Embryonal rhabdomyosarcoma of the urinary bladder in childhood. Report of five own cases

Malignant lesions of the bladder in infancy and early childhood arise from mesenchymal tissue. Among the mesenchymal tumors embryonal rhabdomyosarcoma is more common in young children than in adults. The gross appearance of rhabdomyosarcoma of the bladder is typically that of polypoid grapelike clusters of tissue, termed "sarcoma botryoides". Histologically, the lesion is consistent with embryonal rhabdomyosarcoma. The growths originate in the submucosal layer of the trigone and the internal urethral orifice. Rhabdomyosarcoma (RMS) of the bladder in children tends to remain within the disease. Deep extension and metastasis to regional lymph nodes and to distant sites is a relatively late occurrence. The symptoms are characteristically those of obstruction and strangury. Hematuris is uncommon. The intravenous urogram shows characteristic lobulated filling defects. Treatment of choice is almost always total cystectomy. Suprapubic cystotomy must be prohibited both as a method of drainage and as an approach for biopsy since tumors ulcerating through the abdominal wall after cystotomy and local excision have been observed. With concomitant administration of radiation therapy in selected cases and the combination of surgery with multiple drug chemotherapy, promising results have been obtained in the therapy of embryonal RMS of the bladder in children.     

Indirect computed tomography lymphography of subdiaphragmatic lymph nodes using iodinated nanoparticles in normal dogs

RATIONALE AND OBJECTIVES: We evaluated the imaging characteristics of an iodinated particulate contrast agent for indirect computed tomography (CT) lymphography of normal subdiaphragmatic lymph nodes in dogs. METHODS: Four milliliters of a 15% (wt/vol) iodinated nanoparticle suspension was injected into the gastric, colonic, rectal, or cervical submucosa, loose paraprostatic fascia, or metatarsal subcutaneous tissues in 10 healthy beagles. Endoscopic, CT, or ultrasound guidance was used when necessary to facilitate contrast agent delivery. CT and radiographic images were obtained prior to contrast administration and at 4 hr, 24 hr, and 7 days postcontrast injection. Postmortem examinations were then conducted. RESULTS: CT images showed enhancement of regional lymph nodes draining the various injection sites. The mean attenuation of opacified nodes was 678 +/- 463 Hounsfield units 24 hr after injection and remained elevated 7 days later. Lymph node opacification on CT images correlated well with the node location observed on postmortem examinations. CONCLUSION: Subdiaphragmatic lymph nodes can be effectively opacified using an iodinated nanoparticle contrast agent for indirect CT lymphography.     

Penetration of intravesical doxorubicin in human bladders

The bladder wall penetration kinetics of intravesical doxorubicin were examined in radical cystectomy patients, to provide insight into drug concentrations at target tumor sites. The dosing solution (40 mg/20 ml) was instilled just prior to the start of surgery and maintained for 60-115 min until just prior to bladder excision. The data showed considerable inter-patient variability in the peak plasma concentration (24-fold), urine concentration (7- fold), and tissue concentration (28-fold). The urine concentration at the time of tissue harvest was about 17% of the concentration in the dosing solution. This was due to the dilution by post-catheterization residual urine and urine produced during treatment. The doxorubicin concentration dropped by 32-fold across the urothelium, and declined semi-logarithmically with respect to depth in the capillary-perfused tissues beneath the urothelium with a 50% decrease over about 500 micromole. In three of six patients from whom tumor tissue was obtained, the doxorubicin concentration was higher than the adjacent non-tumor-bearing tissues of comparable tissue depth, whereas the reverse was seen in the remaining three tumors. The plasma concentrations were 0.02, 0.03, 0.05, 0.27, and 0.69% of the concentrations in the tumors, urothelium, lamina propria, superficial and deep muscle layers, respectively. These data indicate: (a) a considerable intra- and inter-patient variability in bladder tissue concentrations, in part due to the variability in the urine concentration; (b) the urothelium is an effective barrier to doxorubicin penetration; and (c) a targeting advantage of intravesical therapy for the treatment of superficial bladder cancer yielding superficial bladder tissue concentrations at least 2000-fold higher than in the systemic circulation. A comparison of the data of doxorubicin with our previously published data on mitomycin C shows similar bladder tissue pharmacokinetics for the two drugs, suggesting that there is no pharmacokinetic preference for either drug.     

