Therapeutic efficacy of T cells derived from lymph nodes draining a poorly immunogenic tumor transduced to secrete granulocyte-macrophage colony-stimulating factor.

We examined the host immune response to the poorly immunogenic B16-BL6 melanoma, which was transduced to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) (450 ng/10(6)/24 h). Tumor growth after subcutaneous inoculation was not significantly altered, although an influx of neutrophils and monocytes/macrophages was evident within tumors and draining lymph nodes (LNs). Immunization with irradiated transduced cells did not induce systemic immunity to the parental tumor. However, vaccination with transduced tumors significantly augmented in vivo sensitization of draining LN cells. These tumor-draining LN (TDLN) cells, when secondarily stimulated in vitro with anti-CD3 monoclonal antibodies and expanded in interleukin-2 (10 U/ml), exhibited greater release of GM-CST and interferon-gamma against tumor compared with TDLN cells from animals with parental tumor. In adoptive immunotherapy, activated LN cells draining transduced tumors mediated significant reductions of the numbers of established pulmonary metastases compared with LN cells draining parental tumor, which were ineffective. In addition, the therapeutic efficacy of LN cells draining transduced tumors was significantly better than LN cells primed in vivo with tumor cells admixed with Corynebacterium parvum, which we have previously described as an approach to generate immune cells. Thus, GM-CSF appears to be an important adjuvant in the induction of tumor immunity.

Immune responsiveness to a murine mammary carcinoma modified to express B7-1, interleukin-12, or GM-CSF.

This report characterizes the immunological host response to a syngeneic murine mammary carcinoma along with variants genetically modified to express B7-1 or secrete GM-CSF and interleukin-12 (IL-12). MT-901 is a subline of a mammary adenocarcinoma that was chemically induced in the Balb/c host. It was found to be weakly immunogenic by immunization/ challenge experiments, and it induced tumor-specific T-cell responses in lymph nodes (LN) draining progressive subcutaneous tumors. Tumor clones expressing B7-1 or secreting GM-CSF exhibited reduced tumorigenicity without completely abrogating tumor growth, whereas IL-12 elaboration lead to complete tumor growth inhibition. In vivo subcutaneous inoculation of a transgenic cell clone secreting GM-CSF (240 ng/10(6) cells/24 hours) resulted in significantly enhanced T-cell reactivity of tumor-draining lymph node (TDLN) cells as compared to wild-type TDLN cells. This finding was obtained from observations assessed by several different methods, including: 1) in vitro cytotoxicity, 2) in vitro interferon-gamma release, and 3) adoptive transfer in mice with established tumor. Moreover, the transfer of activated LN cells derived from mice inoculated with GM-CSF-secreting tumor cells resulted in the prolonged survival of animals with macroscopic metastatic disease, which was not evident utilizing LN cells from mice inoculated with wild-type tumor. By contrast, clones that expressed B7-1 or IL-12 (4 ng/10(6) cells/24 hours) did not elicit enhanced tumor-reactive TDLN cells compared with wild-type tumor when assessed in the adoptive transfer model. The autocrine secretion of GM-CSF by transduced tumor cells was found to serve as an effective immune adjuvant in the host response to this weakly immunogenic tumor.

Genetic approaches to adoptive cellular therapy of malignancy.

The rapid strides made in recombinant gene technology have been the impetus for equally dramatic developments in our understanding of various aspects of cancer biology. These areas include genetic events leading to carcinogenesis, growth factors, cellular proliferation, differentiation, apoptosis, and metastasis. Novel therapeutic approaches that take advantage of this new knowledge involve biological alteration of the host target cell at a genetic level. Many of these approaches are discussed in greater detail in other sections of this issue. This article focuses on genetic approaches to the adoptive immunotherapy of malignancy. This form of cellular therapy refers to the infusion of tumor reactive immune cells to mediate regression of established tumor. This review is divided into several areas, each one involving different methods of genetic manipulation to generate immune cells for subsequent adoptive transfer and include: (1) the use of gene-modified tumors to serve as immunogens to generate antitumor T cells, (2) genetic manipulation of effector cells to enhance antitumor reactivity; and (3) genetic construction of immunocompetent effector cells from naive cells.

Laparoscopic repair of lumbar hernias.

