Somatic mutations of the APC, KRAS, and TP53 genes in nonpolypoid colorectal adenomas

Colorectal adenomas are macroscopically visible morphological changes of the mucosa that can develop focal carcinoma in the absence of surgical intervention. The successive molecular changes proposed to occur at different stages in the adenoma-carcinoma sequence were primarily based on DNA studies of exophytic, polypoid-type adenomas. Not all colorectal lesions, however, display an exophytic phenotype and a presumed distinct colorectal neoplasm, the nonpolypoid adenoma, was subsequently described as a precursor of colorectal cancer. The low incidence of KRAS mutations in nonpolypoid colorectal adenomas reported previously suggested a different genetic basis for the transformation process in these lesions. We have pursued the identification of genetic changes in benign sporadic nonpolypoid colorectal adenomas in a selected Swedish patient group with no family history of colorectal cancer. Mutation screening of the adenomatous polyposis coli (APC), KRAS, and TP53 genes was conducted using the protein truncation test, heteroduplex-single-strand conformation polymorphism analysis, and denaturing gradient gel electrophoresis on PCR-amplified fragments. Fourteen mutations in the APC gene were characterized in 10/20 samples. Mutations in the KRAS and TP53 genes were identified in 3/57 and 4/51 adenomas, respectively. The mutation frequencies and distribution of mutations in APC correlate with published data on exophytic adenomas. The low mutation frequency of the TP53 gene is consistent with the benign nature of the research material. KRAS activation (an early event in polypoid colorectal adenomas) apparently does not play a significant role in nonpolypoid adenoma development but may result in the development of a polypoid configuration. Genes Chromosomes Cancer 27:202-208, 2000.     

Clinical significance of alterations of chromosome 8 detected by fluorescence in situ hybridization analysis in pathologic organ-confined prostate cancer

Loss of 8p22 and gain of 8q24 are known to be common chromosomal alterations in prostate cancer. We have previously demonstrated that concurrent 8q24 overrepresentation and 8p22 loss were associated with a poor prognosis in patients with high-grade, locally advanced prostate cancer. We evaluated the alteration of 8p22 and 8q24 in a large cohort of pathologic organ-confined prostate cancer using fluorescence in situ hybridization (FISH) analysis. All 195 patients with Gleason scores > 5, pathologic stage T(2)N(0)M(0) (pT(2)N(0)M(0)) prostate cancer, who underwent a radical prostatectomy at the Mayo Clinic between 1987 and 1991, and for whom blocks were available, were selected for this study. The median follow-up period was 9.5 years, and endpoints of this study were biochemical and clinical disease progression. The latter includes local as well as systemic disease progression. FISH analysis using paraffin-embedded tissues was performed for 8p22 (LPL), centromere 8 (8cen), and 8q24 (MYC) and was successful for 156 tumors (80.0%). Of these tumors, 104 (66.6%) had one or more numeric alterations of the 3 loci evaluated. An increased copy number of 8q24 was observed in 66 (42.3%) tumors, of which 20 (12.8%) had an additional increase (AI) of 8q24, and 46 (29.5%) had a gain of 8q24 with an equivalent gain of 8cen. Losses and gains of 8p22 were detected in 81 (51.9%) and 20 (12.8%) tumors, respectively. An AI of 8q24 was significantly associated with the tumor Gleason score (P = 0.042). Univariate analysis indicated that loss of 8p22 was a significant predictor of biochemical and clinical disease progression (P = 0.025 and P = 0.011, respectively). Furthermore, the group with loss of 8p22 concurrent with an AI of 8q24 (Loss 8p22-any 8cen-AI 8q24) had an increased rate of biochemical disease progression (P = 0.052). Multivariate analysis demonstrated that neither individual nor the Loss-any-AI combination of alterations was a significant independent predictor of disease progression when adjusting for Gleason score, preoperative PSA levels, and DNA ploidy. These data suggest that loss of 8p22 is associated with a poor prognosis, specifically when it is accompanied by AI of 8q24 in pT(2)N(0)M(0) prostate cancer.     

