Prevention of breast tumour development in vivo by downregulation of the p185neu receptor.

Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial cells develop breast tumours at an average of 44 weeks of age. In this study, intraperitoneal injection of a monoclonal anti-receptor antibody specific for the rat neuT oncogene product dramatically affected tumour development in these transgenic mice in a dose-dependent manner. A significant proportion (50%) of mice, when injected with anti-receptor antibodies, did not develop tumours even after 90 weeks of age. The phosphotyrosine levels of the membrane fraction of breast tissues in the anti-receptor antibody-treated mice were almost completely abolished when a higher dose of antibodies was used. This study demonstrates, for the first time, that immunologic manipulation of an oncogene product can effectively prevent the development of tumours in a rodent transgenic model.

Identification of an I-J+ antigen-presenting cell required for third order suppressor cell activation.

We have found that an I-J+ I-A- antigen-presenting cell (APC) is required for Ts3 activation in vivo. Together with the I-J restriction previously reported for Ts3 induction (Takaoki, M., M.-S. Sy, A. Tominaga, A. Lowy, M. Tsurifiji, B. Benacerraf, R. Finberg, and M. I. Greene, 1982, J. Exp. Med., 156:1325), it appears that this I-J+ APC is responsible for I-J restriction in the triggering of Ts3. This restriction may be exerted via a pre-Ts3 associative recognition of antigen and I-J encoded determinants, analogous to the T helper recognition of antigen in the context of I-A and I-E determinants.

Down-regulation of HER2/neu expression induces apoptosis in human cancer cells that overexpress HER2/neu.

The HER2/neu oncogene is overexpressed in a significant fraction of human tumors; such overexpression is thought to play a role in the aberrant proliferation of cancer cells. The effects of HER2/neu-specific phosphorothioate antisense oligodeoxyribonucleotides on HER2/neu expression, tumor cell proliferation, and activation of apoptotic cell death pathways have been examined. Antisense treatment down-regulates HER2/neu expression in a dose-dependent and sequence-specific manner. HER2/neu antisense treatment specifically inhibits the growth of tumor lines that overexpress HER2/neu, but it has little effect on the growth of tumor cells that express low levels of HER2/neu. Down-regulation of HER2/neu expression is not only cytostatic, but it also results in the activation of apoptotic cell death pathways in cells that overexpress HER2/neu. These results suggest that, in addition to stimulating tumor cell proliferation, HER2/neu overexpression in cancer cells acts as an antiapoptotic cell survival factor.

Suppression of beta-catenin inhibits the neoplastic growth of APC-mutant colon cancer cells.

Mutations involving the adenomatous polyposis coli (APC) tumor suppressorgene/beta-catenin signaling pathway have been identified in the majority of colon carcinomas. However, the role of aberrant beta-catenin signaling in the neoplastic growth of APC-mutant colon cancer cells has not been directly studied. To address this question, antisense oligonucleotides have been used to specifically down-regulate beta-catenin expression in APC-mutant human colon carcinoma cells. Antisense-mediated suppression of beta-catenin inhibits the in vitro proliferation, anchorage-independent growth, and cellular invasiveness of APC-mutant human colon carcinoma cells. The systemic administration of beta-catenin antisense oligonucleotides down-regulates beta-catenin expression in vivo in human colon cancer xenografts in nude mice. Such treatment inhibits the tumorigenic growth of colon cancer xenografts and can completely eradicate tumors in some treated animals. These studies formally demonstrate the critical role of beta-catenin signaling in the neoplastic growth of APC-mutant colon cancer cells and suggest that strategies targeting beta-catenin may be of use in the therapy of colon cancer.

Lymph node evaluation and survival after curative-intent resection of duodenal adenocarcinoma: A matched cohort study.

BACKGROUND: Lymph node (LN) metastasis in patients with duodenal adenocarcinoma is associated with poor prognosis; however, the optimal extent of LN assessment and the interaction between LN assessment and adjuvant systemic therapy is poorly understood. METHODS: Resected non-metastatic duodenal adenocarcinoma patients (n = 1743) were identified in the National Cancer Database (1998-2011). Logistic regression analysis identified covariates associated with LN metastasis. The influence of increasing LN cut-off points on overall survival (OS) was analysed using the log-rank test and Cox proportional hazards modelling. Adjuvant chemotherapy (AC) and surgery alone cohorts were matched (1:1) by propensity scores based on the likelihood of nodal metastasis or survival hazard on Cox modelling. OS in the matched cohort was compared by Kaplan-Meier estimates. RESULTS: LN metastases were present in 865 (49.6%) patients. Increasing LN assessment was associated with an increased likelihood of nodal involvement (P = 0.008). In node-negative patients, increasing LN assessment was associated with a decreased risk of death, with the largest actuarial survival differences observed for ≥15 LN (hazard ratio [HR] 0.63, 95% confidence interval [CI] 0.48-0.82, P = 0.001). In the propensity score-matched cohort of node-negative patients, AC was associated with non-significant improvements in 5-year actuarial (66.1% versus 58.7%, HR 0.79, 95% CI 0.53-1.18, P = 0.249), and did not vary by adequacy of LN counts (<15 LNs: HR 0.79, 95% CI 0.51-1.24, P = 0.305; ≥15 LNs: HR 0.90, 95% CI 0.35-2.30, P = 0.900). CONCLUSIONS: The extent of LN identification has prognostic significance in resected node-negative duodenal adenocarcinoma, but cannot be implicated in the selection of node-negative patients for AC.

