Intrachromosomal genomic instability in human sporadic colorectal cancer measured by genome-wide allelotyping and inter-(simple sequence repeat) PCR.

We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loci exist for intrachromosomal genomic instability in human colorectal cancer.

Hepatic lymphatic mapping: a pilot study for porta hepatis lymph node identification.

The status of the porta hepatis lymph nodes in patients with hepatic metastases from colorectal cancer affects their prognosis and management. Lymphatic mapping with isosulfan blue dye is well established in breast cancer and melanoma. An animal model consisting of three dogs receiving general anesthesia was utilized. Each dog underwent a laparotomy and increasing doses of isosulfan blue dye were injected into the right medial segment of the liver. Intraoperatively, the presence of blue dye in the porta hepatis region was determined and the lymph node identified. Continuous physiological monitoring was performed. Serum determination of liver function tests, amylase levels, and white blood cell count were performed preoperatively and on postoperative days 1, 2, 4, and 7. The animals were sacrificed on day 7. A portal lymph node was identified in each case and there was no perioperative morbidity or mortality. There were no significant alterations in blood pressure or heart rate in the animals. There was a dose-responsive decrease in the O2 saturation as measured by transcutaneous monitoring, but arterial blood gas analysis showed that pO2 levels remained stable. There were no significant changes in the liver function tests, amylase levels, or white blood cell counts. There was a small increase in alkaline phosphatase, which normalized by postoperative day 7. Hepatic injection of isosulfan blue dye appears to be safe and effective in identifying porta hepatis lymph nodes in the animal model and sets the basis for further study in human subjects.

p53 tumor suppressor gene mutations predict decreased survival of patients with sporadic colorectal carcinoma.

BACKGROUND: Mutations of the p53 tumor suppressor gene play an integral role in sporadic colorectal carcinogenesis but prior studies have failed to show their prognostic significance consistently. METHODS: Fifty-six consecutive sporadic colorectal tumors were analyzed for their p53 status. Polymerase chain reaction amplification with primers for exons 5-9 was conducted and these products were subjected to single strand conformation polymorphism analysis. Suspected mutations were confirmed with DNA sequencing. p53 status was entered into a colorectal clinical database and these patients then were followed prospectively. Patient status with regard to disease recurrence and survival was updated every 6 months. Survival and disease free survival were calculated according to the method of Kaplan and Meier. The association between p53 status and clinical and pathologic factors with survival and recurrence was statistically determined using univariate analysis and the Cox proportional hazards model for multivariate analysis. RESULTS: p53 mutations were detected in 28 of 56 patients (50%). The median follow-up time was 45 months (range, 3-72 months). There were 33 patients (59%) who were alive at last follow-up. Fifteen of the 23 patients who died (65%) had p53 mutations and 8 (35%) had wild-type p53. Thirteen patients developed a disease recurrence, 9 of whom (69%) had tumors with p53 mutations. Overall 4-year survival rates for patients with wild-type p53 and mutant p53 were 71% and 54%, respectively (P = 0.05). The 4-year disease free survival rates for patients with wild-type p53 and mutant p53 were 83% and 62%, respectively (P = 0.09). p53 status and stage were found to be independent significant predictors for survival (p53 negative: P = 0. 02; stage: P = 0.0002.) Stage was found to be the sole significant predictor for disease free survival (P = 0.006). CONCLUSIONS: In this group of colorectal carcinoma patients, p53 mutations were a significant negative prognostic indicator for overall survival. This finding holds prognostic and therapeutic implications for the management of colorectal carcinoma patients.

The onset and extent of genomic instability in sporadic colorectal tumor progression.

Cancer cell genomes contain alterations beyond known etiologic events, but their total number has been unknown at even the order of magnitude level. By sampling colorectal premalignant polyp and carcinoma cell genomes through use of the technique inter-(simple sequence repeat) PCR, we have found genomic alterations to be considerably more abundant than expected, with the mean number of genomic events per carcinoma cell totaling approximately 11,000. Colonic polyps early in the tumor progression pathway showed similar numbers of events. These results indicate that, as with certain hereditary cancer syndromes, genomic destabilization is an early step in sporadic tumor development. Together these results support the model of genomic instability being a cause rather than an effect of malignancy, facilitating vastly accelerated somatic cell evolution, with the observed orderly steps of the colon cancer progression pathway reflecting the consequences of natural selection.

Clinicopathologic effects of cryotherapy on hepatic vessels and bile ducts in a porcine model.