Evaluation of autologous fat implantation in the rat urinary bladder submucosa

OBJECTIVES: Autologous fat has been used as a bulking implant material for stress urinary incontinence. There is considerable controversy as to the ultimate fate of the grafted fat. This study was conducted to determine the fate of autogenous fat implanted into the bladder of rats. METHODS: Two groups of adult female rats were studied. In the test animals (group 1, n = 20), mesenteric adipose tissue (1 mL) was harvested and homogenized with an equal weight of sterile saline. Using a 25-gauge needle, 0.5 g of saline-fat mixture was injected into the dorsal bladder neck submucosa. Control animals (group 2, n = 12) were injected with sterile saline only. A subset of animals from each group were killed after 7, 35, 105, and 1 50 days, and the bladder and urethra were fixed. The fixed tissue was examined microscopically and photographed at each follow-up period. RESULTS: Seven days after injection of fat, there was a pronounced acute inflammatory reaction with numerous polymorphonuclear leukocytes and macrophages at the site of fat injection. There was minimal inflammatory reaction at the site of saline injection. By day 35, most of the fat had been eliminated by these phagocytes because of severe acute and chronic inflammation. By day 105, the submucosa tissue of the experimental rats had returned to normal visually and to a flat surface, lacking the appearance of a sizable "bulge" as shown at days 0 and 7. Histopathologic findings were also similar to the control rats. Inflammatory cells were no longer present by day 105. CONCLUSIONS: The implantation of homogenized, autologous fat in the rat urinary bladder submucosa causes acute and chronic inflammation and fat necrosis. The severe phagocytosis at the implant sites eliminates the vast majority of the devitalized implanted fat during the first month.     

Prospective randomized evaluation of preoperative endoscopic vital staining using CH-40 for lymph node dissection in gastric cancer

BACKGROUND: CH-40 is a suspension of activated carbon particles that was developed in Japan to carry anticancer drugs to regional nodes and peritoneal seedings of gastric cancer. METHODS: Forty-five consecutive patients who had surgical resection and D2 lymph node dissection for gastric cancer over a 2-year period were randomly assigned to preoperative endoscopic submucosal injection of CH-40 (group A) or no staining (group B). A total of 21 patients in group A and 24 in group B were available for analysis. RESULTS: The number of resected nodes per patient was significantly higher (t = 6.06; 40 df; P < .0001) in group A (mean+/-S.E. = 35.3+/-1.24) than in group B (mean+/-S.E. = 25.5+/-1.02). The rate of metastatic nodes resected was significantly higher (chi2 = 6.903 ; 1 df; P = .009) in stained (22.5%) than in non-stained (14.7%) nodes of group A and also (chi2 = 6.906; 1 df; P = .009) in stained nodes of group A than in group B (15.8%). CONCLUSIONS: Preoperative endoscopic vital staining with CH-40 proved to be rapid, safe, and effective in all cases in this series. Its use allowed surgeons to resect a higher number of lymph nodes. and to identify and examine more metastatic nodes. It also permitted identification of nodal micrometastases on routine histopathologic examination.     

Experimental and clinical study on intraoperative local infusion chemotherapy (ILIC) for advanced gastric carcinoma

An intensive chemotherapy combined with surgery, termed "intraoperative local infusion chemotherapy (ILIC)", was devised. The ILIC procedure is to infuse 50 mg of cisplatin through the feeding artery to the tumor, which has been isolated from blood flow by clamping the stomach and blood vessels, before radical surgery. An experiment in dogs showed considerable free Pt transfer into thoracic lymph after ILIC. Sum total Pt in the thoracic lymph after ILIC (294.5 micrograms/3 h) was 4.3 times that after IV administration (68.3 micrograms/3 h). In ILIC, AUC of free Pt in the thoracic lymph was about 5.8 times that in peripheral blood. ILIC was applied in 24 advanced gastric cancer patients, consisting of 6, 2, 4 and 12 in pTNM stage II, IIIA, IIIB and IV, respectively. Pt concentration of the tissues was high in the tumor (23.22 micrograms/g) and the regional lymph nodes (2.95 micrograms/g) compared to that in the serum (0.45 microgram/ml). The survival rate (Kaplan-Meier) in the ILIC patients was significantly higher than that of control patients (matched pair method) from among patients treated by surgery alone. No fatal complications were encountered in any patient treated by ILIC.     

