Identification and characterization of a suite of tumor targeting peptides for non-small cell lung cancer.

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

Transumbilical single-incision laparoscopic distal gastrectomy for early gastric cancer.

BACKGROUND: Laparoscopic gastrectomy is a widely accepted procedure for treating early gastric cancers. This procedure is less invasive than conventional open approaches, and the oncologic outcomes are comparable. Single-incision laparoscopic surgery, developed to reduce the invasiveness of traditional laparoscopy, is applied to various abdominal surgical procedures. However, its application to laparoscopic gastrectomy for the treatment of gastric cancer has not been reported, mainly because of difficulties achieving adequate lymphadenectomy and reconstruction. The authors report their initial clinical experience with single-incision laparoscopic gastrectomy for early gastric cancer. METHODS: A single vertical 2.5-cm intraumbilical incision was made, and three laparoscopic trocars were placed within the umbilicus. A 2-mm mini-loop retractor was inserted in the left upper and middle abdomen, and a roll of gauze was attached to its tip. This instrument is an atraumatic and useful tool for retracting various organs. Gastric mobilization and adequate dissection of lymph nodes were performed. The stomach and duodenum then were transected intracorporeally using linear staplers. Intracorporeal anastomosis was performed for reconstruction. RESULTS: All seven single-incision laparoscopic distal gastrectomies with lymphadenectomy were performed without the use of additional trocars or conversion to laparotomy. The median time for gastric mobilization with lymphadenectomy was 155 min (range, 130-183 min). The median operative time was 344 min (range, 282-385 min), and the median estimated blood loss was 25 ml (range, 0-100 ml). A median total of 67 lymph nodes were retrieved. No serious perioperative complications occurred, and no mortalities were observed in this case series. CONCLUSIONS: The authors' initial experience with single-incision laparoscopic distal gastrectomy showed that it is a feasible and safe procedure for early gastric cancer and gives a favorable cosmetic result. To the authors' knowledge, this is the first report describing successful single-incision laparoscopic gastrectomy for gastric cancer.

Synthesis and characterization of a high-affinity {alpha}v{beta}6-specific ligand for in vitro and in vivo applications.

The α(v)ß(6) integrin is an attractive therapeutic target for several cancers due to its role in metastasis and its negligible expression in normal tissues. We previously identified a peptide from a phage-displayed peptide library that binds specifically to α(v)ß(6). The tetrameric version of the peptide has higher affinity for its cellular targets than the corresponding monomers. However, the inefficient synthesis limits its clinical potential. We report here a convergent synthesis producing the tetrameric peptide in high yield and purity. The ease of the synthesis allows for rapid optimization of the peptide. We have optimized this α(v)ß(6) integrin-binding peptide, determining the minimal binding domain and valency. Importantly, the half-maximal binding affinity of the optimal peptide for its target cell is in the 40 to 60 pmol/L range, rivaling the affinity of commonly used antibody-targeting reagents. This peptide mediates cell-specific uptake, is functional in diagnostic formats, is stable in sera, and can home to a tumor in an animal. We anticipate that this high-affinity ligand for α(v)ß(6) will find clinical use as a diagnostic and therapeutic reagent.

Totally laparoscopic sigmoid colectomy: a simple and safe technique for intracorporeal anastomosis.

BACKGROUND: Current techniques of laparoscopic colectomy require an abdominal incision for extraction of the specimen. Although this incision is smaller than that for open laparotomy incision, it may reduce the advantages of laparoscopic surgery. In totally laparoscopic sigmoid colectomy, intracorporeal anastomosis is technically difficult. A safe and simple technique for circularly stapled intracorporeal anastomosis is described. METHODS: After mobilization of the colon and division of the mesentery, a semicircumferential colotomy is made at the anterior colonic wall just proximal to the transection site. The anvil of a circular stapling device, secured with a Prolene suture, is introduced via the colotomy. The suture is advanced anteriorly so that the center rod of the circular stapling device penetrates the colonic wall. The colon is staple-transected at this point to secure the anvil on the proximal colon. A grasping forceps is brought through the rectum, and the specimen is extracted through the colotomy made at the distal staple line. After the colotomy is reclosed with a linear stapler, anastomosis is established using a hemidouble stapling technique. RESULTS: Totally laparoscopic sigmoid colectomies were performed for 16 patients with colon cancers. All the patients were treated laparoscopically without any complications. The average operation time was 180 min. Although one patient experienced wound infection, no major complications occurred. There was no mortality in this series. CONCLUSIONS: The procedure of totally intracorporeal anastomosis combined with transanal extraction of the specimen can be performed easily, enabling surgeons to achieve minimal invasiveness comparable with that of hybrid natural orifice translumenal endoscopic surgery (NOTES).

