgery Score (PSS): PSS ≤ 1 limited surgery group (LSG), PSS >1 extensive surgery group (ESG). Survival of lymph node (LN) negative and positive patients was analyzed separately. RESULTS: 40 patients were in LSG and 65 in ESG. Mean time from diagnosis to CRS/HIPEC was 6 and 17 months for LSG and ESG, respectively (p = 0.004). Groups were well balanced in peritoneal cancer index, complete cytoreduction rate, and LN status. One, 3, and 5-year OS among LN negative patients was 95, 83, and 75% for the LSG (n = 22) group and 87, 55, and 32% for the ESG (n = 35), group respectively (p = 0.026). One, 3, and 5-year OS among LN positive patients was 69, 50, and 17% for the LSG (n = 18) group and 80, 21, and 14% for the ESG (n = 30), group respectively (p = 0.613). For all patients 1, 3, and 5-year OS was 84, 65, and 54% for the LSG (n = 40) group and 86, 43, and 26% for the ESG (n = 65) group, respectively (p = 0.029). CONCLUSION: Extensive surgical treatment before CRS/HIPEC is associated with delay of CRS/HIPEC and poorer OS overall, especially among LN negative patients. We recommend early referral of PMCA patients to a peritoneal surface malignancy center.

Systemic chemotherapy (SC) before cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) in patients with peritoneal mucinous carcinomatosis of appendiceal origin (PMCA).

BACKGROUND: The role of SC before CRS/HIPEC for patients with PMCA is unclear. This study explores the effect of SC prior to CRS/HIPEC on overall survival (OS) in patients with PMCA. METHODS: 72 patients with recently diagnosed PMCA who underwent CRS/HIPEC were identified from a prospective database. Thirty patients had SC before CRS/HIPEC (Group 1) and 42 did not (Group 2). Patients who were referred to our center after multiple lines of SC were excluded from this analysis. OS was estimated. RESULTS: Median follow-up was 3.2 years. Groups were similar regarding lymph node positivity, postoperative SC and rate of complete cytoreduction. Twenty-four (80%) patients in Group 1 and 21 (50%) in Group 2 had high grade histology (HG) (p = 0.01). OS from CRS/HIPEC at 1, 2, and 3 years was 93, 68, 51% in Group 1 and 82, 64, 60% in Group 2, respectively (p = 0.74). Among HG patients 3-year survival was 36% in the SC group vs. 35% in the group without SC (p = 0.67). The 3-year OS for patients with low grade (LG) tumors was 100% in the SC group vs. 79% in the group with no prior SC (p = 0.26). Among patients with signet ring cell (SRC) histology, 1, 2 and 3-year survival was 94, 67 and 22% in the SC group vs. 43, 14, 14% in the group with no SC, respectively (p = 0.028). There were only 6 patients with LG PMCA who received prior SC. CONCLUSIONS: Preoperative SC could improve the prognosis of patients with high-grade PMCA with SRC histology.

The role of VEGFR-2 expression in outcomes and survival of patients with peritoneal carcinomatosis from appendiceal cancer.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a prognostic factor and target treatment for metastatic colorectal and ovarian cancer. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) has improved survival on peritoneal carcinomatosis (PC) from appendiceal cancer. We hypothesize that tumoral high expression of VEGF receptor 2 (VEGFR-2) is a negative prognostic factor for survival in patients with PC from appendiceal cancer. METHODS: A retrospective study of a prospective database revealed 89 patients with PC from appendiceal cancer who underwent 127 CRS/HIPECs. Surgical specimens from 59 patients were tested to identify high vs. low VEGFR-2 expression. Patient outcomes and survival were analyzed. RESULTS: There were 26 males and 33 females. Mean age was 51 years. Forty-seven VEGFR-2 high expressers and 15 low expressers were identified. Mean follow-up of high and low expressers was 25.1 and 26.6 months, respectively (p = 0.806). At follow-up, 33 (70%) high expressers were alive and 14 (30%) deceased, while 11 (92%) low expressers were alive and 1 (8%) deceased. Recurrence, use of bevacizumab, CC score, PCI, and LN status showed no differences between high and low expressers. OS for high expressers was 90.5%, 59.8%, and 47.1% at 1-, 3-, and 5-years, respectively, while OS for low expressers remained stable at 91.7% at 1-, 3-, and 5-years (p = 0.133). CONCLUSION: There is a trend towards better outcomes and survival in patients with PC from appendiceal cancer who have low expression of VEGFR-2 compared to high expression. More studies are encouraged to confirm this trend.