Radiotherapy of bladder cancer: relevance of bladder volume changes in planning boost treatment

PURPOSE: This study aims to evaluate tumor motion with controlled changes of the bladder volume, and to assess the reproducibility of bladder (and tumor) position using a urinary catheter balloon as an immobilization device. METHODS AND MATERIALS: First, three patients with tumor growths in three different bladder regions (trigone, left lateral wall, anterior wall) were evaluated. Three-dimensional CT-based reconstructed images were used to measure the displacement of the tumors when 100 cc were removed from a bladder originally filled with 170 cc of contrast. The 3D calculated boost beam arrangements and field sizes for the three tumors in the partially emptied bladders were used to simulate treatment of the same tumors in the maximally filled bladders. Dose-volume histograms were obtained. Second, verification of an ellipsoid model for bladder volume changes was undertaken in 41 patients. Third, in eight additional patients a urinary catheter balloon filled with 80-cc sterile saline solution was used in an attempt to reproduce the shape and spatial coordinates of the bladder during the boost treatment. A pair of orthogonal films with the 80-cc balloon filled with contrast material were taken at simulation and repeated twice at weekly intervals during radiotherapy. The reproducibility was quantified by sequentially calculating the common surface of the bladder images in each orthogonal view. RESULTS: Target motion, especially in the craniocaudal axis, appeared to be more relevant for tumors arising in the bladder walls (15 mm) than in the trigone (5 mm). Underdosage (<95% of the prescribed dose to the target volume) was observed in 20, 20, and 50% (with 1 cm margins around the tumor) and in 10, 10, and 15% (with 1.5 cm margins around the tumor) of the tumors arising in the trigone, left lateral wall, and anterior wall, respectively. The ellipsoidal model was validated with a strong correlation coefficient allowing to establish a predictive model for bladder wall displacements as a function of bladder volume. In the balloon reproducibility study, mean reproducibility factors of 0.84 (+/-0.06) and 0.82 (+/-0.07) were obtained for both anteroposterior and lateral views, respectively. CONCLUSIONS: Changes in bladder volume and shape related to bladder filling can result in clinically significant displacements of the target volume. A minimum of 2-cm margins around the target may compensate for extreme bladder volume changes during boost treatment. An ellipsoidal model for the bladder is consistent with these observations. Although an 80-cc urinary catheter balloon helped to immobilize the bladder, reproducibility was less than perfect.     

Leiomyoma of the bladder: report of two cases

We report 2 cases of leiomyoma of the urinary bladder. A 41-year-old female visited our hospital with the complaint of pollakisuria. A solid tumor of the urinary bladder was found by ultrasonography. A large shadow defect at the left-anterior wall was shown by drip infusion pyelography (DIP). Computed tomographic scan (CT) and magnetic resonance imaging (MRI) also revealed a large tumor. T1-weighted image revealed a homogeneous low intensity tumor and T2-weighted image disclosed heterogeneous low intensity tumor. Cystoscopy revealed a large submucosal tumor. Partial cystectomy was performed, and she has had neither recurrence nor metastasis for 36 months. A 32-year-old male was referred to our hospital with the complaint of macrohematuria. A solid tumor of the urinary bladder was found by ultrasonography. A shadow defect was not clearly detected by DIP. A large tumor was detected on the anterior wall by MRI. T1-weighted image showed a homogeneous low intensity tumor and T2-weighted image disclosed a high intensity tumor. Cystoscopy revealed a submucosal tumor on the anterior wall. Urine cytology did not suggest a malignancy. The biopsied specimens revealed only an inflammatory change in the mucosa. Partial cystectomy was carried out. He has had neither recurrence nor metastasis for 29 months. Histological diagnosis in both cases was leiomyoma of the urinary bladder.     