Isolated hepatic cholinergic denervation impairs glucose and glycogen metabolism

BACKGROUND: Hepatic innervation plays an essential role in insulin extraction and glucose production, but the specific role of hepatic cholinergic innervation remains unclear. We sought to establish a model of isolated hepatic cholinergic denervation (IHCD), and to assess whether glycogen storage or the control of net hepatic glucose production (HGP) was altered by IHCD. MATERIALS AND METHODS: Sprague-Dawley rats underwent either hepatic vagotomy or sham operation. Liver tissue was stained for vesicular acetylcholine transporter (VAChT) and (nonspecific neural) protein gene product 9. 5 (PGP) for verification of IHCD. Liver glycogen content was quantified in fed and fasted IHCD or sham-operated animals. HGP was determined after single-pass isolated liver perfusion, during which a 30-min 12 ng/ml glucagon infusion was begun after equilibration, and after 10 min, a 200 microU/ml insulin infusion was added. RESULTS: Uniform staining of PGP and absence of VAChT staining in hepatic vagotomized rats demonstrated the validity of our model. Glycogen content of sham-operated livers (n = 8) increased from 6.0 +/- 1.7 in the fasting state to 10.6 +/- 1.8 mg/g liver, after feeding (P < 0.05). IHCD livers (n = 8) showed no comparable increase (3.5 +/- 0.6 to 4.0 +/- 0.7 mg/g liver). Perfusion with glucagon alone resulted in less HGP in IHCD livers (n = 12) compared with sham-operated livers (n = 10) (integrated HGP 3.3 +/- 0.3 mg/g liver min(-1) vs 5.1 +/- 0.5 mg/g liver min(-1), P < 0.05). Insulin infusion revealed impaired responsiveness to insulin after IHCD; the ratio of HGP in the final 10 min of perfusion (glucagon and insulin) to HGP in the initial 10 min (glucagon alone) was 90.3 +/- 2.4% for IHCD livers versus 68.1 +/- 4.4% for sham-operated controls, respectively (P = 0.0002). CONCLUSIONS: Our study shows that IHCD results in significant impairment in liver glycogen storage and impaired hepatic sensitivity to glucagon and, possibly, to insulin. We conclude that hepatic cholinergic integrity is essential to normal hepatic glucose metabolism.     

Effect of age and gender on sudomotor and cardiovagal function and blood pressure response to tilt in normal subjects

Normative data are limited on autonomic function tests, especially beyond age 60 years. We therefore evaluated these tests in a total of 557 normal subjects evenly distributed by age and gender from 10 to 83 years. Heart rate (HR) response to deep breathing fell with increasing age. Valsalva ratio varied with both age and gender. QSART (quantitative sudomotor axon-reflex test) volume was consistently greater in men (approximately double) and progressively declined with age for all three lower extremity sites but not the forearm site. Orthostatic blood pressure reduction was greater with increasing age. HR at rest was significantly higher in women, and the increment with head-up tilt fell with increasing age. For no tests did we find a regression to zero, and some tests seem to level off with increasing age, indicating that diagnosis of autonomic failure was possible to over 80 years of age. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1561–1568, 1997     