Repeated cytoreductive surgery and hyperthermic intraperitoneal chemotherapy in peritoneal carcinomatosis from appendiceal cancer: analysis of survival outcomes.

BACKGROUND: Cytoreductive surgery (CRS)/hyperthermic intraperitoneal chemotherapy (HIPEC) is the procedure of choice in patients with peritoneal dissemination from appendiceal cancer. Although recurrence rates are 26%-44% after first CRS/HIPEC, the role of repeated CRS/HIPEC has not been well defined. We hypothesize that patients undergoing multiple CRS/HIPEC's have meaningful long term survival. METHODS: A retrospective study of a prospective database of 294 patients with peritoneal carcinomatosis (PC) was conducted, of these 162 had PC of appendiceal origin. Twenty-six of these patients underwent 56 CRS/HIPEC. Survival and outcomes was analyzed. RESULTS: The percentage of patients with pre-surgical PCI scores ≥ 20 for the first, second, and third CRS/HIPEC was 65, 65, and 25%, respectively. Complete cytoreduction (CC 0-1) at first, second, and, third surgeries was 96, 65 and 75%, respectively. The mean operating time was 10.1 h. There was no 30-day peri-operative mortality. Following the first, second, and third CRS/HIPEC 27, 42, and 50% experienced grade III complications, respectively. Mean follow up was 51, 28, and 16 months from the first, second, and third CRS/HIPEC, respectively. Overall survival rate for the first CRS/HIPEC was 100, 83, 54, and 46% at years 1, 3, 5 and 10, respectively; from the second CRS/HIPEC 91, 53, and 34% at 1, 3, and 5 years, respectively; and from the third CRS/HIPEC was 75% at one year. CONCLUSION: Repeat CRS/HIPEC can lead to meaningful long term survival rates in patients with appendiceal peritoneal carcinomatosis with morbidity and mortality similar to those of the initial CRS/HIPEC.

Clinical utility of elevated tumor markers in patients with disseminated appendiceal malignancies treated by cytoreductive surgery and HIPEC.

BACKGROUND: Appendiceal malignancies with peritoneal spread have been successfully treated with Cytoreductive Surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). The aim of this study is to clarify the utility of common tumor markers in selecting patients for the combined treatment. METHODS: Data on 56 patients with appendiceal neoplasms treated with CRS and HIPEC were prospectively collected. Chi square test was used to analyze a link between common tumor markers and completeness of cytoreduction score (CC score) and preoperative peritoneal cancer index score (PCI score). Cox proportional hazard model was used to perform survival analysis. RESULTS: Forty-two patients were alive after 3 years of follow-up. Hazard ratio of disease related death was 5.6 (95% CI, 1.8-17.2) among patients with high CC score as compared to those with low CC score. Number of abnormal tumor markers (0 vs 1/2/3) correlated with PCI score 16.2 vs 32.5 (p < 0.001) but not with completeness of cytoreduction or survival. The 3-year survival rates in patients with normal vs abnormal CA 125 levels were 83% vs 52%(p = 0.003). CONCLUSIONS: Multiple abnormal tumor markers were not useful as an exclusion criterion for patients undergoing CRS. Elevation in CA 125 was an important indicator of survival in these patients. Complete cytoreduction was crucial for long-term survival.

Radioimmunoguided-intraoperative radiation therapy in colorectal carcinoma: a new technique to precisely define the clinical target volume.