Effects of intra-abdominal pressure on pharmacokinetics and tissue distribution of doxorubicin after intraperitoneal administration

Increased hydrostatic pressure in solid tumor nodules decreases the penetration of chemotherapy into cancerous tissue. This is true for both i.v. and i.p. chemotherapy. The purpose of this study was to determine the influence of increasing intra-abdominal pressures on the pharmacokinetics and tissue distribution of doxorubicin administered i.p. Four groups of 10 Sprague Dawley rats were given i.p. doxorubicin (4 mg/kg) during 60 min combined with no pressure (control), 10, 20 and 30 mm Hg pressures. During the course of i.p. chemotherapy, peritoneal fluid and blood were sampled. Two other groups of 10 rats received the same dose of i.p. doxorubicin during 10 min combined with no pressure and 30 mm Hg pressure. At the end of experiments animals were sacrificed and tissue samples were collected. Doxorubicin concentrations in peritoneal fluid, plasma and tissues were determined by HPLC. Pharmacokinetic studies showed that increased intra-abdominal pressures of 10, 20 and 30 mm Hg did not alter peritoneal fluid AUCs, the plasma AUCs and the peak ratios of i.p. doxorubicin when compared to the control group (no pressure). A subset analysis of high intra-abdominal pressure groups (20 and 30 mm Hg) versus control group showed statistically significant differences in peritoneal fluid AUCs, plasma AUCs and AUC (peritoneal fluid/plasma) ratios. For all groups, the highest tissue concentrations of doxorubicin were found in tissues associated with the parietal peritoneum: the bladder, the abdominal wall and the diaphragm. After 10 min of i.p. chemotherapy, the group treated with 30 mm Hg pressure showed a significant increase of doxorubicin concentrations in these tissues as compared to the control group. This significant increase of tissue doxorubicin concentrations was not found after 60 min of pressure with i.p. chemotherapy; prolonged intra-abdominal pressure was associated with a high incidence of intestinal ischemia. In conclusion, intra-abdominal pressure of 20 and 30 mm Hg significantly decreased the AUC ratios of i.p. doxorubicin but concomitantly increased tissue uptake of doxorubicin in bladder, diaphragm and abdominal wall during the first 10 min of i.p. administration. These findings may have significance in the design of improved strategies to increase tissue concentrations of chemotherapy delivered by an i.p. route.     

Lymphoscintigraphy with double tracer technique in carcinomas of the head-neck region

BACKGROUND: Lymphoscintigraphy has been used since the early 1960s to demonstrate lymphatic drainage of head and neck tumors, but did not prove satisfactory. With the increasing importance of highly sophisticated neck dissection procedures, lymphoscintigraphy may have greater diagnostic impact. This assumes that lymphoscintigraphy will allow an accurate correlation of lymphatic drainage with anatomic structures. In this paper, we report on a method of lymphoscintigraphy with simultaneous body contouring. METHODS: Double-tracer lymphoscintigraphy was performed in 78 patients with squamous cell carcinoma. Patients received 100 MBq 99mTc-colloid in 0.1-0.2 ml in 3-4 peritumoral localizations. Ten patients were injected during surgery. Two milliliters of perchlorate solution were given orally in order to block the thyroid. Twenty minutes later patients received 50 MBq 99mTc-pertechnetate i.v. for body contouring. Planar images were obtained over 5 min each at 30 min and 4-6 h after injection from anterior, right lateral and left lateral using a LFOV-gamma camera. RESULTS: The thyroid was not visualized in any of the patients. In 28 of 78 patients (36%), the injection site was the only focal activity seen. In 50 of 78 patients (64%), lymph drainage was observed. Thirty-six of 78 patients (46%) showed unilateral lymphatic drainage, and 14 of 78 (18%) showed bilateral drainage. In all 50 patients showing lymphatic drainage, lymph nodes could be easily assigned to the six cervical lymph node compartments described. CONCLUSIONS: Double-tracer lymphoscintigraphy enables an accurate correlation of cervical lymph nodes and anatomic structures of the head and neck region. These findings suggest that the impact of these studies on the preoperative planning for neck dissection should be reevaluated.     

Role of local lymphatic stimulation in the prevention of early complications after stomach surgeries

Experiments were conducted on 120 albino rats and 16 dogs with the use of scanning and electron microscopy as well as the radionuclide method of microcirculation study to substantiate the method of regional lymphostimulation after operations on the stomach for the prevention of early postoperative complications. Some lymphagogues (lidase, heparin, procaine hydrochloride) were infused in the early postoperative period through a catheter introduced into the hepatoduodenal ligament. This zone is represented by a large group of lymph nodes receiving lymph from the stomach and the adjacent organs and the intercellular space of the hepatoduodenal ligament. The study showed that lymphostimulation improves the circulation of lymph and blood which are disturbed as the result of the operation and improves the local homeokinesis in the wall of the stomach after its resection and vagotomy. Lymphostimulation increases the resorption and transport activity of the lymphatic system and improves drainage of the edema fluid and the products of disturbed metabolism from the interstitium of the gastric mucosa. The results of using the method of regional lymphostimulation in 441 patients after various operations on the stomach for peptic ulcer showed the possibility of reducing the number of early postoperative complications, such as inadequacy of the sutures of the gastroduodeno--entero--anastomosis, anastomositis, infectious-inflammatory complications, and others.     

