A simple technique for preventing bar displacement with the Nuss repair of pectus excavatum.

BACKGROUND/PURPOSE: The most common complication of the minimally invasive technique for repair of pectus excavatum (MIRPE) is bar displacement, which has been reported to occur in 9.5% of all cases, particularly in teenaged patients. The use of a lateral stabilizing bar has improved stability but has not eliminated the occurrence of this problem. The authors report a new technique added to the standard MIRPE that creates an additional third point of fixation of the pectus bar to prevent displacement. METHODS: The technique requires the simple placement, via a spinal needle, of a nonabsorbable suture next to the sternum, encircling a rib and the bar, using a single 3-mm stab wound and thoracoscopic guidance. The suture simply is buried under the skin. Since 1998, this technique has been applied to 20 patients who underwent MIRPE. RESULTS: The average age was 14 years; 80% were boys. Average operating time was 75 minutes, and all patients had thoracoscopy with the MIRPE. A lateral stabilizing bar also was used in 14 patients. Four patients had 2 struts placed. Average length of stay was 5.5 days. There were no early complications. Mean follow-up was 12 months. Bar displacement occurred in 1 patient early in the series in which an absorbable suture was used for fixation. One patient had a prolonged hospital stay of 7 days because of postoperative pain. CONCLUSIONS: This modification to the original technique of MIRPE creates a 3-point fixation system that minimizes the risk of bar shifting even in teenaged patients. It does not add any significant time or cost to the operation, and it is fairly simple to perform. The authors believe that this technique decreases the occurrence of bar displacement, and they recommend its use for all patients with pectus excavatum considered candidates for the Nuss repair.

Restoration of p53 function in anaplastic Wilms' tumor.

BACKGROUND/PURPOSE: Recent studies have reported a high incidence of p53 mutations in anaplastic Wilms' tumors (WT). Restoration of the normal p53 state by current gene therapy techniques is thus an attractive potential mode of therapy for this tumor, which is poorly responsive to standard therapy. The purpose of this study is to determine whether gene delivery of normal p53 is possible and to characterize the subsequent effect of restoring the wild-type p53 state. METHODS: Anaplastic WT RM1 cells (mutant p53) were transduced with replication-deficient adenoviral vectors containing either the wild-type p53 gene (rAd-p53) or the gene encoding a green fluorescent protein (rAd-GFP). The transduction efficiency of adenovirus for RM1 cells was determined by flow cytometric analysis of rAd-GFP-transduced cells. The effect of p53 transduction on cell viability was evaluated using a colorimetric proliferation assay. Apoptosis was evaluated by labeling DNA breaks using a TUNEL assay (Apo-Direct kit). RESULTS: Cells treated with increasing concentrations of viral particles relative to tumor cells (multiplicity of infection-MOI) showed a dose-dependent increase in the number of cells transduced. Twenty-four hours after viral treatment, the percentage of cells transduced for MOIs of 10, 50, 100, and 500 was 29.5, 60.9, 74.6, and 92.4, respectively; at 48 hours the percentage of cells transduced increased to 70.8, 90.7, 93. 7, and 96.3, respectively. Viral treatment at an MOI of 50 reduced cell proliferation by 10% at 17 hours and 97% at 5 days; at an MOI of 100, the relative reduction in proliferation was 15% and 99.8%, respectively. When assayed, 30% of cells became apoptotic at an MOI of 50, and 48% at an MOI of 100. CONCLUSIONS: Highly efficient delivery of the p53 tumor suppressor gene by adenoviral vector to anaplastic WT is possible. Subsequent restoration of the normal p53 state results in reduced viability and increased apoptosis. Gene replacement of p53 may represent a novel therapeutic agent for anaplastic Wilms' tumors.

Effectiveness of beta-glucan collagen for treatment of partial-thickness burns in children.