BACKGROUND: Lumbar hernias are rare defects in the posterolateral abdominal wall that may be congenital or acquired. Repairing these defects is difficult by virtue of their location and the inherent weakness of the surrounding tissues. We report a series of seven patients who had their lumbar hernias repaired laparoscopically at two institutions. STUDY DESIGN: We retrospectively reviewed all lumbar hernias repaired laparoscopically in our institutions within the last 16 months (August 1996 to November 1997). Postoperative followup was 1-15 months. RESULTS: Seven patients underwent laparoscopic repair. Five hernias were acquired defects and two were congenital. One to three defects were found per patient. The average size of the hernia defect was 77.8 cm2. We used a polypropylene or a polytetrafluoroethylene mesh in all patients; the average size of the mesh used was 336.4 cm2. The average length of hospital stay was 1.7 days. One patient returned with an abscess over the mesh, which necessitated removal of the graft. Otherwise, there were no complications, and the remaining six patients had no recurrences after followup of 1-14 months. CONCLUSIONS: The laparoscopic approach is safe and effective for repairing lumbar hernias. Advantages of this approach include excellent operative visualization, decreased hospital stay postoperatively, and a solid repair without recurrence during shortterm followup.

The significance of pH and manometric testing after laparoscopic fundoplication.

Laparoscopic antireflux surgery has become the standard operation for gastroesophageal reflux disease (GERD). This study examined the outcomes of laparoscopic antireflux surgery, hypothesizing that both subjective symptoms and objective pH would correlate with manometric parameters to reflect the absence of reflux after fundoplication. We evaluated 56 patients who underwent laparoscopic antireflux surgery. Preoperative and postoperative symptoms were documented by chart reviews and confirmed by telephone interviews with the patient. Preoperative pH probe and esophageal manometry studies were compared with postoperative studies performed 3 to 6 months after fundoplication. Subjective symptoms were correlated with objective measurements of acid reflux and lower esophageal sphincter pressure (LESP). The follow-up period was 3 to 29 months. Symptomatic improvement was seen in 91% of patients, and good to excellent improvement in preoperative symptoms was cited. Postoperatively, there was significant improvement in percentages of upright supine times when esophageal pH was less than 4 (p <0.001). There was an increase in LESP from an average of 16.9 mmHg preoperatively to 22.7 mmHg postoperatively (p <0.001). There was no correlation between postoperative LESP and symptoms or LESP and 24-h pH results. However, there was a predictive correlation between LESP and postoperative heartburn symptoms (p <0.001). These findings imply that symptom follow-up evaluation is adequate in the asymptomatic patient after laparoscopic fundoplication, and that routine physiologic testing is not necessary.

Biopsy techniques for skin, soft-tissue, and bone neoplasms.

This article discusses important aspects of the biopsy of neoplasms in skin, soft tissues, and bone. There are a variety of clinical conditions in which specific biopsy techniques are indicated. Inappropriate biopsy of tumors in these sites may compromise subsequent definitive therapy.

Laparoscopic repair of recurrent ventral hernias.

Break down after repair of recurrent ventral hernias can exceed 50 per cent. Laparoscopic techniques offer an alternative. This study evaluated the efficacy of the laparoscopic approach for recurrent ventral hernias. A retrospective review on all patients with a recurrent ventral hernia who underwent laparoscopic repair at our institution from August 1995 to June 1997 was performed. Demographic, operative, postoperative, and follow-up data were collected. Thirty-one patients underwent an attempted laparoscopic ventral hernia repair. Sixteen were for recurrent hernias; 15 were successfully repaired laparoscopically. The patients were typically obese (mean body mass index, 30 kg/m2), had an average of 2.4 previous open repairs (range, 1-7), and six patients had previously placed intra-abdominal mesh. An average of 3.5 (range, 1-16) defects were found per patient with a mean total hernia size of 130 cm2 (6-480 cm2). In all cases, expanded polytetrafluoroethylene mesh (average, 299 cm2) was secured with transabdominal sutures. Postoperatively patients required an average of 19 mg of narcotics (MSO4 equivalent). Bowel function returned in 1.7 days. Length of stay averaged 2.0 days (1-4 days). There were two complications: cellulitis, which resolved with antibiotics, and skin break-down, which required mesh removal. With follow-up averaging 18 months (7-29 months), there is one recurrence; the case in which the mesh was removed. Laparoscopic repair of recurrent ventral hernia seems promising. Decreased hospital stays, postoperative pain, wound complications, and a low rate of recurrence are benefits of this technique.