Monoclonal antibodies specific for the neu oncogene product directly mediate anti-tumor effects in vivo.

We have produced a panel of monoclonal antibodies which bind cell surface domains of the 185 Kd tumor antigen (p185) encoded by the neu oncogene. All of these antibodies stain neu-transformed cells in immunofluorescence assays and immunoprecipitate p185 from metabolically labeled cell lysates. All of the anti-p185 monoclonal antibodies, regardless of isotype, exert a selective cytostatic effect on the growth of neu-transformed cells suspended in soft agar, demonstrating their ability to directly inhibit the transformed phenotype. Anti-p185 antibodies of the IgM, IgG2a, and IgG2b isotypes exert a cytolytic effect on neu-transformed cells in the presence of complement. Only one IgG2a monoclonal antibody is also able to mediate minimal levels of antibody-dependent cellular cytotoxicity (ADCC) (Roussel et al., 1984) in the presence of non-immune spleen cells. In vivo administration of anti-p185 antibodies of the IgG1, IgG2a, and IgG2b isotypes exerts a profound inhibitory effect on the tumorigenic growth of neu-transformed cells. This tumor inhibitory effect is unaffected by depleting tumor bearing animals of complement, and is only minimally affected by depleting tumor bearing animals of macrophages. This suggests that neither complement-mediated killing nor ADCC are necessary for the anti-tumor effects of p185-specific monoclonal antibodies. The results presented here demonstrate that monoclonal antibodies reactive with cell surface domains of an oncogene-encoded protein can directly inhibit tumor growth in vitro and in vivo. Such antibodies may prove useful in the therapy of certain malignancies.

Monoclonal antibodies reactive with distinct domains of the neu oncogene-encoded p185 molecule exert synergistic anti-tumor effects in vivo.

The neu oncogene encodes a 185,000 dalton transmembrane glycoprotein, p185. The current study examined the effects of p185-specific monoclonal antibody administration on the tumorigenic growth of neu-transformed NIH3T3 cells implanted into nude mice. Treatment with anti-p185 monoclonal antibodies of the IgG1, IgG2a, IgG2b subclasses was able to profoundly inhibit the growth of neu-transformed cells. Furthermore, while none of these antibodies individually was able to cause complete eradication of tumors, the administration of mixtures of antibodies reactive with two distinct regions on the p185 molecule resulted in synergistic anti-tumor effects and complete eradication of tumors in a substantial fraction of the treated animals. The effect was oncogene specific, since the growth of ras transformed cells was not influenced by anti p185 antibodies. These results demonstrate that antibodies reactive with multiple domains of a tumor antigen can exert synergistic anti-tumor effects, and suggest that therapy with monoclonal antibodies specific for the neu oncogene product may play a role in cancer treatment.

Molecular cloning and chromosomal localization of the human homologue of a B-lymphocyte specific protein tyrosine kinase (blk).

A cDNA encoding the human homologue of the murine protein tyrosine kinase, blk, has been cloned from a human B-lymphocyte cDNA library by cross-species hybridization using the murine blk cDNA as a probe. The sequence of the 2608 bp human blk cDNA clone contains an open reading frame encoding a predicted 505 amino acid protein with SH3, SH2 and catalytic domains that contain consensus sequences of the src protein tyrosine kinase family. Comparison of human and murine blk sequences indicated that they share 86% amino acid identity, the most conserved region being the catalytic domain (93% identity). Like the murine blk gene human blk is expressed only in B lymphocytes. The human blk gene was mapped to chromosome 8 at p22-23.

HER2/neu antisense targeting of human breast carcinoma.

