- Email: [email protected]
Alternatively spliced FGFR-1 isoforms attenuate the malignant phenotype of FGF-1 transformed pancreatic ductal cells1
SPORADIC COLORECTAL CARCINOMA MICRODISSECTION: A NOVEL LABORATORY TECHNIQUE TO IDENTIFY TUMOR SUPPRESSOR GENES Authors:
Conclusion: These data may indicate that alternative receptor splicing may modulate the transforming effect of fibroblast growth factors in pancreatic cancer.
LM Rodriguez RB Penetrante MM Vaughan HK Slocum TB Shows NJ Petrelli Buffalo, NY
SYMPTOMATIC OUTCOMES AFTER LAPAROSCOPIC ANTIREFLUX SURGERY Authors:
Purpose: Multiple tumor suppressor genes have been found by loss of heterozygozity (LOH) analysis. Surgical samples are frequently contaminated with normal tissue elements, making molecular analysis with polymerase chain reaction– based techniques problematic to carry out and analyze accurately. Therefore, the aim of this study was to use a laser dissecting microscope to remove contaminating tissues, re-evaluate pathologic staging, and identify tumor suppressor genes after microdissection. This technique is sensitive but expensive, costing $50,000 to $75,000 for the microscope alone. Methods: At Roswell Park Cancer Institute, all colorectal tumors (CRC) are stored in a tumor data bank. The pathologist identified tumor tissue that was frozen in liquid nitrogen and archived it for research analyses. The remainder of the tissue was used for pathologic staging. Sixty-seven CRC were microdissected with a 15-blade scalpel, a 22-gauge needle, and an inverted dissecting microscope. Each tumor was weighed, placed in a cryogel, and cut into 30-m shavings using a Cryostat machine. The tumors were kept at ⫺24 C. Hematoxylin and eosin (H&E)-stained sections were taken at the first, center, and last shavings and labeled A, B, and C, respectively. The shavings between A and B were collected into a Petri dish and those between B and C into a second dish. A pathologist analyzed the H&E slides. Invasive carcinoma was classified as H&E with greater than 95% invasive carcinoma. Results: Invasive carcinoma was found in 34 samples (51%) and villous adenoma in 17 (25%); 8 samples had less than 50% carcinoma and 8 contained no carcinoma. Conclusions: These results indicate that tumors should be microdissected and serially stained with H&E to give a representation of the carcinoma present through the tumor prior to any biologic analysis. This technique is inexpensive, sensitive, and specific. Furthermore, it allows picking the best shavings from AB or BC prior to any LOH investigation, which is crucial when looking for tumor suppressor genes. (Funded by National Institutes of Health Grant T32 CA09581.)
K Gray, MD K Draper, MD WO Richards, MD M Holzman, MD K Sharp, MD Nashville, TN
Purpose: This study was designed to evaluate symptomatic outcomes following laparoscopic antireflux surgery. Methods: Patients referred for antireflux surgery completed a selfadministered 19-question gastrointestinal (GI) survey. The survey evaluates 4 GI symptom complexes: gastroesophageal reflux disease (GERD), abdominal pain, dysphagia and irritable bowel. The GERD symptoms are broken down into GI and respiratory symptoms. Questions are scored on a Likert scale with 0 ⫽ no symptoms and 100 ⫽ severe symptoms. All patients who had an antireflux procedure and completed pre- and postoperative surveys were included in the study. Results: The 40 patients studied included 21 men and 19 women of mean age 47 ⫾ 15 years. Analysis of pre- and postoperative scores using the paired Student’s t-test was as follows (values expressed as mean ⫾ SEM): Symptom complex
Preoperative score*
Postoperative score*
p Value
GERD Abdominal pain Dysphagia Irritable bowel
54.3 ⫾ 3 40.7 ⫾ 4 43.8 ⫾ 6 19.1 ⫾ 2
20.7 ⫾ 4 21.4 ⫾ 4 20.0 ⫾ 4 19.0 ⫾ 3
⬍.0001 .001 .002 ⬎.97
Analysis of the GI components (heartburn, regurgitation, eructation) and respiratory components (difficulty falling asleep, insomnia, feeling rested on awakening) of the GERD symptom complex reveals that both types of symptoms improved
Gastrointestinal GERD score Respiratory GERD score ALTERNATIVELY SPLICED FGFR-1 ISOFORMS ATTENUATE THE MALIGNANT PHENOTYPE OF FGF1 TRANSFORMED PANCREATIC DUCTAL CELLS Authors:
Methods: Nontumorigenic ARIP cells were transformed, producing a malignant phenotype (ie anchorage-independent growth, growth in reduced serum, resistance to immunologically stress-mediated death, ONOO⫺) via retrovirally delivered chimeric secreted form of human FGF-1 (ARIP-K cell). ARIP-K cells were transfected (CaPO4) with FGFR-1␣ receptor (P106 vector) and evaluated for evidence of successful gene transfer, growth, phenotype morphology, and response to ONOO⫺-induced death. Results: Biochemical assessment (RT-polymerase chain reaction, Western blotting) confirmed the expression and functionality of the transgene in the ARIP (P106) cells. Compared with ARIP-K cells, ARIP (P106) cells demonstrated a reversal of transformed phenotype, evidenced by a 2-fold decrease (p ⬍ .001 test) in growth rate in reduced serum (0.05%) and a return of nonfoci anchorage– dependent growth. Finally, these cells demonstrated marked reversal in sensitivity to peroxynitrite (ONOO⫺ 1 mM), 85% of ARIP-K cell survival versus 20% cell survival in ARIP (P106) (p ⬍ .001).