Ultrasonographic assessment of bladder neck mobility in continent bitches and bitches with urinary incontinence attributable to urethral sphincter mechanism incompetence

OBJECTIVE: To determine bladder neck positional changes between standing and recumbent positions in bitches and whether change is related to continence status or general anesthesia, or both, and to evaluate reproducibility of measurements. ANIMALS: 45 continent animals and 46 incontinent bitches with urethral sphincter mechanism incompetence (SMI). PROCEDURE: Distance between the bladder neck and perineal skin was measured ultrasonographically via the perineum while each dog was conscious in standing and right lateral recumbency and in right lateral recumbency under general anesthesia. Measurements of the bladder neck position obtained in right lateral recumbency under anesthesia were compared with radiographic measurements of the same parameter. Reproducibility of the ultrasonographic measurements of the distance between bladder neck and perineum was assessed on 3 occasions for each position in 50 dogs. RESULTS: In all dogs, differences in bladder neck position between standing and recumbent conscious-associated positions were not significant. However, caudal bladder neck movement between standing conscious- and recumbent anesthesia-associated positions and between recumbent conscious- and anesthesia-associated positions was significant for all dogs. Incontinent bitches had greater degree of caudal bladder movement during anesthesia. Mean difference in bladder neck positions between recumbent conscious- and recumbent anesthesia-associated positions was 0.24 cm in continent, compared with 0.73 cm in incontinent, bitches. Radiographic measurements were significantly greater than ultrasonographic measurements. Differences between repeated measurements for standing position were not significant, but those for recumbent conscious- and recumbent anesthesia-associated positions were significant. CONCLUSIONS: Additional vesicourethral support mechanisms in continent dogs, are deficient in bitches with SMI, allowing the bladder neck to move further caudad.     

Carboplatin-based versus cisplatin-based chemotherapy in the treatment of surgically incurable advanced bladder carcinoma

BACKGROUND: The carboplatin-based chemotherapeutic regimen M-CAVI (methotrexate, carboplatin, and vinblastine) is active against bladder carcinoma and can be administered to patients who are ineligible to receive cisplatin or doxorubicin. The authors designed a randomized study to evaluate whether M-CAVI offers a therapeutic advantage over the cisplatin-based regimen M-VAC (methotrexate, vinblastine, doxorubicin, and cisplatin) in the treatment of patients with surgically incurable advanced bladder carcinoma. METHODS: Patients with surgically incurable advanced bladder carcinoma were enrolled on a randomized trial comparing M-CAVI, which consists of carboplatin (300 mg/m2 on Day 2, adjusted using Calvert's formula for an area under the curve of 5), methotrexate (30 mg/m2 on Days 1, 15, and 22), and vinblastine (3 mg/m2 on Days 2, 15, and 22) administered every 28 days, versus standard M-VAC. The eligibility criteria included histologically proven bladder carcinoma, surgically incurable disease, and no prior chemotherapy. Patients were treated until disease progression or unacceptable toxicity occurred. RESULTS: From January 1989 to January 1994, 47 assessable patients were included. Seventeen patients had lymph node disease and 30 had distant metastatic disease. Twenty-three patients were randomized to receive M-CAVI and 24 to receive M-VAC. Patient characteristics in the two groups were similar. Overall response rates were 39% (95% confidence interval [CI], 20-62%) for M-CAVI and 52% (95% CI, 30-73%) for M-VAC (P = 0.3), with 3 complete responses observed among patients treated with M-VAC and none among those in the M-CAVI group. M-VAC was associated with more gastrointestinal toxicity, stomatitis, alopecia, and Grade 4 neutropenia than M-CAVI. One toxicity-related death occurred in the M-VAC group. There was a statistically significant difference in median disease-related survival time favoring M-VAC (16 months; range, 6 to 22+) versus M-CAVI (9 months; range, 6 to 14+) (P = 0.03). CONCLUSIONS: M-CAVI is less toxic but less active than M-VAC in the treatment of patients with advanced bladder carcinoma. Carboplatin-based regimens in which carboplatin is administered at the dose range used in the current study should be reserved for patients who cannot tolerate cisplatin treatment. Further research is required to assess the impact of high dose carboplatin in the treatment of this disease.     