BACKGROUND/PURPOSE: Beta glucan collagen matrix (BGC), which combines the carbohydrate beta-glucan with collagen, has been used as a temporary coverage for adult partial thickness burns with reported good results. Observed advantages of BGC coverage include reduction of pain, improved healing, and better scar appearance. Potentially even more important in children is the elimination of painful daily dressing changes to the burned epithelial surface, as well as decreased fluid loss. This report details the authors' 2-year experience with BGC in a pediatric burn center. METHODS: Retrospective chart review of 225 consecutive pediatric patients treated at our institution between 1997 and 1999 identified 43 patients (19%) with suspected partial thickness burns treated with BGC as the primary wound dressing. BGC was applied to a debrided burn wound and secured with steri-strips, kerlix, and an ace wrap. After 24 hours, adherence of the BGC was confirmed and then left open to air. RESULTS: The most common cause of burn injury was scald (61%), followed by flame (37%), and contact (2%). The average age of patients was 5.5 years (range, 6 weeks to 16 years) and mean percent total body surface area burned was 9.3% (1% to 35%). Thirty-four patients (79%) had the BGC remain intact while the wound healed underneath, with excellent cosmetic results, minimal analgesic requirements, and no need for repetitive dressing changes. Nine patients (21%) had the BGC removed before wound healing: 6 patients lost the BGC because of progression of the burn to full thickness, 2 had BGC nonadherence over a joint, and 1 had an unexplained nonadherence. CONCLUSIONS: Partial-thickness burns in children can be effectively treated with BGC with good results, even in infants and toddlers. BGC markedly simplifies wound care for the patient and family and seems to significantly decrease postinjury pain.

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias.

Leukemic blasts from patients with acute phase chronic myeloid leukemic and refractory acute myeloid leukemia are highly resistant to a number of cytotoxic drugs. To overcome multi-drug resistance, we engineered a diphtheria fusion protein by fusing human interleukin-3 (IL3) to a truncated form of diphtheria toxin (DT) with a (G4S)2 linker (L), expressed and purified the recombinant protein, and tested the cytotoxicity of the DTLIL3 molecule on human leukemias and normal progenitors. The DTLIL3 construct was more cytotoxic to interleukin-3 receptor (IL3R) bearing human myeloid leukemia cell lines than receptor-negative cell lines based on assays of cytotoxicity using thymidine incorporation, growth in semi-solid medium and induction of apoptosis. Exposure of mononuclear cells to 680 pM DTLIL3 for 48 h in culture reduced the number of cells capable of forming colonies in semi-solid medium (colony-forming units leukemia) > or =10-fold in 4/11 (36%) patients with myeloid acute phase chronic myeloid leukemia (CML) and 3/9 (33%) patients with acute myeloid leukemia (AML). Normal myeloid progenitors (colony-forming unit granulocyte-macrophage) from five different donors treated and assayed under identical conditions showed intermediate sensitivity with three- to five-fold reductions in colonies. The sensitivity to DTLIL3 of leukemic progenitors from a number of acute phase CML patients suggests that this agent could have therapeutic potential for some patients with this disease.

Outcome analysis of minimally invasive repair of pectus excavatum: review of 251 cases.

BACKGROUND/PURPOSE: Since the first report in 1997 by Dr Nuss of the technique for minimally invasive repair of pectus excavatum (MIRPE), the popularity and demand for this operation has increased dramatically. Many pediatric surgeons became familiarized with MIRPE and have applied it to a large number of patients. Outcomes and complications have not yet been defined. METHODS: A comprehensive survey of APSA members was conducted to review technical problems, complications, and outcomes of this new technique. RESULTS: Of the 74 survey responders, 31 (42%) currently use the MIRPE as their procedure of choice, and 251 cases were reviewed. A total of 74.2% of surgeons relied on direct observation and written documentation to obtain training in MIRPE. Less than 60% used the chest index in the preoperative assessment. A total of 98% used the Walter Lorenz bar for the MIRPE. The most common complication was bar displacement or rotation requiring reoperation (9.2%). Pneumothorax requiring tube thoracostomy was reported in 4.8%. Less common problems included infectious complications (2%), pleural effusion (2%), thoracic outlet obstruction (0.8%), cardiac injury (0.4%), sternal erosion (0.4%), pericarditis (0.4%), and anterior thoracic artery pseudoaneurysm (0.4%). Three patients (1.2%) required early strut removal. Reoperation using the open modified Ravitch approach was performed in 2 patients (0.8%). Most surgeons indicated that teenaged patients (>15 years old) were at higher risk for complications. Thoracoscopy in combination with MIRPE was used by 61% of the surgeons. Overall patient satisfaction was rated as excellent or good (96.5%). CONCLUSIONS: The relatively high incidence of problems with MIRPE is probably related to the learning curve associated with the introduction of this new technique. Awareness of technical details, careful patient selection, use of a stabilizing bar, and thoracoscopy likely will result in decreased complications. Long-term results are yet to be determined. The development of a national registry is of great importance for further outcome analysis of MIRPE.