Alternative site entry for laparoscopy in patients with previous abdominal surgery.

Laparoscopic procedures are increasingly performed in patients who have undergone prior abdominal surgery. Safe entry into the peritoneum includes avoidance of underlying viscera often tethered to the abdominal wall from surgical adhesions. Our group describes an alternative site technique utilizing the open Hasson procedure in a previously unoperated field, thus avoiding potential underlying adhesions. During the past 24 months this technique has been performed successfully in 95 patients, and no open conversions due to visceral or vascular injuries were necessary. Previous abdominal surgery should not be an absolute contraindication to minimally invasive procedures.

The mini-laparoscopic intragastric resection of a gastroesophageal stromal tumor: a novel approach.

The laparoscopic wedge resection of gastric stromal tumors has been shown to be safe and effective. The removal of these tumors at the gastroesophageal junction is not amenable to a typical local resection because of anatomic inaccessibility and potential luminal restriction after resection. Also, an esophagogastrectomy is not tenable in the setting of benign disease. The recent advances in miniaturized laparoscopic instrumentation (2-mm diameter) have expanded the laparoscopic options, including intraluminal resection. The first intragastric mini-laparoscopic resection of the gastric stromal tumor at the gastroesophageal junction is reported.

Migrating calcified enterolith and chronic anemia: an unusual case presentation of a Meckel's diverticulum.

Meckel's diverticulum is the most common congenital anomaly of the small intestine, occurring in about 2 % of the population. The most common complications associated with a Meckel's diverticulum include obstruction, bleeding, and inflammation (7, 9, 11, 18-20). The estimated lifetime risk of developing symptoms with a Meckel's diverticulum is 4-6 % (16), with the risks of complications decreasing with age. Stones within Meckel's diverticulum are recognized as a rare complication in the adult population (13,15). However, it has not been reported in the pediatric age group. The authors describe a 19-month-old male who presented with intermittent abdominal pain and vomiting, chronic microcytic anemia and a calcified stone in the lower abdomen, who was found to have a Meckel's enterolith.

[Minimally invasive procedures of the pancreas]. / Minimal-invasive Verfahren am Pankreas.

Laparoscopic pancreatic surgery is technically difficult and demanding owing to its retroperitoneal location and its complex anatomic relationships. Performing these operations requires familiarity with the anatomy of the retroperitoneum and advanced laparoscopic skills. Intracorporeal suturing skills are also mandatory. Due to the small number of operations performed in centers, an evaluation period of at least 5 years is necessary to determine the role of laparoscopic procedures in pancreatic disease processes. However, laparoscopic procedures are beginning to represent an undeniable part of the surgical repertoire against pancreatic pathology.

Diagnostic procedures and pretreatment evaluation of soft tissue sarcomas.

The diagnosis and preoperative evaluation of patients with a suspected soft tissue sarcoma involve several important considerations to ensure optimal treatment outcomes. Biopsy techniques must involve the retrieval of adequate tissue to establish the histologic diagnosis and grade of the tumor without compromising the subsequent definitive surgical resection. CT or MRI techniques represent the gold standard to evaluate the local extent of disease which is necessary to ascertain surgical resectability and/or the need for radiation therapy. CT scanning of the lungs is mandated for all patients with sarcomas to evaluate for metastatic disease and, for patients with abdominal or retroperitoneal tumors, scanning of the liver should also be included. Accurate staging of these patients will help define overall survival.

Laparoscopic adrenalectomy for cancer.