Somatically acquired genetic alterations in flat colorectal neoplasias

Somatically acquired mutations in several genes have been reported as playing an important role during colorectal tumorigenesis. Two alternative groups of carcinomas, termed LOH⁺ and RER⁺, have been defined on the basis of their genetic anomalies, a biallelic inactivation of the APC or the TGF-βRII genes, occurring as an alternative, in LOH⁺ or RER⁺ tumors. It is a generally accepted hypothesis that most of colorectal cancers (CRC) develop from a pre-existing adenomatous polyp. Such benign lesions are usually exophytic polyps, a small proportion of adenomas having been described as flat lesions. The latter histological category has thus been proposed to bear specific genetic alterations. In order to examine this hypothesis, we have characterized a series of 44 flat colorectal neoplasias for their RER status and for somatic APC, KRAS and TGF-βRII genes mutations. Flat colorectal neoplasias were found to be of the RER⁺ subtype in 22% of cases, all of them exhibiting a TGF-βRII mutation. A mutation of the APC and KRAS genes has been found in 42% and 4% of tumors, respectively, none of these tumors being of the RER⁺ subtype. With the exception of a low KRAS mutation rate, flat adenomas appear to follow tumorigenesis pathways very similar to those identified in exophytic adenomas and carcinomas. Int. J. Cancer 77:366–369, 1998. © 1998 Wiley-Liss, Inc.     

Y-specific sequences in Turner syndrome]

Turner Syndrome (TS) is the only one monosomy that occurrs+ in humans. The cytogenetics of TS is very well known from years. It has been estimated that almost 98-99% of TS foetuses end in abortion. It was suggested that the monosomy arises relatively late during embryonal development and survived TS individuals could be mosaics. It has been proved that mosaic karyotype mos 45,X/46X, + mar(Y) occurrs++ in 2% to 11% of TS patients. The patients having additional cell line containing der(Y) are at increased risk of gonadoblastoma development. In these cases gonadectomy should be considered. Therefore detection of mosaic and establishing the origin of marker chromosome (specially containing Y-specific sequences) is of special importance. The aim of present study was to detect the small mosaics, containing mar(Y) in TS patients, by using PCR and FISH techniques. Eight Y sequences for the PCR analyses as well as bicolor in situ hybridisation with painting probes for Y and X chromosomes have been applied. The positive amplification for Y-specific sequences has been detected in 7% of TS patients. Our results support the thesis that searching for the Y sequences should be introduced to routine genetic TS diagnosis.     

Acellular dermal matrix reduces capsule formation in two‐stage breast reconstruction

Acellular dermal matrix (ADM) is commonly employed to create an inferior pocket for the tissue expander in two‐stage breast reconstruction. The authors sought to determine whether placement of ADM during the first stage of reconstruction decreases the amount of capsule formation at implant exchange. Patients who underwent mastectomy and tissue expander reconstruction were included in this study. Two biopsies were obtained at the time of implant exchange, one from the pocket adjacent to the ADM and the other from the area adjacent to the pectoralis muscle. Pathology analysis was performed on each sample. Ten patients underwent immediate breast reconstruction with Alloderm during the 3‐month study period. Capsule thickness was significantly greater in the areas where the expander was in direct contact with the pectoralis muscle (782 ± 194 µm) compared to those in contact with human acellular dermal matrix (hADM) (47·91 ± 110·82 µm; P < 0·05). Analysis of the sub‐pectoral capsule demonstrated diffuse deposition of collagen, neutrophils, contractile myofibroblasts and synovia‐like metaplasia, characteristic of a foreign body response. Conversely, within the inferior pocket where the hADM was in direct contact with the expander, we noted migration of host epithelial cells, fibroblasts, mesenchymal cells and angiogenesis, indicating host tissue regeneration. Acellular dermal matrix, when placed at the first stage of breast reconstruction, significantly reduces thickness and inflammatory character of the capsule in comparison to the patient's native tissue.     

Long-term prognostic significance of primary Gleason pattern in patients with Gleason score 7 prostate cancer: impact on prostate cancer specific survival