A new method of treatment for complete tracheal rings in an infant: endoscopic laser division and balloon dilation.

PURPOSE: The authors describe a new technique for management of complete tracheal rings in infants. METHODS: The procedure consists of rigid bronchoscopy with KTP laser division, in the posterior midline, of the complete rings and gradual advancement of the bronchoscope aided by endoscopic balloon dilation. CONCLUSIONS: The laser division, coupled with balloon dilation, allows for controlled separation of the cartilages posteriorly. The anterior esophageal wall buttresses the posterior tracheal separation.

Recurrent pancreatitis in a child with pancreas divisum. Endoscopic therapy of a Santorinicele.

Pancreas divisum is a rare congenital anomaly of the pancreatic ducts that has been implicated in pancreatitis. In addition, the finding of a Santorinicele, which is a cystic dilatation of the dorsal duct, suggests that there is an obstruction associated with a congenital or acquired weakness of the mucosa. We used an endoscopic technique to treat a child with recurrent pancreatitis who was found to have pancreas divisum and a large Santorinicele.

Toxicology and pharmacokinetics of DTGM, a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human granulocyte-macrophage colony-stimulating factor, in cynomolgus monkeys.

We developed a fusion toxin consisting of the catalytic and translocation domains of diphtheria toxin linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF) (DTGM) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to determine the toxicity and pharmacokinetics of DTGM in cynomolgus monkeys (Macacca fascicularis), which possess cross-reactive GM-CSF receptors. Four groups of young adult monkeys (6 males and 12 females) were treated with five daily bolus iv infusions of 1, 5, 7.5, and 10 microgram/kg DTGM. Monkeys (2 males and 2 females) treated at 1 microgram/kg/day showed no significant side effects. Monkeys (2 males and 2 females) treated at 5 microgram/kg/day showed Grade 1-2 thrombopenia (NCI common toxicity criteria) on day 9. In contrast, monkeys (6 females) treated at 7.5 microgram/kg/day developed Grade 3 neutropenia, Grade 1-2 thrombopenia, Grade 1-3 anemia, and Grade 1-3 hypoalbuminemia. The neutropenia developed by day 4 in the 7.5 microgram/kg/day monkeys and by day 3 or 5 in the 10 microgram/kg/day monkeys and resolved in both groups by day 9, but the thrombopenia, anemia, and hypoalbuminemia persisted until day 16. Monkeys (2 male and 2 female) treated with 10 microgram/kg/day showed Grade 4 neutropenia that resolved by day 8 and Grade 2-3 anemia, hypoalbuminemia, and thrombopenia. Three of the animals developed sepsis. DTGM plasma half-life was 30 min with a peak concentration of 0.1 microgram/mL or 2 nM (1000-fold higher than the IC50 in vitro for AML blasts). Immune responses were minimal in all animals tested at 14 and 28 days with anti-DTGM levels <1 microgram/mL. All four animals at 10 microgram/kg died or were euthanized, and necropsies were performed. Animals necropsied on days 4 and 6 showed marked apoptosis and hypoplasia in the marrow, which was completely resolved for animals necropsied on day 9. No injury to other organs, including kidney, heart, liver, central nervous system, or lung, was seen. The drug was selectively toxic to malignant or differentiated myeloid cells with little toxicity to myeloid progenitors or other organs. Minimal effects in nontarget tissues make DTGM a promising candidate chemotherapeutic agent.

Diphtheria toxin fused to granulocyte-macrophage colony-stimulating factor and Ara-C exert synergistic toxicity against human AML HL-60 cells.