PURPOSE: The clinical target volume (CTV) to be irradiated by intraoperative radiation therapy (IORT) after resection is generally based on the surgeon's estimation of close margins. We have developed a new technique, radioimmunoguided-intraoperative radiation therapy (RIG-IORT), that uses an intraoperative hand-held gamma-detecting probe to define areas of residual microscopic disease containing radiolabeled monoclonal antibodies to tumor associated antigen, to more precisely delineate the CTV for IORT. METHODS AND MATERIALS: Patients were injected i.v. with 2 mCi 125I- radiolabeled CC49 antibody approximately 3 weeks before surgery. They then underwent radioimmunoguided surgery (RIGS) with maximal resection of tumor. A hand-held gamma-detecting probe (Neoprobe 1000) was used intraoperatively to detect and resect areas of high radioactivity, representing tumor. Areas with persistently high probe counts after resection were the areas of occult residual disease, and represented the CTV to be irradiated. The IORT was given with either 6-9 MeV electron beam from a dedicated linear accelerator, or with high-dose-rate brachytherapy from a remote afterloader. If all RIGS-positive tissue had been resected, or if widely disseminated disease remained, the patient was not considered for IORT. RESULT: This technique was used in 31 patients with colorectal adenocarcinoma recurrent into the pelvis (n = 23) or paraortic nodes (n = 8). The CTV for IORT was delineated by increased RIGS count in 13 of 19 patients (68%) with microscopic residual, and in 11 of 12 patients (92%) with gross residual. In the other 7 patients, the tumor area did not accumulate the radiolabeled antibody; therefore, these tumor beds were irradiated based on the surgeon's estimation of close margins. Hence, overall, the RIG-IORT technique was used to define the tumor bed for IORT in 24 of 31 patients (77%). This technical report focuses on the development of the RIG-IORT technique and does not address the outcome results of the treated patients. CONCLUSION: A new technique, RIG-IORT, which uses radiolabeled monoclonal antibodies to precisely determine the CTV for IORT, is described. Whether the use of this technique will lead to improved tumor control will only be known upon the outcome analysis of RIG-IORT-treated patients compared with those obtained using traditional IORT techniques.

Iodine-125 brachytherapy in the treatment of colorectal adenocarcinoma metastatic to the liver.

BACKGROUND: The liver is the site of distant failure in > 33% of patients with colorectal adenocarcinoma. Liver resection is the only potentially curative option in these patients. Patients with incompletely resected liver lesions (due to the proximity to critical vascular structures) are at high risk of dying of progressive disease in the liver. This pilot study was performed to determine whether the intraoperative implantation of iodine-125 (I-125) seeds could reduce the recurrence and improve the survival of patients with incompletely resected liver metastases. METHODS: Fifty-six patients with unresectable or residual disease after surgical resection of liver metastases from colorectal carcinoma underwent permanent implantation with I-125 seeds to deliver 160 gray to the periphery of the target volume. RESULTS: The 1-, 3-, and 5-year actuarial control rates of liver disease were 41%, 23%, and 23%, respectively. The 5-year actuarial control of liver disease was better for patients with a solitary metastasis (39%) than for those with multiple metastases (9%) (P = 0.04). The 1-, 3-, and 5-year actuarial overall survival rates were 71%, 25%, and 8%, respectively (median, 20 months; 95% confidence interval, 17-23). The radiation-related complications were minimal. CONCLUSIONS: I-125 liver brachytherapy is feasible with minimal radiation-related morbidity. Good prognostic factors for long term liver control and survival are the presence of a solitary metastasis, postresection minimal residual disease requiring smaller volume implants, and no prior liver resections. Future prospective trials should be directed toward this patient population, which has the highest probability of obtaining improved results from the local dose escalation provided by brachytherapy. Adjuvant regional chemotherapy clearly is needed due to the high rate of liver recurrence and ultimate death from liver failure observed in spite of liver resection and brachytherapy.

Staging of colorectal cancer: biology vs. morphology.

PURPOSE: An accurate determination of the extent or staging of a disease is critical, because it provides the basis for making therapeutic decisions. Staging is a collaborative effort by the surgeon and the pathologist. Radioimmunoguided surgery has been evaluated for its ability to help surgeons determine the extent of disease during surgery, when management decisions have the most impact on patient care. This study was done to compare radioimmunoguided surgery "biostaging" with traditional pathologic staging (TNM) as predictors of survival in patients undergoing curative resections for colorectal cancer. METHODS: Ninety-seven patients with colorectal cancer were prospectively enrolled in radioimmunoguided surgery protocols. Evaluation of follow-up survival data was performed. All patients underwent exploratory laparotomy and radioimmunoguided surgery with resection of their primary colorectal tumor. Survival data were analyzed with the Kaplan-Meier method with log-rank comparisons. RESULTS: Of 97 patients enrolled in the study, 59 were evaluable and completely resectable by radioimmunoguided surgery. Mean follow-up was 62 months, with a range of 34 to 89 months. By traditional staging 13 patients were pStage I, 18 patients were pStage II, and 28 patients were pStage III. By radioimmunoguided surgery biostaging, 24 patients were radioimmunoguided surgery-negative whereas 35 patients were radioimmunoguided surgery-positive. Survival rates by pathologic stage approached a significant difference, but did not, as of the conclusion of the study period, reach it (P = 0.12). Survival rates based on radioimmunoguided surgery status demonstrated a highly significant difference (P = 0.0002). CONCLUSIONS: Radioimmunoguided surgery biostaging provides new information intraoperatively on cancer staging that has not been available before. This may lead to new strategies for therapy that can be individualized and optimized for each patient with cancer.