PURPOSE: We determined the long-term clinical significance of primary Gleason pattern in patients with Gleason score 7 prostate cancer. MATERIALS AND METHODS: We reviewed the records of all patients who underwent bilateral pelvic lymph node dissection and radical retropubic prostatectomy for Gleason score 7 prostate cancer at our institution. All patients who underwent adjuvant hormonal or radiation therapy were excluded from analysis. Patients were monitored for biochemical failure, that is PSA progression, systemic recurrence and cancer specific survival. RESULTS: We identified 1,688 patients who met admission criteria, of whom 1,256 (74.4%) had primary Gleason pattern 3 and 432 (25.6%) had primary Gleason pattern 4. Median followup was 6.9 years. At 10 years primary Gleason pattern 3 was associated with increased biochemical recurrence-free survival (48% vs 38%, p <0.001), lower systemic recurrence (8% vs 15%, p <0.001) and higher cancer specific survival (97% vs 93%, p = 0.013) for Gleason primary grades 3 and 4, respectively. All of these end points remained significant on multivariate analysis when controlling for preoperative PSA, seminal vesicle involvement, margin status, DNA ploidy and TNM staging. PSA doubling time was shorter in patients with primary Gleason pattern 4 (1.64 vs 1.01 years). Systemic recurrence and cancer specific survival were associated with a PSA doubling time of less than 1 year. CONCLUSIONS: Gleason score 7 prostate cancer is a heterogeneous entity. We should continue to stratify patients according to primary Gleason pattern. Patients with Gleason score 4 + 3 prostate cancer have more aggressive disease and experience higher rates of biochemical failure, systemic recurrence and cancer specific death.     

Defining prostate specific antigen progression after radical prostatectomy: what is the most appropriate cut point?

PURPOSE: The most appropriate definition of biochemical progression after radical prostatectomy and radiation therapy is uncertain. We analyzed the effect of using various prostate specific antigen (PSA) end point definitions for defining biochemical progression after radical prostatectomy and attempted to determine the best PSA cut point to use. Aspects of the American Society for Therapeutic Radiology and Oncology (ASTRO) definition of biochemical failure after radiation therapy are also analyzed in our radical prostatectomy cases. MATERIALS AND METHODS: A total of 2,782 men with clinically localized prostate cancer (cT1-T2) who had undergone radical prostatectomy between 1987 and 1993 were reviewed. All patients had regular PSA determinations from surgery through followup. Analysis was limited to patients who did not receive adjuvant treatment within 90 days of radical prostatectomy. Biochemical, PSA progression-free percent after radical prostatectomy was determined by the Kaplan-Meier method using several PSA cut points, including 0.2, 0.3, 0.4 and 0.5 ng./ml. or greater, as well as 0.4 ng./ml. or greater and increasing. Progression-free percent was also assessed using the ASTRO definition, which is 3 increases in PSA. To determine which PSA level was most appropriate to define progression after radical prostatectomy, the percentage of patients with a continued PSA increase after reaching each cut point was determined. The relationship between the maximum PSA within 3 years of surgery and subsequent development of clinical disease was also assessed. RESULTS: Progression-free percent was dependent on the PSA cut point used. Biochemical progression-free percentages for cut points 0.2, 0.3, 0.4 and 0.5 ng./ml. or greater were 62%, 72%, 76% and 78% at 5 years, and 43%, 54%, 59% and 61% at 10 years, respectively. A subsequent increase in PSA was noted in 49%, 62% and 72% of patients who had PSA 0.2, 0.3 and 0.4 ng./ml., respectively. Subsequent clinical progression (local or systemic) was directly related to the maximum PSA attained within 3 years of radical prostatectomy (p=0.0001). Progression-free percent for definitions requiring multiple increases in PSA were dependent on when the event was said to occur. Backdating of events at or before the first PSA (ASTRO definition) resulted in poorer, short-term progression-free percent (78% at 5 years), with little apparent likelihood of long-term failure (78% at 10 years). Coding the event at the last PSA increase when all event criteria had been met resulted in more realistic progression-free percent estimates (85% at 5 and 59% at 10 years). CONCLUSIONS: Biochemical, PSA progression rates vary markedly depending on the method used to define PSA failure. Methods that require multiple increasing PSA values, for example the ASTRO definition, give misleading results, especially if the event time is backdated. Standards for defining PSA progression would allow more consistent and comparable progression estimates after radical prostatectomy. PSA 0.4 ng./ml. or greater may be the most appropriate cut point to use since a significant number of patients with lower PSA do not have a continued increase in it.