Human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GM-CSF) sensitized wild-type and Bcl2-overexpressing HL60 human leukemia cells to intoxication by Ara-C based on proliferation and clonogenic assays. The toxin/drug combination showed dramatic synergistic toxicity with combination indices of < 0.1. Synergy was not seen with two other protein synthesis inhibiting drugs--ricin and cycloheximide nor with GMCSF alone. No changes in Ara-C incorporation into cellular DNA or cell cycle occupancy were seen. As compared to exposure to DT388-GM-CSF or Ara-C alone, co-treatment produced significant increases in cytosolic accumulation of cytochrome c, a higher percentage of cells with loss of mitochondrial membrane potential and an increase in reactive oxygen species and morphologic changes of apoptosis, and a greater induction of poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor 45 (DFF45) cleavage activities of caspase 3. Co-treatment did not significantly alter Bcl2, Bcl-xL, Bax or Fas receptor (FasR), but modestly increased Fas ligand (FasL) protein. These finding suggest that co-treatment with DT388-GM-CSF may lead to a lowered apoptotic threshold and clonogenic survival of human AML blasts due to Ara-C. These observations also suggest that clinical trials of combination therapy may be warranted in patients with AML.

Primary laparoscopic-assisted endorectal colon pull-through for Hirschsprung's disease: a new gold standard.

OBJECTIVE: To describe the surgical technique and early clinical results after a one-stage laparoscopic-assisted endorectal colon pull-through for Hirschsprung's disease. SUMMARY BACKGROUND DATA: Recent trends in surgery for Hirschsprung's disease have been toward earlier repair and fewer surgical stages. A one-stage pull-through for Hirschsprung's disease avoids the additional anesthesia, surgery, and complications of a colostomy. A laparoscopic-assisted approach diminishes surgical trauma to the peritoneal cavity. METHODS: The technique uses four small abdominal ports. The transition zone is initially identified by seromuscular biopsies obtained laparoscopically. A colon pedicle preserving the marginal artery is fashioned endoscopically. The rectal mobilization is performed transanally using an endorectal sleeve technique. The anastomosis is performed transanally 1 cm above the dentate line. This report discusses the outcome of primary laparoscopic pull-through in 80 patients performed at six pediatric surgery centers over the past 5 years. RESULTS: The age at surgery ranged from 3 days to 96 months. The average length of the surgical procedure was 2.5 hours. Almost all of the patients passed stool and flatus within 24 hours of surgery. The average time for discharge after surgery was 3.7 days. All 80 patients are currently alive and well. Most of the children are too young to evaluate for fecal continence, but 18 of the older children have been reported to be continent. CONCLUSION: Laparoscopic-assisted colon pull-through appears to reduce perioperative complications and postoperative recovery time dramatically. The technique is quickly learned and has been performed in multiple centers with consistently good results.

High-level expression and purification of the recombinant diphtheria fusion toxin DTGM for PHASE I clinical trials.

A genetically engineered fusion toxin targeted to acute myeloid leukemic (AML) blasts was designed with the first 388 amino acid residues of diphtheria toxin with an H-M linker fused to human granulocyte-macrophage colony-stimulating factor. The cDNA was subcloned in the pRK bacterial expression plasmid and used to transform BL21 (DE3) Escherichia coli harboring pUBS500 plasmid. Transformants were grown in Superbroth and induced with IPTG. Inclusion bodies were isolated, washed, and denatured in guanidine hydrochloride with dithioerythritol. Recombinant protein was refolded by diluting 100-fold in cold buffer with arginine and oxidized glutathione. After dialysis, purified protein was obtained after anion-exchange, size exclusion on FPLC, and polymixin B affinity chromatography. The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Fifty-four 3-liter bacterial culture preparations were made and pooled into 27 batches. The final product was characterized by Coomassie Plus protein assay, Coomassie-stained SDS-PAGE, limulus amebocyte lysate endotoxin assay, human AML HL60 cell cytotoxicity assay, HPLC TSK3000, N-terminal sequencing, E. coli DNA contamination, C57BL6 mouse toxicity, and immunohistochemistry. Yields were 23 mg/liter bacterial culture of denatured fusion toxin. After refolding and chromatography, final yields were 24 +/- 4% or 5 mg/liter. Vialed product was sterile and 1.7 +/- 0.4 mg/ml in PBS. Purity by SDS-PAGE was 99 +/- 1%. Aggregates by HPLC were <1%. Potency revealed a 24-h IC50 of 2.7 +/- 0.5 pM on HL60 cells. Endotoxin levels were 1 eu/mg. The N-terminal sequence was confirmed, and E. coli DNA was <113 pg/mg. The LD10 in mice was 110 microg/kg/day x5. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 3 months at -80 and -20 degrees C. Further, the drug was stable at 4, 25, and 37 degrees C in human serum for 48 h. Drug reacted only with human monocytes, granulocytes, and myeloid precursors in frozen human tissue sections by immunohistochemistry. The synthesis of this protein drug should be useful for production for clinical phase I/II clinical trials and may be suitable for other diphtheria fusion toxins indicated for clinical development. This is the first report of the scaleup of a recombinant fusion toxin for clinical trials.