Experimental pyeloureterectomy and calyceal neck transection with autotransplantation and bladder advancement

OBJECTIVE: To determine whether Dexon mesh, closely applied to the kidney, provides purchase for sutures to permit bladder/parenchymal apposition on autotransplantation and that, if this line of apposition were some distance from but surrounding renal papillae, urothelium would proliferate to cover exposed parenchyma to form a widely patent lumen; this should facilitate removal of the whole of an upper tract collecting system, retaining renal parenchyma alone. MATERIALS AND METHODS: To test this possibility and explore the practicability of the concept, nine dogs underwent bilateral nephrectomy followed by unilateral autotransplantation: the other kidney was discarded. Because the canine renal pelvis is intrarenal, the ureter was stretched maximally before passing fine scissors into the renal hilum to transect the collecting system as close to the kidney as possible in six of the nine dogs. In the remaining three dogs, partial nephrectomy was performed with division of the calyceal necks under vision. Thinned bladder wall was sutured to Dexon mesh some distance from the collecting tubules; omentum was applied to the suture line. RESULTS: Three dogs were killed prematurely at < 2 weeks because of perioperative complications. Four were killed at 2, 4, 5 and 8 weeks and two at 12 months. Dexon mesh proved to be an effective anchoring fabric, providing close apposition of bladder wall and parenchyma. There was no adhesion of the kidney to peritoneal contents. Urothelial proliferation to cover exposed parenchyma occurred early and by 12 months, a thin stroma was interposed between parenchyma and epithelium. The kidney was preserved in all but one removed electively, this dog having both cystitis and pyelonephritis at 12 months. CONCLUSIONS: This study showed that autotransplantation of a kidney after removal of its collecting system and advancement of thinned bladder wall to renal parenchyma is practicable, with regenerated urothelium bridging the deficiency by covering exposed parenchyma, to create a widely patent lumen.     

Isolated lung perfusion with doxorubicin prolongs survival in a rodent model of pulmonary metastases

BACKGROUND: We developed a rodent model of unilateral pulmonary metastases to evaluate long-term survival after isolated lung perfusion with doxorubicin. METHODS: In the model development study, on day 0, two groups of F344 rats (n = 15) underwent transient right pulmonary artery occlusion for either 5 or 10 minutes at the time of intravenous injection of methylcholantrene-induced sarcoma cells. On day 14, all animals were sacrificed and lung nodules counted. In the survival study, on day 0, 21 rats received intravenous injection of sarcoma cells with concomitant 10-minute right pulmonary artery occlusion. On day 7, eight rats underwent left isolated lung perfusion with doxorubicin (6.4 mg/kg); five rats underwent perfusion with buffered Hespan; six untreated rats were studied as controls. RESULTS: Ten of fifteen animals (67%) in the model study with 5-minute pulmonary artery occlusion had right-sided tumor nodules. Ten-minute occlusion resulted in a tumor-free right lung in all animals. In the survival study, all animals in the Hespan and control groups died of massive tumor replacement of the left lung, with median survival times of 20 and 18 days, respectively. The median survival time of 36 days for the animals undergoing isolated lung perfusion with doxorubicin was significantly longer (p < 0.00001). The left lung of two of the doxorubicin perfused rats was tumor-free at 6 weeks. CONCLUSIONS: Isolated lung perfusion with doxorubicin results in a durable response and prolongs survival in the treatment of experimental sarcoma pulmonary metastases.     