Overexpression of the HER2/neu oncogene is observed in approximately 30% of human breast carcinoma specimens. HER2/neu overexpression is a negative prognostic factor in breast cancer patients. Cancer cells that overexpress HER2/neu may also be less sensitive to chemotherapy. In order to further define mechanisms by which HER2/neu overexpression drives neoplastic cell growth and chemoresistance, antisense oligonucleotides (ODNs) have been utilized to selectively down-regulate HER2/neu expression in human breast cancer cells. Such antisense ODNs suppress HER2/neu mRNA and protein levels in a dose-dependent, sequence-specific manner. Down-regulation of HER2/neu expression in HER2/neu overexpressing breast cancer cells inhibits cell cycle progression in G0/G1 and results in apoptotic cell death. In tissue culture studies, combined treatment of HER2/ neu overexpressing breast cancer cells with HER2/neu antisense ODNs and conventional chemotherapeutic agents results in synergistic inhibition of cancer cell growth and activation of apoptotic cell death mechanisms. These studies have been extended to demonstrate synergistic antitumor effects following systemic treatment with antisense ODNs plus doxorubicin in nude mice bearing human breast carcinoma xenografts. Collectively these findings demonstrate that HER2/neu overexpression stimulates anti-apoptotic cell survival mechanisms and suggest that HER2/neu antisense ODNs may be of use in cancer therapeutics.

Suppressor T cells and the immune response to tumors.

In this paper we have attempted to define the role of suppressor T cells in many well-defined murine tumor systems. We have knowingly omitted a blocking antibodies, suppressor B cells as mediators of tumor immunosuppression in various murine tumor systems; these have been well reviewed elsewhere. Also, we have focused on the importance of two different types of antigen-presenting cells in the induction and suppression of cell-mediated immunity and on some of the different modalities employed to inhibit Ts function. Finally, we have discussed the acquired immunodeficiency syndrome and the possible role of a defective helper pathway and enhanced suppressor pathway in its pathogenesis. We and others believe that the suppressor pathway is preferentially activated by tumor antigen(s) in the cases of many immunogenic murine tumors--possibly due to the release of tumor antigen(s) from tumor cells, their subsequent trafficking to specific areas of the spleen and other organs, and, ultimately, their presentation by certain APC to Ts. Ts may then act directly upon helper Lyt 1+2- T cells as these cells interact with tumor antigen(s) on I-A+ APC. Alternatively, if the effector pathway were somehow impaired--e.g., by ultraviolet radiation or a virus--then the suppressor pathway may be activated in an unregulated manner, often to the detriment of the host. Biochemical characterization of the tumor antigens that stimulate Ts generation and, presumably, tumor growth and definitive documentation of a role of APC in the processing and presentation of these tumor antigens to Ts need to be done. Then selective stimulation of the effector immune response, along with inhibition of the suppressor response, to tumor antigens with drugs, monoclonal antibodies, and soluble mediators or their analogues may be possible in the near future.

Synergistic antitumor effects of HER2/neu antisense oligodeoxynucleotides and conventional chemotherapeutic agents.

BACKGROUND: The HER2/neu oncogene is overexpressed in a substantial fraction of human tumors. HER2/neu overexpressing tumors may be intrinsically resistant to chemotherapy. The present study examined the ability of antisense-mediated downregulation of HER2/neu expression to enhance the antitumor effects of conventional chemotherapeutic agents against human tumor cells that overexpress HER2/neu. METHODS: The effects of HER2/neu antisense oligodeoxynucleotides (ODNs) on the growth inhibitory and proapoptotic activity of several distinct chemotherapeutic agents were examined in vitro. In vivo effects of HER2/neu antisense ODNs in combination with doxorubicin hydrochloride were assessed by examining the growth of human tumor xenografts implanted into nude mice. RESULTS: The proliferation of tumor cell lines that overexpress HER2/neu was inhibited by antisense ODNs in combination with conventional chemotherapeutic agents in an additive or synergistic fashion. Such combination therapy also demonstrated synergistic activation of apoptosis. HER2/neu antisense ODNs in combination with doxorubicin hydrochloride demonstrated synergistic antitumor effects in vivo as well. CONCLUSIONS: Downregulation of HER2/neu expression can enhance the sensitivity of human cancer cells, which overexpress HER2/neu to the cytotoxic effects of chemotherapy. Antisense ODNs targeting the HER2/neu gene may play a role in cancer therapy.

Management of diagnostic dilemmas of the pancreas by ultrasonographically guided laparoscopic biopsy.

INTRODUCTION: Pancreatic lesions may be difficult to diagnose because of small size or inaccessibility. Such lesions are being seen with increasing frequency because of advances in pancreatic imaging techniques. In the past 18 months we have evaluated 14 patients whose pancreatic lesions could not be diagnosed by traditional means, including percutaneous biopsy. METHODS: With the patient under general anesthesia, the anterior surface of the pancreas was exposed by a three-trocar laparoscopic technique. The lesion was located by laparoscopic ultrasonography. A core biopsy needle was inserted into the lesion under simultaneous visual and ultrasonographic guidance using picture-in-picture techniques. RESULTS: The main diagnostic dilemma encountered was the differentiation of pancreatic cancer from pancreatitis. Other conditions were lymphoma and renal cell carcinoma. Excellent tissue samples were obtained, allowing diagnosis and planning of treatment in all cases. Operative time ranged from 1 to 4 hours, and length of stay ranged from 1 to 3 days. Blood transfusions were not required, and there were no complications. Alcohol nerve block was performed laparoscopically in one patient in this group after the diagnosis was made by frozen section. CONCLUSIONS: Direct ultrasonographically guided laparoscopic biopsy provides rapid, safe diagnosis of pancreatic lesions.