"One-stop" surgery: implications for anesthesiologists of an expedited pediatric surgical process.

BACKGROUND: "One-stop surgery" (OSS) allows pediatric patients to undergo initial surgical evaluation, anesthesia, surgery, and discharge home, on the same day. METHODS: Patients referred for umbilical hernia repair, circumcision, or central venous catheter removal completed a screening questionnaire, after which they were scheduled for initial surgical and anesthesia evaluation if eligible and had surgery if indicated on the same day. RESULTS: Three patients had comorbidity precluding OSS, two patients refused indicated surgery, two patients did not require surgery, and 12 patients did not keep their appointment. Eighty patients had surgery without complications. Average total time was significantly shorter for OSS than non-OSS for circumcision (120 vs 142 min) and umbilical hernia repair (139 vs 165 min) but similar for catheter removal (100 vs 109 min). All families were satisfied with OSS. CONCLUSIONS: One-stop surgery appears to be a safe, efficient, and convenient alternative to the traditional process for patients and their families.

Impact of segmental grafts on pediatric liver transplantation--a review of the United Network for Organ Sharing Scientific Registry data (1990-1996).

PURPOSE: The aim of this study was to assess the relative impact of segmental grafts from cadaveric and living donors on outcomes in 3,409 pediatric transplants (<18 years) between 1990 and 1996. METHODS: Analysis of the United Network for Organ Sharing (UNOS) Scientific registry data from 1990 to 1996 was performed. RESULTS: Liver grafts consisted of 2,636 whole grafts (WLG), 246 liver donor grafts (LDG), 89 split liver graft (SLG), and 438 reduced-size grafts (RSG). Although the number of pediatric transplants were unchanged between 1990 and 1996, segmental grafts made up an increasing proportion from 14.5% to 29.2%, and WLG decreased proportionately. The increase among segmental grafts occurred for LDG (threefold), followed by SLG (53%) and RSG (50%). One-year graft and patient survival rates for 3,409 transplants were 69.7% and 81.9%, respectively and were significantly higher (P<.001) in nonhospitalized patients than in hospitalized patients (79.8% and 91.3% v 61.0% and 73.7%). LDG graft survival (75.9%) was comparable with WLG(70.9%) but significantly better at 1 year than SLG (60.3%, P = .007) and RSG (61.1%, P = .001), even after excluding retransplants and ICU patients. Patient survival rates were not different statistically between groups. A separate analysis of outcomes in recipients less than 1 year of age suggested significantly better graft and patient survivals for LDG (83.3% and 89.4%) than for WLG (62.3% and 76.5%) and RSG (62.7% and 75%). CONCLUSIONS: Segmental liver grafts from cadaveric and living donors constitute an increasing proportion of pediatric transplants. Survival rates of cadaveric segmental graft are inferior to those of live donor segmental grafts even after adjustment for medical condition. Live donor grafts demonstrate consistently superior graft and patient outcomes in pediatric recipients less than 1 year of age, and should be promoted aggressively as a solution to the critical shortage of size matched grafts in small recipients.

One-stop surgery: evolving approach to pediatric outpatient surgery.

PURPOSE: Maximizing patient satisfaction is of prime importance in today's competitive outpatient surgery market. The authors recently devised a system, one-stop surgery, which simplifies outpatient surgery for pediatric patients and their families by combining the traditionally separate preoperative evaluation and subsequent operation into one visit. This report describes our initial experience with one-stop surgery. METHODS: Umbilical hernia repair, circumcision, and portacath removal were considered surgical procedures appropriate for our one-stop surgery pilot study. Medical information obtained by phone or fax from referring physicians was used to identify potential candidates. Families were contacted, precertified for their surgical procedure, and given nothing by mouth instructions. The day of surgery the child was evaluated by the attending pediatric surgeon. If the diagnosis was confirmed, and no contraindications to surgery were identified, the child immediately underwent the prescheduled surgical procedure. RESULTS: From April through October 1997, 61 children were scheduled for one-stop surgery. Nine patients (15%) were no shows, and one additional family opted not to proceed with circumcision. The remaining 51 children (83%) underwent their one-stop surgical procedure: umbilical hernia repair (n = 23), circumcision (n = 19), portacath removal (n = 8), and inguinal hernia repair (n = 1). No child had an anesthetic contraindication to surgery, and only one minor postoperative complication (wound hematoma) occurred. CONCLUSIONS: This pilot study has demonstrated that with appropriate patient screening and cooperation of the entire surgical team, a variety of outpatient surgical procedures can be handled using this one-stop surgery method. By combining one-stop surgery with our previously reported phone follow-up system, many minor surgical procedures can be managed with only one visit to the hospital. Decreasing the "hassle factor" of outpatient surgery for children and their families, who frequently live far from their closest children's hospital, while providing the highest quality of specialized surgical and anesthetic care, may potentially be a very powerful marketing tool for pediatric surgical specialists.