Adenocarcinoma of the pancreas: detection of occult metastases in regional lymph nodes by a polymerase chain reaction-based assay

BACKGROUND: Stage I (T1-2NOM0) adenocarcinoma of the pancreas is associated with a 5-year survival rate of 15-25%. Despite apparently curative resection and pathologic staging indicating localized disease, these cancers recur. The authors hypothesized that there exists microscopic regional disease that is not detected by surgical exploration or routine histopathology. METHODS: Because 90-95% of pancreatic cancers exhibit codon 12 K-ras mutations, the authors examined regional lymph nodes for mutated K-ras as a marker of metastasis. DNA was extracted from paraffin embedded archival specimens (primary tumors and histologically negative lymph nodes) of patients with Stage I pancreatic adenocarcinoma. The target region of K-ras was amplified by polymerase chain reaction (PCR) and tested for codon 12 mutation by BstN1 restriction digestion (restriction fragment length polymorphism [RFLP]) that recognized normal but not mutated sequences. Cell lines that harbored normal or mutated K-ras and resected jejunum or gallbladder were used as controls. The regional lymph nodes of 22 patients whose tumors harbored mutated K-ras were tested. RESULTS: Dilution experiments with normal and mutant control cell line DNA demonstrated an assay sensitivity for mutated K-ras of 0.1%. Mutated K-ras was found in at least 1 regional lymph node in 16 (73%) of 22 patients with pathologic Stage I pancreatic adenocarcinoma, which suggested metastases not detected by routine histopathology. DNA sequence analysis was performed in four patients and confirmed identical point mutations in the primary tumor and accompanying PCR/RFLP positive lymph nodes. CONCLUSIONS: Pathologic examination of regional lymph nodes in pancreatic adenocarcinoma specimens fails to detect metastases in many patients. Lymph node micrometastasis is one reason for the poor survival rates observed among patients with Stage I cancers. PCR/RFLP may have a role in staging early pancreatic cancers.     

Therapeutic drug monitoring of doxorubicin in paediatric oncology using capillary electrophoresis

A method for the determination of doxorubicin and its main metabolite doxorubicinol in human plasma is described. Two different sample preparation procedures are applied depending on the expected concentration: To monitor the peak plasma levels, 10 microL of plasma are deproteinated with acetonitrile. After centrifugation, the supernatant is directly applied to the capillary by hydrodynamic injection. For the determination of lower amounts of doxorubicin and its main metabolite doxorubicinol 100 microL of plasma is extracted by liquid-/liquid extraction with chloroform. After evaporation of the organic phase, the sample is reconstituted in acetonitrile/water (95/5 v/v) and injected into the capillary by electrokinetic injection. Idarubicin serves as the internal standard. Laser-induced fluorescence detection with an Ar-ion laser emitting at 488 nm and a 520 nm cut-off filter is used for detection. The accuracy of the method was calculated to be 3.0% at higher concentrations and 15.0% at the limit of quantification. Reproducibility data are in accordance to the generally accepted criteria for bioanalytical methods. The limit of quantification is 2 microg/L, enabling us to monitor doxorubicin plasma levels for several days after application. Noninvasive blood sampling (from the fingertip) using heparinized capillaries was found to be a simple and convenient procedure and provides reproducible data. Initial results show high interindividual variability in doxorubicin peak plasma levels.     

Evaluation of smooth muscle and collagen subtypes in normal newborns and those with bladder exstrophy

PURPOSE: Many patients who undergo bladder exstrophy closure as newborns, subsequent epispadias repair and later bladder neck reconstruction become completely continent yet complications can occur. After successful initial exstrophy closure and later epispadias repair some patients may fail to gain sufficient capacity for bladder neck reconstruction or satisfactory capacity and continence after bladder neck reconstruction. In an attempt to understand the pathogenesis of these failures we compared bladder biopsies from normal neonates and those with exstrophy. MATERIALS AND METHODS: Bladder biopsies obtained from the midline of the bladder wall just above the base of the trigone from 12 newborns with exstrophy were compared to bladder sections from 9 neonatal cadavers. All bladder specimens were stained with monoclonal antibodies against type I, III or IV collagen and a subset was further stained with Masson's trichrome to define the extracellular matrix. All specimens were then analyzed using a color digital image analysis system. RESULTS: At initial examination of the extracellular matrix there was an increase in the collagen-to-smooth muscle ratio from 0.38 in controls to 1.2 in newborns with exstrophy, comprising an increase in collagen and decrease in smooth muscle. The collagen component of the extracellular matrix was then further defined to quantitate the amount of each collagen type (I, III and IV) deposited. We then evaluated the ratio of collagen type-to-total collagen sampled. Compared to control bladders there was no statistical difference in the amount of type I or IV in the bladders of newborns with exstrophy at initial closure. However, there was a 3-fold increase in type III collagen (0.14 +/- 0.05 to 0.46 +/- 0.2%, p < 0.001) in the bladders of neonatal controls versus newborns with exstrophy. CONCLUSIONS: This alteration in collagen makeup may represent an earlier developmental stage of the exstrophy bladder at birth, which then remodels and changes after successful initial closure. Further studies are underway to examine the collagen composition of bladders at bladder neck reconstruction, failed closures and augmentation.     