We will review the literature on the operative techniques and patient outcomes of laparoscopic adrenalectomy for cancer. Further, in our own study, an analysis of the preoperative assessment, operative, and hospital course, and postoperative follow-up was performed on all patients undergoing a laparoscopic adrenalectomy for cancer or metastasis from October 1996 through February 1998. Twelve laparoscopic resections were performed in 11 patients. There were six males and five females with an average age of 62 years (range, 40 to 79). The mean American Society of Anesthesiologists (ASA) score was 3.1 (range, 2 to 4). All of the tumors except one were due to metastatic cancer. The metastatic sources included renal cell cancer (four), lung cancer (two), colon cancer (two), adrenal cancer (one), and melanoma (one). Seven patients required a left adrenalectomy, three underwent a right adrenalectomy, and one was bilateral. The approach was transperitoneal in eight cases and retroperitoneal in four. The mean size of the tumors was 5.9 cm (range, 1.8 to 12 cm). Operative time averaged 181 minutes (range, 100 to 315 minutes), and blood loss was 138 cc (range, 20 to 1,300 cc). Average hospital stay was 2.3 days (range, < 1 to 6 days). One patient required conversion to an open approach due to local invasion of the tumor into the lateral wall of the vena cava, which was resected with the specimen. This procedure resulted in the largest blood loss of the series (1,300 cc). All specimens had negative surgical margins. There was one complication (9%), a laceration of the epigastric artery, which was controlled laparoscopically. At a mean follow-up of 8.3 months (range, 0.5 to 19 months), there have been no port site or local recurrences. One patient has developed a new hepatic nodule, which is being worked up for metastatic disease. Ten of the 11 patients (91%) are currently alive; one has died of expansive cerebral metastases from melanoma.

Concurrent induction of CD4+ and CD8+ T cells during tumor growth with antitumor reactivity in adoptive immunotherapy.

We have previously reported that the poorly immunogenic D5 melanoma transduced to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) will elicit immunity in tumor-draining lymph node (TDLN) cells after subcutaneous inoculation. After in vitro activation with anti-CD3 and interleukin-2 (IL-2), these cells acquire in vivo antitumor reactivity to wild-type tumor in the adoptive immunotherapy of pulmonary metastases. Using monoclonal antibodies, depletion of CD4+ or CD8+ T cells immediately after the adoptive transfer of activated TDLN cells revealed that both subsets could mediate the regression of tumor in the absence of exogenous IL-2 administration. CD8+ cells were more potent than CD4+ cells in mediating tumor regression on a per cell basis. We found that the exogenous administration of IL-2 enhanced the antitumor efficacy of CD4+ T cells. Purified CD4+ and CD8+ TDLN cells that were activated separately in culture released GM-CSF and interferon-gamma in response to wild-type tumor in vitro and mediated tumor regression in vivo. Last, the induction of either immune CD4+ or CD8+ T-cell subset during growth of the GM-CSF-secreting melanoma was found to be unaffected by the depletion of the alternate T-cell subset before tumor inoculation. These findings demonstrate that both CD4+ and CD8+ T cells can independently acquire therapeutic reactivity and presumably recognize two separate epitopes involved in tumor rejection.

Histologic and morphologic effects of rotary atherectomy on human cadaver arteries.

Intimal splitting and medial dissection caused by balloon angioplasty and thermal burns and subintimal vacuolization caused by laser angioplasty have been proposed as potential causes of failure from these respective procedures. The purpose of this study was to evaluate the histologic and morphologic effects of a new high speed rotary atherectomy device (the Rotoblator) on human cadaver arteries. Fifteen stenotic human cadaver superficial femoral and popliteal arteries were harvested and atherectomized using the Rotoblator. Histologically, on cross-section, the atherectomized arteries exhibited a denuded endothelial layer, a smooth rounded luminal contour, minute plaque separation from the media, absence of plaque fragmentation, and variable remaining rim of atheromatous plaque. The media showed variable loss of the internal elastic lamina, normal remaining elastin, and normal smooth muscle cells. The adventitia was normal and completely intact in all sections with no media/adventitia separation or perforations. Scanning electron microscopy revealed endothelial peeling, etching from the atherectomy devices within the smooth muscle layer, and a tapered atherectomy distal endpoint. The arterial branches were preserved without evidence of any disruption of the branch artery orifice. Compared to balloon angioplasty and laser angioplasty, the Rotoblator atherectomy device appears to leave a smoother, more rounded luminal surface that may result in less risk of embolic complications and arterial wall damage.

Laparoscopic cryoablation of hepatic metastases.

Cryosurgery for liver metastases may improve survival for unresectable hepatic metastases. The laparoscopic approach to managing these tumors is a novel method fostered by increasing surgeon and patient interest in minimally invasive surgical techniques and the development of laparoscopic ultrasound and cryoprobes. A retrospective review of our patients who underwent laparoscopic cryoablation of hepatic tumors from April 1996 to December 1997 was conducted. We report on this experience and comment on the feasibility and safety of the procedure based on this early trial.