Acute chest syndrome in the postoperative sickle cell patient.

BACKGROUND/PURPOSE: Acute chest syndrome (ACS), a phenomenon of pulmonary sequestration in sickle cell disease (SCD) patients, is frequently missed in the postoperative SCD child. The constellation of symptoms range from fever and respiratory distress to abdominal discomfort. In its most fulminate state, the syndrome has been reported in some series to carry almost a 25% to 50% mortality rate in the postoperative patient. The incidence in pediatric patients in the era of minimally invasive surgery is unknown. METHODS: Since December 1995, 63 episodes of ACS have been documented in the nearly 500 SCD children seen at our institution. Six of 63 episodes occurred within 2 weeks after a surgical procedure under general anesthesia. During this period, 59 operations were performed by the pediatric surgery service on SCD patients with an ACS incidence of 10.2%. Careful review of the preoperative, intraoperative, and postoperative management of these patients was performed. RESULTS: All six received preoperative oxygen saturation monitoring and intravenous fluid (IVF) hydration. One half of these patients required transfusion to achieve a hemoglobin level of greater than 10 mg/dL. Documentation of intraoperative temperature, hypoxia, volume status, and hypercarbia as well as any atypical perioperative events were monitored and reviewed. All patients received postoperative oxygen supplementation and IVF hydration. Onset of ACS ranged from 1 hour to 7 days postoperatively. Only one of six was thought to be of microbial etiology (elevated mycoplasma titers), and all patients received prophylactic antibiotic and aggressive pulmonary therapy. Overall length of hospitalization was increased with an average stay of 6.1 days. There were no postsurgical ACS deaths. CONCLUSIONS: Despite close attention and avoidance of known risk factors for development of postoperative SCD complications, ACS occurred with an incidence much higher than previously reported in the literature (0.4% v 10.2%). Interestingly, five of six cases were after laparoscopic procedures suggesting that the advantages of laparoscopy, such as reduced postoperative pain, do not extrapolate to decreased incidence of ACS.

Pediatric laparoscopic biliary tract surgery.

Laparoscopic cholecystectomy is being performed with increasing frequency in children. The authors discuss the presentation, surgical technique, overall results, and potential complications associated with pediatric laparoscopic biliary tract surgery, citing a large personal experience as well as that reported in the literature.

A new technique for laparoscopic splenectomy with massively enlarged spleens.

Splenectomy is indicated in several hematological disorders and it can be particularly challenging in children with sickle cell disease, splenomegaly, and recurrent sequestration. Over the last 6 months, we have developed a new technique for laparoscopic splenectomy (LS) for hypersplenism and splenomegaly in five children with sickle cell disease. The average age of our patients was 6 years (range, 2-11), and the average weight was 18.7 kg (range, 13.2-30.1). On preoperative ultrasound, spleen size index ranged from 0.42 to 0.76. For the LS, four trochars were placed. One patient, who also underwent a laparoscopic cholecystectomy, had six trochars placed, two of which were used for both cholecystectomy and splenectomy. After laparoscopic mobilization of the spleen and hilar vascular stapling, a Steiner electromechanical morcellator was inserted through the 12-mm port to extract cores of splenic tissue until complete splenectomy was achieved. No patient required conversion to an open procedure or creation of a larger incision to remove the massively enlarged spleen. Operative time averaged 190 minutes; the combined LS and cholecystectomy took 245 minutes. Postoperative length of stay was <2 days for all patients. There were no complications, and no patient required postoperative transfusion. Based on these early findings, we conclude that intracorporeal coring of splenic tissue allows for safe and complete laparoscopic removal of very large spleens in small children. It provides expedient recovery and minimal postoperative pain and scarring. This new technique should enable surgeons to perform LS even in patients with massive splenomegaly, eliminating the need for large and cumbersome intracorporeal bags or the creation of additional incisions to remove the spleen.