BACKGROUND: The proximity of a hepatic tumor to major vessels and bile ducts limits the use of cryotherapy because of the potential damage to these structures. However, the effects of cryotherapy on major hepatic vessels and bile ducts are not well understood. METHODS: Nine pigs underwent laparotomy and intraoperative ultrasound to identify hepatic vessels larger than 5.0 mm. Cryotherapy consisting of two freeze-thaw cycles was performed, incorporating the identified vessel. In four pigs the Pringle maneuver was performed to determine the effects of partial vascular occlusion on the hepatic parenchyma and structures undergoing cryotherapy. The animals were sacrificed 30 days postoperatively, and the livers were processed for histologic examination. RESULTS: Eight of the nine livers had vessels larger than 5.0 mm incorporated into the iceball, with all vessels having evidence of infarction but remaining patent. All the livers had major bile ducts incorporated in the iceball, with eight having evidence of infarction. The Pringle maneuver had no real effect on the degree of vessel and bile duct infarction. There was no incidence of hepatic bleeding, liver fracture, bile leak, or hemobilia. CONCLUSIONS: Cryotherapy results in the infarction of major hepatic vessels and bile ducts but can be safely performed in the porcine model. Proximity of tumors to major vascular and biliary structures may not be a contraindication to the use of cryotherapy. Further studies are necessary to determine whether cryotherapy can be used in humans.

Absence of frequent involvement of modifier of Min(APC) in sporadic colorectal cancer.

BACKGROUND: Mutations in the multiple intestinal neoplasia (Min) gene, the mouse homologue of the APC gene, result in the development of intestinal tumors. The degree of tumor expression is suppressed by the modifier of Min (MOM). Alterations in the MOM gene result in markedly increased tumor expression in the mouse. The human homologue of the MOM gene has been mapped to a locus on chromosome 1p35-36, but the role of the MOM gene in the development of human sporadic colorectal cancers has not been defined. METHODS: The microsatellite marker D1S199 has been previously mapped to the region of the MOM gene and was used as a primer for PCR amplification. The PCR products were subjected to denaturing electrophoresis and analyzed for loss of heterozygosity (LOH) and the mismatch repair phenomenon (RER) of each tumor compared to its mucosal control. RESULTS: 48 consecutive sporadic colorectal cancers and normal adjacent mucosa were analyzed. LOH was noted in 2 of 48 tumors and the RER phenomenon was noted in 6 of 48 tumors. Thus, 8 of 48 tumors (16.7%) showed alterations in the region of the locus of the MOM gene. There was no association between alterations in this region and TNM stage, disease-free survival, overall survival, or p53 mutation. CONCLUSIONS: Although mutation of the APC gene is an integral component of sporadic colorectal carcinogenesis, alteration in the region including the MOM gene does not appear to play a significant role in the development or clinicopathologic behavior of human sporadic colorectal tumors.

p53 tumor suppressor gene status and the degree of genomic instability in sporadic colorectal cancers.

BACKGROUND: Genomic instability reflects the propensity and the susceptibility of the genome to acquire multiple alterations and, in turn, is believed to be a driving force behind multistep carcinogenesis. Although the molecular basis of genomic instability in sporadic colorectal cancers remains largely a mystery, mutation of the p53 tumor suppressor gene (also known as TP53) has been proposed to play an integral role in this process. However, a dilemma exists in that p53 mutation appears to be a late event in the progression of sporadic colorectal tumors, whereas genomic instability, serving as a facilitator of tumor progression, is envisioned as occurring early in this process. PURPOSE: We evaluated the relationship between p53 mutation and the major form of genomic instability in sporadic colorectal tumors, namely, that involving DNA breakage, which leads to chromosomal translocations, insertions, deletions, and gene amplification. METHODS: Fifty-eight sporadic colorectal tumors that had been previously evaluated for genomic instability were analyzed for p53 mutations. These tumors were from consecutively diagnosed patients. Genomic instability was quantified by use of inter-simple sequence repeat polymerase chain reaction analysis that employed (CA)8RG and (CA)8RY primers (R = purine [A or G]; Y = pyrimidine [C or T]); a genomic instability index (a measure of the number of alterations in tumor DNA in comparison with normal DNA, expressed as a percent) was calculated for each tumor. Mutation of the p53 gene in exons 5-9 was determined by use of single-strand conformational polymorphism-polymerase chain reaction analysis and DNA sequencing. Chi-squared analysis was used to determine the statistical significance of differences between groups of tumors. Reported P values are two-sided. RESULTS: p53 mutations were identified in 29 (50%) of the 58 tumors. The median genomic instability index value was 3.3%. Nineteen (65.5%) of the 29 tumors with p53 mutations had genomic instability indices that were less than the median value (range, 0%-2.6%); the remaining 10 (34.5%) tumors had genomic instability indices that were greater than the median (range, 3.9%-13.0%). Eleven (37.9%) of the 29 tumors with wild-type p53 genes had genomic instability indices that were less than the median value (range, 0%-2.6%), whereas the remaining 18 tumors had genomic instability indices above the median (range, 3.9%-11.7%). There was a statistically significant association between a lesser degree of genomic instability and the presence of p53 mutations (P = .032). CONCLUSIONS AND IMPLICATIONS: Tumors with no or minimal evidence of genomic instability are more likely to harbor p53 mutations than tumors with evidence of substantial genomic instability. p53 mutations play an important role in the development of cancers but do not appear to initiate or promote genomic instability in sporadic colorectal tumors.