Novel clinical approaches in monoclonal antibody-based management in colorectal cancer patients: radioimmunoguided surgery and antigen augmentation.

Surgery, the most effective treatment for colon and rectal cancer, is based on empirical knowledge of the patterns of tumor spread, gross findings at laparotomy, and histologic confirmation of tumor-free margins. In spite of the many technical improvements in surgery, there has not been a significant change in cure rates for colon and rectal cancers. In fact, one-half of affected patients will not survive 5 years. It is in this arena of treatment for primary colon and rectal cancer patients that radioimmunoguided surgery (RIGS) technology may provide the most benefit. RIGS is an intraoperative procedure for detection of carcinoma lesions that are targeted with a radiolabeled monoclonal antibody (MAb) to provide the surgeon with immediate intraoperative definition of tumor margins and identification of occult disease. To optimize this technique, our studies were designed to increase tumor uptake by higher affinity CC-49 (a second-generation MAb) and to increase tumor antigen expression using biological response modifiers (BRMs). The ability of BRMs, such as interferons (IFNs), to enhance the expression of tumor-associated antigens, may play an important role in an adjuvant setting for MAb-based treatment. Preclinical and clinical data provided evidence for the use of IFN as an adjuvant to enhance MAb-targeting of human carcinoma lesions. A combination protocol with IFN and RIGS is ongoing at our institution.

[Lymphadenectomy in colorectal carcinoma]. / Lymphadenektomie beim colorectalen Carcinom.

Recurrence of colorectal carcinomas occurs in about 50% of the cases with localized neoplasia. It is understood that the tumor recurrence is due to residual micrometastases not found during surgery or extraregional (peripheral blood or bone marrow). We developed a procedure to detect non-visible, abdominal metastases using a radiolabeled anti-tumor cell antibody injected before the operation (radioimmunoguided surgery RIGS). However, even with the best technique, it is not possible to remove all micrometastasis if a hematogenic dissemination happens. Based on the knowledge of disturbing humoral immune reaction is mounted against shed tumor associated antigens (TAA), we developed a new method to reduce and correct the B cell response and B cell recruitment due to chronic TAA immun complex presentation on follicular dendritic cells (immune corrective surgery, ICS). This method is based on a selective lymphadenectomy. The target lymph nodes were those loaded with TAA-immune complex. The detection method used was the injection of radiolabeled antibody able to recognize the immune complex. From 20 patients (stage I, II and III) treated with ICS, 17 survived more than 5 years 'showing a statistically significant increase of survival compared to patients treated with standard procedures. In conclusion, these data show that surgery of colorectal cancer should be selectively extended to specific anatomical regions in order to remove hidden micrometastases, and more importantly, correct postoperative immune processes that could suppress the T cell response against residual tumor cells.

Intraoperative high dose rate brachytherapy in recurrent or metastatic colorectal carcinoma.

BACKGROUND: The survival of patients with recurrent or metastatic colorectal cancer usually is less than 12 months. In an attempt to improve this dismal prognosis, we investigated the role of intraoperative high dose rate brachytherapy (IOHDR) in the management of these patients. METHODS: From April 1992 to December 1996, 26 patients with locally recurrent or metastatic colorectal carcinoma were treated with maximal surgical resection and IOHDR. Intraoperative radiation dose ranged from 10 to 20 Gy, prescribed at 0.5 cm depth. The residual tumor irradiated was microscopic in 16 patients (62%) and gross residual in 10 patients (38%). Six patients received postoperative external beam radiation therapy. RESULTS: After a median follow-up of 28 months (range 6 to 54 months), seven of 15 evaluable patients (47%) failed in the area treated with IOHDR. The median time to local failure was 21 months (range 4 to 52 months). The median survival was 23 months (microscopic 24 months; gross 17 months), with a 4-year actuarial survival rate of 36%. Major morbidity was observed in 7 patients (47%) and usually was surgery-related. CONCLUSION: The use of IOHDR in association with radical resection increases local control in patients with recurrent or metastatic colorectal cancer. Patients with microscopic residual disease achieved a better result than do those with gross residual disease. Future strategies include the addition of limited EBRT dose to IOHDR, even for previously irradiated patients.