384
CURRENT SURGERY
Postoperative
p Value
63.1 ⫾ 3 46.0 ⫾ 5
16.1 ⫾ 4 24.9 ⫾ 5
⬍.0001 .002
Conclusions: Overall, GERD, abdominal pain, and dysphagia symptoms all improve following laparoscopic antireflux surgery; however, irritable bowel symptoms do not improve. Both the GI and respiratory components of GERD symptoms are improved by antireflux surgery.
SM Vickers LA MacMillan-Crow M Green JA Thompson Birmingham, AL
Purpose: Pancreatic cancer continues to be a devastating tumor (280,000 new cases per year; 5% 5-year survival). Pancreatic tumors frequently (50 – 60%) overexpress acidic and basic (FGF-1, FGF-2) fibroblast growth factors and their high-affinity receptor (FGFR-1). Due to alternative receptor splicing, the predominant receptor isoform expressed in normal pancreatic tissue is FGFR-1␣, with FGFR-1 expressed nearly exclusively in pancreatic cancer. We believe the ability of these ligands to modulate cellular transformation and oxidant stress (peroxynitrite)-induced apoptosis is critically linked to the predominantly expressed receptor isoform. The underlying mechanism associated with the aggressive malignant potential of this tumor is not well understood, and this study was designed to evaluate the relationship between the presence of the nonmalignant receptor isoform, FGFR-1␣, in FGF-1/FGFR-1 transformed pancreatic ductal epithelial cells (ARIP-K cells).
Preoperative
INTESTINAL PRODUCTION OF IL-6 INITIATES SYSTEMIC INFLAMMATION IN A MURINE MODEL OF INTESTINAL ISCHEMIA REPERFUSION Authors:
A Stallion, MD TD Kou DS Berger KA Miller, MD DL Dudgeon, MD AD Levine, PhD Cleveland, OH
Purpose: Intestinal ischemia/reperfusion (I/R) injury disrupts the gut barrier and activates systemic inflammation and multisystem organ failure (MOF). Our reported murine model of intestinal I/R demonstrated dose-dependent local injury and mortality [Gastroenterology (1998) 114:A-1587]. We hypothesized that the release of proinflammatory cytokines from the gut modulates systemic inflammation. Methods: Balb/c mice underwent 50 minutes of superior mesenteric artery occlusion with reperfusion. All survived long-term (⬎16 hours). Controls were sham operated. Serum levels of IL-6 and TNF-␣ were measured at 0, 1, 4, and 16 hours by ELISA. Small bowel and liver mRNA were evaluated for production of IL-6 and TNF-␣ by semiquantitative RT-PCR, with glyceraldehyde3-phosphate dehydrogenase (GAPDH) standard and a Student t-test for data analysis (p ⬍ .05). Results: Serum levels of IL-6 were significantly elevated at 4 and 16 hours versus controls (Fig. 1, left). The serum level of TNF␣ was not elevated. There was a significant elevation of IL-6 mRNA at 1 and 4 hours in the jejunum (Fig. 1, center) and TNF␣ mRNA at 4 hours. No significant increase in liver IL-6 (Fig. 1, right) or TNF-␣ mRNA was seen.