Paracrine/autocrine regulation of pancreatic islet cell proliferation and differentiation in the hamster: studies using parabiosis.

Partial obstruction of the pancreatic duct in hamsters leads to new islet formation and reversal of streptozotocin diabetes. The purpose of this study was to delineate the mechanism by which endocrine cell proliferation and differentiation is mediated in this model. Six pairs of parabiotic hamsters were established and partial duct obstruction was inducted in 1 parabiont from each pair. At 6 weeks, the pancreatic weight (mg/100g bw); DNA (microgram/100g bw) and protein content (mg/100g bw) showed 28% (167 +/- 21 vs. 130 +/- 17), 32% (1,052 +/- 206 vs. 795 +/- 159), and 20% (25.4 +/- 6.6 vs. 21.2 +/- 1.9) increases (p < 0.05), respectively, over the non-wrapped parabionts. Morphometric analysis demonstrated the presence of new islets in the wrapped pancreata with a 100% increase in the number of islets/mm2 compared with non-wrapped controls (0.90 +/- 0.5 vs. 1.8 +/- 0.7, p < 0.01). A cytosol extract was prepared from duct-obstructed pancreases, and 4 microliters/g bw injected i.p. twice daily for 2 d produced significant increases in pancreatic weight and DNA content of 12% and 40%, respectively. Cytosol extract from non-wrapped pancreata had no effects compared with saline. When wrapped cytosol extract was injected for 21 d, the labeling index of ductular and islet cells (% cell nuclei labeled with 3H-TdR) was increased 10- and 6-fold respectively over controls (2.42 +/- 0.28 vs. 0.23 +/- 0.01 and 1.17 +/- 0.01 vs. 0.25 +/- 0.04, respectively, p < 0.01). The trophic effects observed in this model of islet cell proliferation and differentiation did not appear to be mediated by a humoral mechanism because the changes induced by partial obstruction were not observed in the non-operated parabiont. Control of pancreatic endocrine cell growth in this model appears to involve paracrine and/or autocrine regulatory mechanisms.

Correlation of perfusion abnormalities on CTAP and immediate postintravenous gadolinium-enhanced gradient echo MRI.

BACKGROUND: The purpose of this study was to evaluate patients with wedge-shaped perfusion defects seen on spiral CT arterial portography for the presence of transient increased wedge-shaped enhancement on dynamic gadolinium-enhanced gradient echo MR images. METHODS: Nineteen patients underwent CTAP and MRI within a 2-week interval. All patients with wedge-shaped perfusion defects on CT arterial portography were evaluated in a separate review session for the presence of transient increased segmental hepatic enhancement on dynamic gadolinium-enhanced spoiled gradient echo (SGE) MR images. RESULTS: Eight patients were identified to have subsegmental, segmental, or lobar wedge-shaped perfusion defects by CT arterial portography. In 8/8 patients, there was transient wedge-shaped increased hepatic enhancement on MR images which corresponded to the perfusion defects identified on CT arterial portography. Transient increased enhancement on MR images was observed on immediate postgadolinium images as high-signal intensity of the involved subsegment, segment, or lobe. This relatively high-signal area faded to near isointensity in all cases on images obtained at 45 s. CONCLUSION: Wedge-shaped perfusion defects demonstrated by CT arterial portography corresponded to wedge-shaped increased hepatic enhancement following gadolinium administration on SGE MR images.

Malignant liver lesions: comparison of spiral CT arterial portography and MR imaging for diagnostic accuracy, cost, and effect on patient management.

We compared two imaging techniques, spiral CT arterial portography (CTAP) and MR imaging, for diagnostic accuracy, procedural cost, and effect on management of 26 patients referred for hepatic surgery for suspected limited malignant liver disease. CTAP and MR imaging were done within a 1-week period (19 within 24 hours); the results of the studies were interpreted prospectively by separate reviewers. Surgical data were evaluated in conjunction with imaging data in 10 patients. Lesion detection and segmental involvement were determined and sensitivity and specificity were calculated. Procedural cost was determined from hospital billing codes. Effect on patient management was determined by the referring oncologic surgeon. CTAP and MR imaging showed 185 and 176 true-positive malignant lesions, 15 and zero false-positive malignant lesions, zero and 18 true-negative malignant lesions, and 13 and 22 false-negative malignant lesions, respectively. CTAP and MR imaging showed 107 and 105 true-positive segments, 11 and zero false-positive segments, 80 and 91 true-negative segments, and four and six false-negative segments, respectively. There was a significant difference in specificity of segmental involvement between MR imaging (1.0 +/- 0) compared with CTAP (0.88 +/- 0.05), P = .03. Total procedural cost was $3,499 for CTAP and $1,224 for MR imaging. CTAP findings did not change patient management over MR imaging findings in any patient, whereas MR imaging findings resulted in a change in patient management over CTAP findings in seven patients (P = .015). The results of our study suggest that MR imaging has higher diagnostic accuracy and greater effect on patient management than CTAP does and is 64% less expensive.