In vitro generation of human cytotoxic T lymphocytes specific for peptides derived from prostate-specific antigen.

BACKGROUND: Protein antigens are presented to cytotoxic T lymphocytes as small peptides (approximately 9-10 amino acids long) bound to class I molecules of the major histocompatibility complex. The identification of tumor-associated antigens and specific peptide epitopes (i.e., antigenic determinants) may be useful in the development of anticancer vaccines. The generation of a cytotoxic T-cell response to one peptide epitope (amino acids 146-154) of human prostate-specific antigen (PSA) has been reported. PURPOSE: Our aim was to identify novel PSA peptides capable of eliciting specific cytotoxic T-cell responses. METHODS: Candidate peptides were identified on the basis of the following criteria: 1) they contained consensus amino acid motifs for binding to HLA-A2, which is the most common type of class I molecule; 2) they lacked strong homology with PSA-related kallikrein proteins; and 3) they were capable of stabilizing HLA-A2 class I molecules on the surface of human T2 (transport deletion mutant) cells, which are defective in antigen presentation. T-cell lines capable of killing (i.e., lysing) T2 target cells that had been pulsed with specific PSA peptides were generated from three different males (two disease-free individuals and one patient with prostate cancer) by incubating peripheral blood mononuclear cells with the peptides and interleukin 2. Specific cell lysis was monitored by the release of radioactivity from target cells that had been labeled with [111In]oxyquinoline. RESULTS: Two novel PSA peptides capable of eliciting cytotoxic T-cell responses were identified; these peptides were designated PSA-1 (amino acids 41-150) and PSA-3 (amino acids 154-163). Four different cytotoxic T-cell lines were generated in response to these peptides-three against PSA-3 and one against PSA-1. Specific lysis of peptide-pulsed T2 cells by the T-cell lines was blocked by the addition of a monoclonal antibody directed against class I molecules. The T-cell lines were also capable of lysing PSA-positive, HLA-A2-positive LNCaP cells (human prostate carcinoma cells). The specificity of LNCaP cell lysis was shown by the following: 1) the inability of added human K562 (chronic myelogenous leukemia) cells to inhibit it, 2) the ability of added anti-HLA-A2 antibodies to block it, and 3) the inability of the T-cell lines to induce substantial lysis of PSA-negative, HLA-A2-positive human cancer cells. IMPLICATIONS: Our studies form a rational basis for the use of PSA peptides or recombinant vectors encoding PSA in the development of anticancer vaccine immunotherapy protocols for patients with prostate cancer.

Phenotypic stability of a cytotoxic T-cell line directed against an immunodominant epitope of human carcinoembryonic antigen.

CTL lines have now been generated against defined peptides of a range of human tumor-associated antigens (TAAs). One of the potential uses of these epitope-specific CTLs is in adoptive transfer immunotherapy. This is a modality, however, that will require long-term in vitro culture of CTLs. To date, little has been reported concerning the phenotypic stability of human epitope-specific CTLs as a consequence of long-term in vitro propagation via peptide stimulation. We report here the serial phenotypic characterization of a CTL line directed against an immunodominant epitope (YLSGANLNL, designated CAP-1) of human carcinoembryonic antigen (CEA). This CTL line was derived from peripheral blood mononuclear cells of a patient with metastatic carcinoma who had been treated with a recombinant CEA-vaccinia vaccine in a Phase I trial; the CTLs were analyzed through 20 in vitro cycle passages of stimulation with CAP-1 peptide and interleukin 2 in the presence of autologous antigen-presenting cells. The CTL line was shown to be phenotypically stable in terms of high levels of cytokine (IFN-gamma, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor) production, expression of homing-adhesion molecules, ability to lyse peptide-pulsed targets, and ability to lyse human carcinoma cells endogenously expressing CEA in a MHC-restricted manner. Vbeta T-cell receptor gene usage was also analyzed. These studies thus present a rationale for the use of long-term cultured epitope-specific human CTLs, directed against a human TAA for potential adoptive transfer immunotherapy protocols.