•
Volume 56 / Numbers 7/8 • September/October 1999
Conclusion: These data may indicate that alternative receptor splicing may modulate the transforming effect of fibroblast growth factors in pancreatic cancer.
LM Rodriguez RB Penetrante MM Vaughan HK Slocum TB Shows NJ Petrelli Buffalo, NY
SYMPTOMATIC OUTCOMES AFTER LAPAROSCOPIC ANTIREFLUX SURGERY Authors:
Purpose: Multiple tumor suppressor genes have been found by loss of heterozygozity (LOH) analysis. Surgical samples are frequently contaminated with normal tissue elements, making molecular analysis with polymerase chain reaction– based techniques problematic to carry out and analyze accurately. Therefore, the aim of this study was to use a laser dissecting microscope to remove contaminating tissues, re-evaluate pathologic staging, and identify tumor suppressor genes after microdissection. This technique is sensitive but expensive, costing $50,000 to $75,000 for the microscope alone. Methods: At Roswell Park Cancer Institute, all colorectal tumors (CRC) are stored in a tumor data bank. The pathologist identified tumor tissue that was frozen in liquid nitrogen and archived it for research analyses. The remainder of the tissue was used for pathologic staging. Sixty-seven CRC were microdissected with a 15-blade scalpel, a 22-gauge needle, and an inverted dissecting microscope. Each tumor was weighed, placed in a cryogel, and cut into 30-m shavings using a Cryostat machine. The tumors were kept at ⫺24 C. Hematoxylin and eosin (H&E)-stained sections were taken at the first, center, and last shavings and labeled A, B, and C, respectively. The shavings between A and B were collected into a Petri dish and those between B and C into a second dish. A pathologist analyzed the H&E slides. Invasive carcinoma was classified as H&E with greater than 95% invasive carcinoma. Results: Invasive carcinoma was found in 34 samples (51%) and villous adenoma in 17 (25%); 8 samples had less than 50% carcinoma and 8 contained no carcinoma. Conclusions: These results indicate that tumors should be microdissected and serially stained with H&E to give a representation of the carcinoma present through the tumor prior to any biologic analysis. This technique is inexpensive, sensitive, and specific. Furthermore, it allows picking the best shavings from AB or BC prior to any LOH investigation, which is crucial when looking for tumor suppressor genes. (Funded by National Institutes of Health Grant T32 CA09581.)
K Gray, MD K Draper, MD WO Richards, MD M Holzman, MD K Sharp, MD Nashville, TN
Purpose: This study was designed to evaluate symptomatic outcomes following laparoscopic antireflux surgery. Methods: Patients referred for antireflux surgery completed a selfadministered 19-question gastrointestinal (GI) survey. The survey evaluates 4 GI symptom complexes: gastroesophageal reflux disease (GERD), abdominal pain, dysphagia and irritable bowel. The GERD symptoms are broken down into GI and respiratory symptoms. Questions are scored on a Likert scale with 0 ⫽ no symptoms and 100 ⫽ severe symptoms. All patients who had an antireflux procedure and completed pre- and postoperative surveys were included in the study. Results: The 40 patients studied included 21 men and 19 women of mean age 47 ⫾ 15 years. Analysis of pre- and postoperative scores using the paired Student’s t-test was as follows (values expressed as mean ⫾ SEM): Symptom complex
Preoperative score*
Postoperative score*
p Value
GERD Abdominal pain Dysphagia Irritable bowel
54.3 ⫾ 3 40.7 ⫾ 4 43.8 ⫾ 6 19.1 ⫾ 2
20.7 ⫾ 4 21.4 ⫾ 4 20.0 ⫾ 4 19.0 ⫾ 3
⬍.0001 .001 .002 ⬎.97
Analysis of the GI components (heartburn, regurgitation, eructation) and respiratory components (difficulty falling asleep, insomnia, feeling rested on awakening) of the GERD symptom complex reveals that both types of symptoms improved
Gastrointestinal GERD score Respiratory GERD score ALTERNATIVELY SPLICED FGFR-1 ISOFORMS ATTENUATE THE MALIGNANT PHENOTYPE OF FGF1 TRANSFORMED PANCREATIC DUCTAL CELLS Authors:
Methods: Nontumorigenic ARIP cells were transformed, producing a malignant phenotype (ie anchorage-independent growth, growth in reduced serum, resistance to immunologically stress-mediated death, ONOO⫺) via retrovirally delivered chimeric secreted form of human FGF-1 (ARIP-K cell). ARIP-K cells were transfected (CaPO4) with FGFR-1␣ receptor (P106 vector) and evaluated for evidence of successful gene transfer, growth, phenotype morphology, and response to ONOO⫺-induced death. Results: Biochemical assessment (RT-polymerase chain reaction, Western blotting) confirmed the expression and functionality of the transgene in the ARIP (P106) cells. Compared with ARIP-K cells, ARIP (P106) cells demonstrated a reversal of transformed phenotype, evidenced by a 2-fold decrease (p ⬍ .001 test) in growth rate in reduced serum (0.05%) and a return of nonfoci anchorage– dependent growth. Finally, these cells demonstrated marked reversal in sensitivity to peroxynitrite (ONOO⫺ 1 mM), 85% of ARIP-K cell survival versus 20% cell survival in ARIP (P106) (p ⬍ .001).