Diphtheria toxin fused to granulocyte-macrophage colony-stimulating factor is toxic to blasts from patients with juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia.

We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor fused to a truncated diphtheria toxin (DT388-GM-CSF) is toxic to patient acute myeloid leukemia progenitors bearing the GM-CSF receptor, but not normal marrow progenitors. We now report that exposure of mononuclear cells from five of seven (71%) juvenile myelomonocytic leukemia (JMML) patients and from 12 of 20 (60%) adult chronic myelomonocytic leukemia (CMML) patients to 10(-9) mol/L DT388-GM-CSF for 48 hours in culture reduces the number of cells capable of forming colonies in semisolid medium (colony-forming units-leukemia) 10-fold to 300-fold (1 to 2.5 log decrease). In contrast, normal myeloid progenitors (colony-forming unit-granulocyte-macrophage) from six different donors treated and assayed under identical conditions were consistently insensitive to the same fusion toxin even when treated as highly purified CD34(+) cells. The leukemic progenitors from the two other JMML patients showed intermediate sensitivity to DT388-GM-CSF and the leukemic progenitors from eight of the 20 (40%) CMML patients were not different from normal progenitors. Parallel measurements of the number and affinity of GM-CSF receptors on cells from the same samples showed no consistent differences between JMML, CMML, and normal light density or CD34(+) bone marrow cells. The increased sensitivity of leukemic progenitors from all JMML progenitors and some CMML patients to the fusion toxin is therefore not likely to be explained by an increased density of GM-CSF receptors on these cells. We also examined the DT388-GM-CSF sensitivity of two murine cell lines transfected with cDNAs encoding varying portions of the human GM-CSF receptor and/or beta chains. These studies showed that high-affinity ligand binding was sufficient for DT388-GM-CSF-induced toxicity, as this could occur even in the absence of functional signal transduction and that the background of the host cell had a major influence on the degree to which this decreased the toxicity of DT388-GM-CSF. The selective sensitivity to DT388-GM-CSF of leukemic progenitors from a majority of JMML and CMML patients suggests that this agent could have therapeutic potential for some patients with these diseases.

Cell-specific modulation of drug resistance in acute myeloid leukemic blasts by diphtheria fusion toxin, DT388-GMCSF.

Radiochemotherapy-resistant blasts commonly cause treatment failure in acute myeloid leukemia (AML), and their resistance is due, in part, to overexpression of multidrug resistance (mdr) proteins. We reasoned that targeted delivery of protein synthesis inactivating toxins to leukemic blasts would reduce the cellular concentrations of relatively short half-life resistance proteins and sensitize the cells to cytotoxic drugs. To test this hypothesis, we employed human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GMCSF). The human AML cell line HL60 and its vincristine-resistant sublines, HL60Vinc and HL60VCR, were incubated in vitro for 24 h with varying concentrations of toxin. Doxorubicin was added for an additional 24 h, and cell cytotoxicity was assayed by thymidine incorporation and colony formation in semisolid medium. DT388-GMCSF sensitized HL60Vinc and HL60VCR but not HL60 to doxorubicin. Combination indices for three log cell kill varied from 0.2 to 0.3. In contrast, pretreatment with doxorubicin followed by toxins failed to show synergy. At least in the case of the vincristine-resistant cell lines, modulation of drug resistance correlated with reduction in membrane P-glycoprotein concentrations based on immunoblots with C219 antibody, flow cytometry with MRK16 antibody, and cell uptake of doxorubicin. These observations suggest clinical trials of combination therapy may be warranted in patients with refractory AML. Further, targeted toxins may represent a novel class of cell-specific modulators of drug resistance for a number of malignancies.

Malignant progenitors from patients with acute myelogenous leukemia are sensitive to a diphtheria toxin-granulocyte-macrophage colony-stimulating factor fusion protein.

We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 x 10(-12) mol/L (range, 5 to >4,000 x 10(-12) mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing > or = 35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 x 10(-9) mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.