Novel approaches to tumor detection and therapy using a combination of monoclonal antibody and cytokine.

Cytokine-based tumor antigen augmentation is one of the approaches researchers and clinicians are using to improve the effectiveness of MAb-directed tumor diagnosis and therapy. Other efforts encompass the use of dose-fractionation for multiple administrations of radioimmunoconjugates, exploitation of genetic engineering to construct antibody molecules with specific biological properties (i.e., altered pharmacokinetics, activation of cellular immune responses, etc.) and use of MAb-directed conjugates that can enhance tumor MAb uptake by altering tumor perfusion. The studies summarized here as well as those from other laboratories have served as the framework for clinical investigations designed to determine the effectiveness of the interferons and other differentiation-inducing agents to alter the tumor antigen phenotype in patients. In an earlier study, patients given IFN-alpha had improved tumor uptake of an antimelanoma MAb. Subsequently, we reported that i.p. IFN-gamma administration substantially upregulated TAG-72 and CEA on the surface of human tumor cells isolated from malignant ascites. A seminal investigation showed significant increase of TAG-72 and CEA levels in tumor biopsies from patients diagnosed with colorectal carcinoma and given systemic IFN-alpha. Those studies led to a clinical trial in which late stage breast cancer patients were administered interferon in combination with therapeutic doses of CC49. Some clinical responses were observed, however, the cytokine and MAb combination may have also enhanced marrow toxicity. Future studies will continue to evaluate the ability to enhance tumor antigen expression in the context of genetically engineered MAbs designed to minimize normal organ toxicity.

Upregulation of DF3, in association with ICAM-1 and MHC class II by IFN-gamma in short-term human mammary carcinoma cell cultures.

This study was designed to determine whether in vitro exposure of isolated short-term human primary and metastatic breast tumor cell cultures to interferon-gamma (IFN-gamma) could enhance expression of the breast tumor associated DF3 antigen in association with the intercellular adhesion molecule 1 (ICAM-1) and MHC class II molecules. Cell cultures were established from primary solid tumors and metastatic cells as previously described (Sgagias et al., 1995). Data show that recombinant human IFN-gamma treatment, in vitro, dramatically increased the breast tumor associated DF3 antigen, in association with ICAM-1, and MHC class II antigens in primary breast cancer cell cultures. All primary breast tumor cell cultures constitutively expressed high levels of HLA-class I antigen. Metastatic breast cancer cell cultures expressed high levels of DF3 and recombinant human IFN-gamma treatment, in vitro, upregulated ICAM-1 and MHC class II antigens before and after passage of the metastatic cells through the nude mouse. Metastatic breast cancer cells similar to primary breast cancer cells constitutively expressed high levels of MHC class I antigens. In addition, three LAK cell lines significantly lysed the primary and the metastatic breast tumor cell cultures to the same degree before and after passage of the metastatic cancer cells through the nude mouse. These data indicate the upregulation of the breast tumor associated DF3 antigen in vitro after IFN-gamma treatment and its persistence in vivo, after passage of the metastatic breast cancer cells through the nude mouse. The ability of IFN-gamma to upregulate the breast tumor associated DF3 antigen in association with the ICAM-1 and HLA class II antigens may play an important role in eliciting an immune response which may contribute to the immunodiagnosis, and immunotherapy of breast cancer.

Macrophage colony-stimulating factor enhancement of antibody-dependent cellular cytotoxicity against human colon carcinoma cells.