Splenic artery aneurysms: methods of laparoscopic repair.

PURPOSE: Surgical therapy for splenic artery aneurysms (SAAs) has traditionally consisted of a laparotomy with resection of the aneurysm and possibly a splenectomy. Our early experience with the laparoscopic approach to treat SAAs is reported. METHODS: A retrospective review of medical records was conducted on all patients who underwent laparoscopic resection of SAAs at the Cleveland Clinic Foundation from May 1996 to August 1997. RESULTS: Four patients with SAAs, three women and one man, with an average age of 55 years (range, 37 to 63 years), underwent successful laparoscopic SAA repair. The average size of the aneurysm was 3.2 cm (range, 2.5 to 5.0 cm). Three patients underwent an aneurysm resection, whereas one patient underwent simple ligation. Intraoperative ultrasound scanning with Doppler was used in three cases as a means of localizing the aneurysm and identifying all feeding vessels; the complete cessation of flow within the aneurysm in the case in which the feeding vessels were simply ligated was also documented. The average intraoperative time was 150 minutes (range, 100 to 190 minutes). The mean estimated blood loss was 105 mL (range, 20 to 300 mL). There were no intraoperative complications. The average hospital stay was 2.2 days (range, 1 to 4 days). CONCLUSION: The laparoscopic approach to splenic artery aneurysm by aneurysmectomy or splenic artery ligation can be safe and effective. The laparoscopic approach affords a short hospital stay and an effective result.

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes.

We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon gamma (IFN gamma) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFN gamma and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFN gamma secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to upregulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor.

Tumor-induced suppression of antitumor reactivity and depression of TCRzeta expression in tumor-draining lymph node lymphocytes: possible relationship to the Th2 pathway.

T lymphocytes from tumor-draining lymph nodes (TDLN), after activation and expansion in vitro, can mediate regression of metastatic tumor in animal models. We have shown that TDLNs are subject to tumor-induced suppression that is tumor specific, T-cell mediated, and dependent on the duration of tumor growth, but the mechanism of this suppression remains largely unknown. Recently, in other model systems, tumor-bearer T cells have been shown to have decreased expression of T-cell receptor-zeta (TCR zeta), a key component in antigen-driven activation pathways. We sought to investigate whether the suppression of TDLN reactivity that accompanies prolonged tumor growth was associated with decreased expression of TCR zeta in fresh and in vitro activated lymph node lymphocytes. Mice bearing subcutaneous tumor deposits of MCA 205 had TDLN cells harvested after various durations of tumor growth, then activated in vitro with anti-CD3 for 2 days (activation phase), followed by expansion with interleukin-2 (IL-2) (10 U/ml) for 3 days (expansion phase). Two-color flow cytometry was used to determine TCR zeta expression in fresh and activated TDLN cells. Antitumor reactivity was assessed by the ability of activated TDLN to mediate regression of lung metastases. There was a time-dependent suppression of the antitumor reactivity of the activated TDLN; activated TDLN from mice bearing tumors 14 days or less were able to mediate the regression of established lung metastases, whereas activated TDLN from animals bearing tumors 21 days or more were ineffective. In addition, TCR zeta expression on T lymphocytes from fresh and activated TDLN was also depressed in a time-dependent manner. Because tumor-induced immunosuppression in our model is known to be T cell mediated, we examined whether the Th2 cytokine IL-4, when added in vitro during activation or expansion, could suppress antitumor reactivity and lead to a depression in TCR zeta expression of TDLN cells in a fashion similar to prolonged tumor growth. The addition of 10 U/ml of IL-4 in vitro had a marked suppressive effect on the antitumor activity of day 14 TDLN; the effect was most pronounced when IL-4 was present during the expansion phase. Fluorescence-activated cell sorter analysis of day 14 TDLN exposed to IL-4 in vitro demonstrated a marked decrease in TCR zeta expression, comparable to that seen in late tumor-bearer TDLN. Thus, TDLN from late tumor-bearers show a consistent decrease in TCR zeta expression that is associated with suppressed antitumor reactivity, and exposure to IL-4 in vitro results in qualitatively and quantitatively similar changes. Our observations suggest a mechanism whereby Th2 cells could mediate immunosuppression by downregulating a critical component of the T-cell-receptor signal transduction machinery.