The "hidden cystic duct" syndrome and the infundibular technique of laparoscopic cholecystectomy--the danger of the false infundibulum.

BACKGROUND: The "classical" biliary injury usually involves misidentification of the common bile duct as the cystic duct. The purpose of this study was to determine if the method of cholecystectomy, specifically the "infundibular technique," might be a contributing factor in this injury. STUDY DESIGN: Twenty-one operative notes of patients who were referred with injury to the common bile duct were examined. Notes were classified as to informativeness. Patient and operative variables potentially related to injury were searched for. RESULTS: Inflammation was the main patient variable associated with injury. The main operative variable was that in most of the injuries the cystic duct was isolated and divided as the first step in the procedure. Often the operative note contained a statement indicating that the surgeon believed that the "cystic" duct (actually the common bile duct) was emanating from the infundibulum of the gallbladder and that this was the anatomic rationale for identification of the cystic duct. In no case was the triangle of Calot completely dissected before injury. CONCLUSIONS: The cystic duct may be hidden in some patients having laparoscopic cholecystectomy, especially in the presence of inflammation. This may lead to the deceptive appearance of a false infundibulum that misleads the surgeon into identifying the common duct as the cystic duct. Biliary injury is more likely when cystic duct identification is made by relying solely on the appearance of the junction of the cystic duct with the infundibulum of the gallbladder, and this technique should be abandoned.

Results of a new strategy for reconstruction of biliary injuries having an isolated right-sided component.

Poor results after repair of biliary injuries are most common when injuries are above the bifurcation of the left and right hepatic ducts or involve aberrant ducts. We have developed a novel approach to the right-sided component of such injuries. Preoperatively all isolated sections of the biliary tree are intubated percutaneously. At surgery the left duct is found by the Hepp-Couinaud approach. Dissection is continued to the right, staying within the coronal plane of the left hepatic duct, and continuing across the gallbladder plate into segment 5 between the hepatic parenchyma and the Wallerian sheath of the right portal pedicle. Hepatic parenchyma, anterior to the sheath, is resected. After a length of portal pedicle is exposed, right-sided bile ducts are opened on their anterior surface, using the percutaneous transhepatic stents as a guide, and hepaticojejunostomy is performed. Twenty-three patients were treated from May 1993 to February 1999. Injury types and (number of patients) were as follows: B (n = 2), C (n = 5), E4 (n = 10), and E5 (n = 6). There were no perioperative deaths. Follow-up ranged from 8 months to 7 years (median 3 years). There have been no cases of restricture, reoperation, or jaundice, and no interventional procedures. Serum bilirubin is normal in all patients. Alkaline phosphatase is normal or less than two times the normal value in 21 of 22 living patients. This novel approach brings the benefits of the Hepp-Couinaud approach to the right hepatic ducts. Very satisfactory results were obtained in the most severe types of biliary injury.

Pancreaticoduodenectomy for lymphoepithelial cyst of the pancreas.

Lymphoepithelial tumors of the pancreas are rare cystic tumors characterized by the presence of a keratinizing squamous epithelium and a dense lymphoid infiltrate on histologic examination. This case report describes the first lymphoepithelial tumor to be resected from the pancreatic head by pancreaticoduodenectomy. This case is also the first in which the cyst was found to be secondarily infected. The radiologic and clinicopathologic features of these unusual tumors are discussed.

Differential binding patterns of monoclonal antibody 2C4 to the ErbB3-p185her2/neu and the EGFR-p185her2/neu complexes.

2C4 (Pertuzumab, Omnitarg) is a monoclonal antibody targeting p185(her2/neu), which is overexpressed in 30% of invasive breast cancer. 2C4 is currently in phase II clinical trials for several types of cancers. This antibody has been reported to disrupt the association between p185(her2/neu) and ErbB3. In our studies of epidermal growth factor receptor (EGFR)-p185(her2/neu) heterodimerization, we noted that 2C4 formed associations with the EGFR-p185(her2/neu) receptor complex. Our data argue against 2C4 as a universal heterodimerization blocker for p185(her2/neu), but indicate that cocktails of monoclonal antibodies binding distinct interaction surfaces of p185(her2/neu) will emerge as the most potent targeted therapy.