A simple and safe method for gastrojejunostomy in laparoscopic distal gastrectomy using the hemidouble-stapling technique: efficient purse-string stapling technique.

AIM: To describe a simple technique for intracorporeal circular-stapled gastrojejunostomy in laparoscopic distal gastrectomy with Roux-en-Y reconstruction. METHODS: After the stomach and duodenum were mobilized, gastrotomy was established in the anterior gastric wall. An anvil, which was secured with a suture needle, was inserted completely through the gastrotomy. The needle was advanced to the greater curvature of the gastric wall to enable penetration of the central rod into the gastric wall. Subsequently, the stomach was cut using a linear stapler to secure the anvil on the stomach and was sequentially transected using another linear stapler to achieve distal gastrectomy. Circular-stapled gastrojejunostomy was then performed intracorporeally using the hemidouble-stapling technique, while handling the shaft of the instrument via the umbilical incision. The jejunal stump was closed using a linear stapler. RESULTS: Gastrojejunostomies were successfully performed in 20 gastric cancer patients using this technique. None of the patients showed anastomotic leakage and/or stenosis. There were no mortalities in this series. CONCLUSIONS: Gastrojejunostomy performed using the above-mentioned technique was safe and simple. The most important feature of the technique was the elimination of the need for purse-string suture placement, as well as the achievement of better cosmesis using the transumbilical approach.

Totally laparoscopic low anterior resection for lower rectal cancer: combination of a new technique for intracorporeal anastomosis with prolapsing technique.

BACKGROUND: Laparoscopically assisted low anterior resection is difficult even for experienced surgeons because of difficulties in selecting the appropriate transection line and completing anastomosis in the narrow pelvic space. The prolapsing technique resolves these problems. We combined our new technique for intracorporeal anastomosis with this prolapsing technique and achieved a totally laparoscopic low anterior resection. METHODS: After the total mesorectal excision, a semi-circumferential colotomy is made at the anterior colonic wall just proximal to the proximal transection site. The anvil of a circular stapling device, secured with a Prolene suture, is introduced via the colotomy. The suture is advanced anteriorly so that the center rod of the anvil penetrates the colonic wall. The colon is staple-transected at this point to secure the anvil on the proximal colon. The distal rectum is everted and pulled transanally outside the body using a grasping forceps inserted from the anus. Staple-closure and transection of the distal rectum is performed under direct vision. Anastomosis is established using the double-stapling technique. RESULTS: Totally laparoscopic low anterior resections using this technique were performed for 7 patients with rectal cancer. There was no anastomotic leakage/stenosis. CONCLUSIONS: Our procedure can be performed easily, which enables surgeons to achieve minimal invasiveness compared with hybrid NOTES.

A simple and safe technique for esophagojejunostomy using the hemidouble stapling technique in laparoscopy-assisted total gastrectomy.

In laparoscopy-assisted total gastrectomy, esophagojejunostomy is technically difficult. We describe a safe and simple technique for circular-stapled esophagojejunostomy. After mobilization of the stomach and the esophagus, a semicircumferential esophagotomy is made at the anterior esophageal wall. An anvil of a circular stapling device, secured with a Prolene suture (Ethicon, Inc, Somerville, NJ), is introduced via the esophagotomy. The suture is advanced anteriorly so that the center rod penetrates the esophageal wall. The esophagus is staple transected at this point. The circular-stapled esophagojejunostomy is then performed using the hemidouble stapling technique. Laparoscopy-assisted total gastrectomies were performed in 10 patients with gastric cancers. All patients were completed laparoscopically without any complications. The time of anvil placement was 9 minutes in median. Although a wound infection occurred in 1 patient, there were no major complications. There was no mortality in this series. Esophagojejunostomy using this technique is safe and simple. Its practical value is the elimination of the need for pursestring suture placement.

Multiple benign metastasizing leiomyomas in the pelvic lymph nodes and biceps muscle: report of a case.

A 47-year-old woman with an earlier history of uterine leiomyoma suffered from multiple recurrent tumors in the retroperitoneal lymph nodes and biceps muscle of the right upper arm. The woman with a right lower abdominal tumor was referred to our hospital. An abdominal computed tomography scan revealed two round nodules with well-defined margins in the retroperitoneum in the pelvis, and echography revealed a similar nodule in the biceps of the right upper arm. A biopsy of the abdominal retroperitoneal tumor demonstrated benign metastasizing leiomyoma (BML). An extirpation of the abdominal tumors was therefore performed. After the operation, false climacteric medical treatment was performed for 3 years and no recurrence has since been observed. This is the first reported case of multiple BML in the lymph nodes and muscle occurring simultaneously.