Antibody-dependent cellular cytotoxicity (ADCC) has been suggested to be an important defense mechanism against tumors. The effects of recombinant human macrophage colony-stimulating factor (rhM-CSF) on ADCC activity of human monocytes were investigated. Human peripheral monocytes were pre-incubated for 72 h with rhM-CSF at various concentrations (50, 100, 200, 400 U/ml) and then used as effector cells in a 24-h 111-Indium release assay. Human carcinoma cell lines LS-174T, CBS, and KLE were used as targets to react with anti-carcinoma monoclonal antibodies (mAbs: murine D612, murine CC49, and chimeric CC49). A significant increase in ADCC activity was observed after monocytes were incubated in 100-400 U/ml of human rhM-CSF. Variation in ADCC activity of monocytes among donors was observed. The enhancement of ADCC activity was blocked by the addition of a neutralizing antibody to rhM-CSF. Less D612 mAb was required for the rhM-CSF-treated monocytes to mediate an equivalent level of ADCC activity as compared to the untreated monocytes. Because of the low levels of rhM-CSF required in these studies to enhance ADCC, treatment of monocytes alone with comparable levels of rhM-CSF did not enhance antibody-independent cytotoxicity. Moreover, it is demonstrated here that recombinant human interleukin-4 (rhIL-4) and rhM-CSF can have a synergistic effect of monocyte-mediated ADCC on human tumor cells. These results thus indicate that rhM-CSF augments ADCC of human peripheral blood monocytes using mAbs to human carcinomas, suggesting a potential role for rhM-CSF in cancer immunotherapy.

Improved tumor radioimmunodetection using a single-chain Fv and gamma-interferon: potential clinical applications for radioimmunoguided surgery and gamma scanning.

Previous studies have shown that (a) single-chain antibody binding proteins, or sFvs, localize experimental tumor xenografts (D.E. Milenic et al, Cancer Res., 51: 6363-6371, 1991) and (b) the administration of gamma-interferon (IFN-gamma) increases the expression of a high molecular weight glycoprotein, tumor-associated glycoprotein 72 (TAG-72), which improves mAb-based tumor targeting as well as radioimmunotherapy (J. W. Greiner et al., Cancer Res., 53: 600-608, 1993). The present experimental study was designed to determine whether exploiting those two observations in combination could augment tumor detection. Initial results revealed significant localization of a single-chain antibody binding protein of CC49 (i.e., CC49 sFv), a second generation anti-TAG-72 mAb, to human colon tumor xenografts (HT-29), which express low constitutive TAG-72 levels. IFN-gamma treatment of mice bearing HT-29 tumors significantly increased TAG-72 levels in the tumor xenografts. Increased TAG-72 expression was accompanied by a 2-4-fold augmentation of CC49 sFv localized to the HT-29 tumors, measured by direct quantitation of 125I-labeled CC49 sFv tumor deposition as well as tumor:normal tissue ratios. Enhanced CC49 sFv tumor localization improved HT-29 tumor visualization by external scintigraphy as well as when using a hand-held gamma-detecting probe to discriminate between normal (i.e., heart, hind leg) and tumor tissue. The gamma-detecting probe was the same as that used intraoperatively with 125I-labeled CC49 IgG to identify occult tumors in patients. The present experimental findings indicate that the efficiency by which 125I-labeled CC49 sFv localizes tumor in vivo can be enhanced with IFN-gamma. Results of the present study suggest that (a) the incorporation of an IFN-gamma treatment schema prior to radioimmunscintigraphy may increase the signal from the tumor site(s), thus providing a better discrimination between tumor and background, and (b) combining 125I-labeled CC49 sFv with IFN-gamma will not only reduce the time interval between antibody injection and surgery, but will also increase the efficiency of tumor localization using the intraoperative gamma-detecting probe.

Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine.