Sarcoplasmic reticulum genes are selectively down-regulated in cardiomyopathy produced by doxorubicin in rabbits

The clinical utility of doxorubicin, an antineoplastic agent, is limited by its cardiotoxicity. Our objective was to determine whether expression of genes encoding proteins that affect Ca2+ homeostasis were altered in the hearts of rabbits chronically treated with doxorubicin. Twelve male New Zealand white rabbits received an injection of doxorubicin (2.5 mg/kg i.v.) once a week for 8 weeks. Eight rabbits were similarly injected with saline as controls. The cardiac function of both groups was evaluated 8 weeks after the final injection, as were the levels of expression of mRNA for Ca2+ transport proteins in the sarcoplasmic reticulum and plasma membrane. The amount of the sarcoplasmic reticulum Ca2+-ATPase and the Ca2+ uptake capacity of the protein were also quantitated. Cardiac output was significantly decreased in the doxorubicin-treated group (71+/-21 ml/min, P<0.05) compared with the control group (118+/-15 ml/min). The mRNA levels for the sarcoplasmic reticulum proteins were significantly diminished in the doxorubicin-treated hearts: ryanodine receptor-2 (relative expression level compared with controls, 0.35+/-0.13, P<0.01), sarcoplasmic reticulum Ca2+-ATPase (0.56+/-0.13, P<0.01), phospholamban (0.62+/-0.20, P<0.01) and cardiac calsequestrin (0. 57+/-0.26, P<0.01). In addition, both relative amount of sarcoplasmic reticulum Ca2+-ATPase protein (doxorubicin-treated group, 69+/-17% of control, P<0.01) and the Ca2+ uptake capacity (46. 9+/-9.8 nmol Ca2+/mg protein-5 min in doxorubicin group v 63.2+/-10. 4 in the control group, P<0.01) were concomitantly decreased with its mRNA expression level. Conversely, the mRNA levels for the plasma membrane proteins did not differ from those of control rabbits: the dihydropyridine receptor (relative expression level, 1. 03+/-0.30, N.S.), plasma membrane Ca2+-ATPase (0.93+/-0.33, N.S.) and the Na+/Ca2+ exchanger (0.87+/-0.34, N.S.). These findings suggest that a selective decrease in mRNA expression for sarcoplasmic reticulum Ca2+ transport proteins is responsible for the impaired Ca2+ handling, and thus, for the reduced cardiac function seen in the cardiomyopathy induced in rabbits by the long-term treatment with doxorubicin.     

Age-related changes in localization of injected radiolabelled lymphocytes in the lymph nodes of antigen-stimulated mice

The localization of i.v. injected syngeneic lymph node cells, radiolabelled with 51Cr or 75Se-L-selenomethionine, was studied in male CBA/H mice aged between 3 and 30 months. The following results were obtained. (1) Localization of cells from young adult donors was greater in the s.c. lymph nodes of old than of young recipients, the main increase being between 15 and 17 months of age. Increases in lymph node weight and DNA-synthesis were also seen at this time; but the rise in cell localization was significant even when calculated per unit of tissue weight. Splenic localization either declined slightly with age or, like the liver, showed no significant change. (2) Local antigenic stimulation by a single injection of sheep erythrocytes into one front footpad, 24 hr before lymph node cell injection, resulted in increased localization in the regional lymph nodes of 3-17 month old, but rarely of 24-30 month old mice. (3) No consistent differences in localization were observed between lymph node cells from 4-month and 25-month old donors. Both age-related and antigen-related increases in cell localization were at least partly attributable to an enhanced rate of entry of lymphocytes from the blood to the lymph nodes. Although the changes underlying the decline in antigen-related localization of cells in old recipients have still to be clarified, it is probable that the defective immune responses of old mice result partly from this decline.