[A case of advanced gastric cancer with peritoneal dissemination responding to S-1/CDDP neoadjuvant chemotherapy].

The patient was a 72-year-old male diagnosed with type III poorly-differentiated adenocarcinoma in the lesser curvature by gastric fiberscopy. An abdominal computed tomography (CT) scan showed the thickness of the gastric wall and the enlarged lymph node around the stomach and laparoscopic examination revealed peritoneal dissemination. The patient received neoadjuvant combined chemotherapy with S-1 and CDDP. S-1 (100 mg/day) was orally administered for 3 weeks followed by 2 drug-free weeks as a course, and CDDP (100 mg/body) was administered by intravenous drip on day 8. After the third course, significant tumor reduction was obtained. Total gastrectomy, splenectomy and D2 nodal dissection were performed. Peritoneal dissemination disappeared, and the histological diagnosis revealed complete disappearance of cancer cells in the ascites and no metastasis in all lymph nodes. The patient has now been in good health with no recurrence for 22 months after surgery. The combined neoadjuvant chemotherapy with S-1 and CDDP can be an effective treatment of choice for advanced gastric carcinoma with peritoneal dissemination.

[A case of stage IV advanced gastric cancer responding to TS-1/CDDP neoadjuvant chemotherapy which leads to a pathological complete response].

A 72-year-old male with advanced gastric cancer (cT3N2M0H0P0CY1, cStage IV) was treated with TS-1/CDDP as neoadjuvant chemotherapy. TS-1 (60 mg/m(2)/day) was orally administered for 3 weeks followed by 2 drug free weeks as a course, and CDDP (60 mg/m(2)) was administered by intravenous drip on day 8. After the fourth course,a significant tumor reduction was obtained. Total gastrectomy, splenectomy, and D 2 type nodal dissection were performed. The histological diagnosis revealed complete disappearance of cancer cells in the stomach and all of the lymph nodes, which is a so-called pathological complete response. The patient has now been in good health without a recurrence for 24 months after surgery. This case suggests that neoadjuvant chemotherapy with TS-1/CDDP is a potential regimen for advanced gastric cancer.

Unresectable gastric cancer with multiple liver metastases effectively treated with combined paclitaxel and doxifluridine chemotherapy.

We report a 72-year-old man who was diagnosed by gastroscopy as having a type III poorly differentiated adenocarcinoma in the lesser curvature, with the longest diameter being 10 cm. An abdominal computed tomography (CT) scan revealed multiple liver metastases, thickening of the gastric wall, and an enlarged paraaortic lymph node. The serum carcinoembryonic antigen (CEA) level was 60 ng/ml and the carbohydrate antigen (CA) 19-9 level was 1355 U/ml. The patient received combined chemotherapy with doxifluridine (800 mg/body per day) and paclitaxel (one course comprised three weekly infusions at a dose of 70 mg/m(2) followed by 1-week rest). After the completion of three courses, the patient achieved a complete response (CR), with complete disappearance of the primary tumor, the metastatic foci in the liver, and the enlarged abdominal lymph nodes; as well, the tumor markers were normalized. Adverse effects included only mild anorexia that was limited to grade 1. He maintained a CR for 1 year and 2 months. Combination chemotherapy with paclitaxel and doxifluridine can be an effective treatment for unresectable advanced gastric carcinoma.

[Combined chemotherapy with weekly Paclitaxel and doxifluridine for advanced and recurrent gastric cancers].

We conducted combined therapy of weekly paclitaxel and doxifluridine (5'-DFUR) for 23 cases of advanced and recurrent gastric carcinomas to investigate their efficacy and safety. Subjects included 7 unresectable cases, 5 noncurative resection cases, and 11 recurrent cases. Twenty of the 23 subjects had a history of prior treatment with another anticancer drug. The treatment regime consisted of one course comprising 70 mg/m(2)of paclitaxel weekly for three consecutive weeks followed by one week rest, combined with 800 mg/day of 5'-DFUR orally. Results revealed a response rate of 17.6% (3/17), with 2 cases of CR, 1 case of PR, 10 cases of NC, and 4 cases of PD. One of the CR cases was an unresectable case involving a primary tumor, liver metastasis, and abdominal lymph node metastasis, while the other was a recurrent case involving abdominal lymph node metastasis. The median survival period was 387 days. The one-and two-year survival rates were 52% and 24%, respectively. In terms of adverse effects, there were only single cases of grade 3 leukopenia and grade 3 neutropenia, with no cases of grade 4 hemotoxicity. Both patients could be treated as outpatients. Combination therapy of weekly paclitaxel and 5'-DFUR can be an effective and safe therapy for advanced and recurrent gastric carcinomas.