384
CURRENT SURGERY
Postoperative
p Value
63.1 ⫾ 3 46.0 ⫾ 5
16.1 ⫾ 4 24.9 ⫾ 5
⬍.0001 .002
Conclusions: Overall, GERD, abdominal pain, and dysphagia symptoms all improve following laparoscopic antireflux surgery; however, irritable bowel symptoms do not improve. Both the GI and respiratory components of GERD symptoms are improved by antireflux surgery.
SM Vickers LA MacMillan-Crow M Green JA Thompson Birmingham, AL
Purpose: Pancreatic cancer continues to be a devastating tumor (280,000 new cases per year; 5% 5-year survival). Pancreatic tumors frequently (50 – 60%) overexpress acidic and basic (FGF-1, FGF-2) fibroblast growth factors and their high-affinity receptor (FGFR-1). Due to alternative receptor splicing, the predominant receptor isoform expressed in normal pancreatic tissue is FGFR-1␣, with FGFR-1 expressed nearly exclusively in pancreatic cancer. We believe the ability of these ligands to modulate cellular transformation and oxidant stress (peroxynitrite)-induced apoptosis is critically linked to the predominantly expressed receptor isoform. The underlying mechanism associated with the aggressive malignant potential of this tumor is not well understood, and this study was designed to evaluate the relationship between the presence of the nonmalignant receptor isoform, FGFR-1␣, in FGF-1/FGFR-1 transformed pancreatic ductal epithelial cells (ARIP-K cells).
Preoperative
INTESTINAL PRODUCTION OF IL-6 INITIATES SYSTEMIC INFLAMMATION IN A MURINE MODEL OF INTESTINAL ISCHEMIA REPERFUSION Authors:
A Stallion, MD TD Kou DS Berger KA Miller, MD DL Dudgeon, MD AD Levine, PhD Cleveland, OH
Purpose: Intestinal ischemia/reperfusion (I/R) injury disrupts the gut barrier and activates systemic inflammation and multisystem organ failure (MOF). Our reported murine model of intestinal I/R demonstrated dose-dependent local injury and mortality [Gastroenterology (1998) 114:A-1587]. We hypothesized that the release of proinflammatory cytokines from the gut modulates systemic inflammation. Methods: Balb/c mice underwent 50 minutes of superior mesenteric artery occlusion with reperfusion. All survived long-term (⬎16 hours). Controls were sham operated. Serum levels of IL-6 and TNF-␣ were measured at 0, 1, 4, and 16 hours by ELISA. Small bowel and liver mRNA were evaluated for production of IL-6 and TNF-␣ by semiquantitative RT-PCR, with glyceraldehyde3-phosphate dehydrogenase (GAPDH) standard and a Student t-test for data analysis (p ⬍ .05). Results: Serum levels of IL-6 were significantly elevated at 4 and 16 hours versus controls (Fig. 1, left). The serum level of TNF␣ was not elevated. There was a significant elevation of IL-6 mRNA at 1 and 4 hours in the jejunum (Fig. 1, center) and TNF␣ mRNA at 4 hours. No significant increase in liver IL-6 (Fig. 1, right) or TNF-␣ mRNA was seen.
•
Volume 56 / Numbers 7/8 • September/October 1999