BACKGROUND: The human carcinoembryonic antigen (CEA), which is expressed in several cancer types, is a potential target for specific immunotherapy using recombinant vaccines. Previous studies have shown that when the CEA gene is placed into vaccinia virus, the recombinant vaccine (rV-CEA) can elicit T-cell responses in both rodents and non-human primates. PURPOSE: Our objective was to determine if rVCEA could elicit CEA-specific T-cell responses in humans with appropriate human leukocyte antigen (HLA) motifs. METHODS: Peripheral blood lymphocytes (PBLs) obtained from patients with metastatic carcinoma, both before and after vaccination with rV-CEA, were analyzed for T-cell response to specific 9- to 11-mer CEA peptides selected to conform to human HLA class I-A2 motifs. RESULTS: While little or no T-cell growth was seen from preimmunization PBLs of patients pulsed with CEA peptides and interleukin 2 (IL-2), T-cell lines were obtained from PBLs of patients after vaccination with one to three cycles of stimulation. Cytolytic T-cell lines from three HLA-A2 patients were established with a 9-amino acid peptide (CAP-1), and the CD8+/CD4+ double-positive T-cell line (V24T) was chosen for detailed analysis. When autologous Epstein-Barr virus (EBV)-transformed B cells were either incubated with CAP-1 peptide or transduced with the CEA gene using a retroviral vector, they were lysed by the V24T cell line, but allogeneic non-A2 EBV-transformed B cells were not. The SW403 human colon carcinoma cell line, which is CEA positive and HLA-A2 positive, was also lysed by the V24T cell line, while two non-HLA-A2 CEA-positive colon carcinoma cell lines were not. To further confirm the class I HLA-A2 restricted nature of the V24T cytotoxicity, the non-HLA-A2 SW837 CEA-expressing colon carcinoma cell line was infected with a recombinant vaccinia virus expressing the HLA class I-A2 gene, and it became susceptible to V24T lysis. Cells infected with vector alone were not lysed. CONCLUSIONS: This study demonstrates for the first time (a) the ability to generate a human cytolytic T-cell response to specific epitopes of CEA, (b) the class I HLA-A2 restricted nature of the T-cell mediated lysis, and (c) the ability of human tumor cells to endogenously process CEA to present a specific CEA peptide in the context of major histocompatibility complex for T-cell-mediated lysis. IMPLICATIONS: These findings have implications in the development of specific second-generation cancer immunotherapy protocols.

Enhanced interleukin-2 production in human tumor-infiltrating lymphocytes engineered by 3'-truncated interleukin-2 gene.

Tumor-infiltrating lymphocytes (TILs), T lymphocytes associated with solid tumors that can be grown with interleukin (IL)-2 in vitro, preferentially accumulate at tumor sites after adoptive transfer. Therefore, TILs can be considered for use as cellular vehicles in gene therapy. We transduced melanoma TILs with the IL-2 gene and clarified functional characteristics of the TIL transductants. TILs transduced with 3'-end-truncated IL-2 gene (480 bp) produced high amounts of IL-2 detected in supernatants when compared to TILs transduced with the native IL-2 gene containing 3'-end adenine-thymidine (AT)-rich sequences (650 bp). The level of IL-2 in supernatants was higher with the addition of anti-Tac antibody (Ab) to block the consumption of IL-2 by the TILs. These TILs could proliferate autonomously in the absence of exogenous IL-2, and the proliferation of TILs could be completely blocked by anti-IL-2 Ab or anti-IL-2 receptor Ab. Thus TILs transduced with IL-2 gene can proliferate through the autocrine loop. However, the expression of IL-2 from TILs transduced with the IL-2 gene was downregulated after 2 to 3 weeks of G418 selection. Our study indicates the feasibility of transduction and expression of a truncated 480-bp IL-2 gene into TILs and the possibility of employing adoptive immunotherapy protocols using TILs modified with this IL-2 gene.

Differential effects of recombinant interferon-alpha and 5-fluorouracil against colon cancer cells or against peripheral blood mononuclear cells.

Comparative studies on the suppressive effects of recombinant interferon-alpha (IFN-alpha), 5-fluorouracil (5-FU), or IFN-alpha + 5-FU have been performed in vitro on colon carcinoma cells (HT-29 cell line) and PHA-stimulated mononuclear cells (MNC) of peripheral blood obtained from healthy donors. IFN-alpha was used at 500 U/ml against HT-29 cells and at 1000 U/ml against MNC on day 1 of culture; 5-FU was used at 250 microM against HT-29 and at 1400 microM against MNC on day 2 of culture. The results show that: (a) IFN-alpha inhibited MNC and HT-29 cells by 13.4% and 32.9%, respectively; (b) 5-FU inhibited MNC and HT-29 cells by 54.7% and 87.0%, respectively; (c) IFN-alpha + 5-FU resulted in a stronger inhibition of HT-29 cells (i.e., 96.1%). In contrast, that combination was significantly less suppressive than 5-FU alone when MNC were used as targets (i.e., 35.9% inhibition). Natural cell-mediated cytotoxic activity relative to 10(6) MNC was not markedly altered by all agents alone or in combination. Moreover, treatment with IFN-alpha, 5-FU or IFN-alpha + 5-FU resulted in a marked increase in the number of HT-29 cells positive for the CEA surface antigen. These data seem to provide further rational support of the clinical use of IFN-alpha + 5-FU in colorectal cancer, based on the differential toxicity of this drug combination on tumor versus normal immunocompetent cells.