Isolation of multiple cell-binding ligands from different phage displayed-peptide libraries.

A technical challenge in the development of biosensor devices for cancer detection and diagnosis is the identification of ligands that recognize cancer cells with high affinity and specificity. Furthermore, it is unlikely that one cell-binding ligand will provide sufficient biological information, thus, multiple ligands for a given cancer type will be needed for confident clinical diagnosis. Biopanning of phage displayed peptide libraries is a route to isolation of specific cell-binding reagents. A potential approach towards isolation of multiple ligands for a single cell type is to pan against the same cell type using different peptide libraries. Here we report the synthesis of a new 20-mer peptide-phage library and its use to select a peptide that binds to the large cell lung carcinoma cell line, H1299. The isolated phage clone binds H1299 cells 80 times better than a control phage and can distinguish between H1299 and normal control cells. The phage clone also binds to the lung pleura epidermoid cell line, Calu-1 but not to all lung carcinoma cell lines. The peptide is functional outside the context of the phage and tetramerization of the peptide on a trilysine core improves the affinity of the peptide. The tetrameric peptide can be used to deliver a fluorescent quantum dot to H1299 cells. Unexpectedly, the peptide shares sequence similarity to a previously isolated H1299-binding peptide isolated from a different 20-mer peptide library. Data suggests that the two peptides target the same cellular receptor. Our results imply that cell-based biopanning can isolate cell-binding ligands that may be of utility for cancer diagnosis, and isolation of cell-targeting peptides from different peptide libraries can expand the repertoire of cell-binding reagents.

Cell-specific delivery of a chemotherapeutic to lung cancer cells.

We report that lung cancer-targeting peptides isolated from a peptide library can be used to deliver an active chemotherapeutic in a cell-specific fashion. The peptides were removed from the context of the phage and placed on a pegylated tetrameric scaffold. The tetrameric peptides were shown to block uptake of their cognate phage. The tetrameric peptides were coupled to doxorubicin, and their cytotoxicity against a panel of different cell lines was tested. Our data demonstrate that these targeting peptides can deliver an active anticancer agent in a cell-specific fashion, resulting in an increase of the therapeutic index of the targeted drug compared to systemic delivery. The efficacy of the peptide conjugate correlates to the affinity of the targeting peptide for a particular cell line. As such, we have demonstrated that cell-specific targeted drugs can be synthesized, even when the cell surface target is unknown.

Antitumor effect to IL-12 administration into the portal vein on murine liver metastasis.

BACKGROUND/PURPOSE: Interleukin (IL)-12 has been shown to possess potent antitumor activity, and an antitumor effect of systemic IL-12 administration has been reported in liver metastasis models. METHODS: In this study, we examined the usefulness of local IL-12 administration into the portal system in the treatment of liver metastasis. First, we confirmed the antitumor effect of recombinant IL-12 (rIL-12) on MC-38 tumors in an intracutaneous model. In the murine liver metastasis model, 1 x 10(5) of MC-38 cells were injected into the portal vein on day 0, and the spleen was transpositioned subcutaneously for administration of rIL-12 continually into the portal system. From days 3 to 7, 0.1 micro g rIL-12 was administered intraperitoneally or intrasplenicly, while Hanks' Balanced Salf Solution (HBSS) was injected intrasplenicly in the control group. RESULTS: The liver weight in the rIL-12 intraperitoneal treatment group (1.88 +/- 0.37 g) and that in the rIL-12 intrasplenic treatment group (1.43 +/- 0.21 g) were significantly less than that in the HBSS group (2.86 +/- 0.74 g; P < 0.05). The numbers of metastatic nodules in the rIL-12 intraperitoneal treatment group (22.3 +/- 17.1) and in the rIL-12 intrasplenic treatment group (12.4 +/- 13.8) were significantly less than that in the HBSS group (137.1 +/- 44.9; P < 0.05). Complete regression of the tumor was observed in one of six mice in the rIL-12 intrasplenic treatment group. This antitumor effect of rIL-12 on MC-38 liver metastasis was not observed in interferon (IFN)-gamma knockout mice. Intraportal administration of IL-12-transduced fibroblasts, which were syngeneic to C57BL/6 mice, had an antitumor effect in the MC-38 liver metastasis model. CONCLUSIONS: These results suggested that the local administration of IL-12 into the portal system would be a useful strategy for the treatment of liver metastasis.