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PubMed2002

Genetic pathways in the evolution of breast ductal carcinoma in situ.

The patterns of allelic loss in 28 cases of pure ductal carcinoma in situ (DCIS) and 25 cases of DCIS associated with invasive ductal carcinoma (IDC) were compared, in order to define whether pure DCIS represented an earlier stage than DCIS associated with IDC in the progression of breast carcinoma. To this aim, the polymerase chain reaction (PCR) was performed on microdissected normal and neoplastic breast tissue, formalin-fixed and paraffin-embedded. Fifteen microsatellite markers were examined, on chromosomes 1p, 3p, 7q, 11q, 12p, 13q, 16q and 17q, mostly focused on regions altered in breast cancer. Loss of heterozygosity (LOH) was greater in pure DCIS than in the DCIS component associated with IDC for 11 out of 15 markers. The difference was statistically significant for D13S260 and D17S800 (p=0.008 and p=0.01, respectively). DCIS associated with IDC showed a lesser degree of alteration than the synchronous IDC component for ten out of 15 markers. In contrast, LOH at D11S1816 and D16S318 was lower in pure DCIS than in DCIS associated with IDC and even greater in the IDC component. These results confirm that DCIS is a possible but not an obligate precursor of invasive breast cancer and suggest that pure DCIS and DCIS associated with IDC may be genetically distinct. The evolution from DCIS to IDC may follow multiple pathways and not a linear model.

Breast Neoplasms↗
PubMed2002

Identification of loci associated with putative recurrence genes in transitional cell carcinoma of the urinary bladder.

Following an earlier study linking monosomy 9 with recurrence of transitional cell carcinomas (TCCs) of the urinary bladder, 109 primary and recurrent TCCs (from 47 patients) were examined to explore genetic alterations at chromosome 9 associated with recurrence. Patient DNA was microdissected and extracted from archival tissue sections and analysed for loss of heterozygosity (LOH) at three regions on chromosome 9 where tumour suppressor genes (TSGs) are known to reside (INK 4A, DBC1, and TSC1). Patients were categorized into two groups, non-recurrent TCC (NR, n=18) and recurrent TCC (REC, n=29). It was noted that 12% of NR tumours, compared with 54% of REC primary tumours (p=0.01), had LOH at all informative markers spanning the TSC1 region. The risk of recurrence was significantly higher in patients with deleted TSC1 than in those who retained the TSC1 region (p=0.035). Levels of LOH at DBC1 or INK 4A were not significantly different in NR tumours than in REC primary tumours and recurrence-free survival was not affected by loss of either of these genes. Loss of all informative markers spanning chromosome 9 was observed in 0% of NR tumours compared with 25% of REC primary tumours (p=0.04). The probability of recurrence was also significantly increased in patients who had LOH at all informative markers spanning chromosome 9 (p=0.016), confirming earlier fluorescence in situ hybridization results. This study provides further evidence that recurrence in bladder cancer is a distinct event, with underlying molecular causes. It also identifies the TSC1 locus as a candidate for a TSG, which drives recurrence in a proportion of TCC patients. Loss of all informative markers, including those residing in the TSC1 region, spanning chromosome 9 was also linked to recurrence.

Carcinoma, Transitional Cell↗
PubMed2003

Profiling and imaging proteins in the mouse epididymis by imaging mass spectrometry.

Different aspects of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) have been used as discovery tools to obtain global and time-correlated information on the local proteomic composition of the sexually mature mouse epididymis from both qualitative and semiquantitative points of view. Tissue sections and laser captured microdissected cells and secretory products were analyzed by MALDI-MS and from the recovered protein profiles, over 400 different proteins were monitored. Over 50 of these, some of which have been identified, displayed regionalized behavior from caput to cauda within the epididymis. Combining the information obtained from high-resolution imaging mass spectrometry and laser captured microdissection experiments, numerous proteins were localized within the epididymis at the cellular level. Furthermore, from the signal intensities observed in the different protein profiles organized in space, semiquantitative information for each protein was obtained.

Amino Acid Sequence↗
PubMed2006

Overexpression and elevated serum levels of phosphoglycerate kinase 1 in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a common malignancy with a very low 5-year survival rate. Currently, there are no valid markers for early detection and targets for therapy. Here, we used 2-DE to analyze the protein profiles of eight PDAC specimens and paired adjacent nontumor tissues. MS was used to identify 15 protein spots that were found to be overexpressed in PDAC tissues versus adjacent controls. One of them was identified as phosphoglycerate kinase (PGK) 1, a secretable glycolytic enzyme known to participate in angiogenesis. Immunohistochemical analysis of 63 PDAC specimens revealed moderate to strong expression of PGK1 in >70% of the tumors. Further Western blotting analysis of cells from tumor and adjacent nontumor tissues obtained by laser capture microdissection confirmed the enhanced expression of PGK1 in tumor cells. Furthermore, the serum levels of PGK1 were significantly higher in PDAC patients (n = 21) than in the control group (n = 25) (p < 0.005), as determined by ELISA. These observations indicate that protein profile analysis using a combination of 2-DE and MS provides an effective strategy for identifying biomarkers that may have diagnostic potential for PDAC, and identify PGK1 as a potential biomarker and/or therapeutic target for PDAC.

Adenocarcinoma↗
PubMed1991

Functionally complex muscles of the cat hindlimb. II. Mechanical and architectural heterogenity within the biceps femoris.

The goal of this study was to analyze the architecture of the cat biceps femoris (BF), a multifunctional hamstring muscle, and to evaluate the relationships between muscle architecture, limb position, and muscle function during natural movement. The BF muscle consists of three neuromuscular compartments: anterior (BFa), middle (BFm) and posterior (BFp). Each compartment is innervated by a separate nerve branch. Nerve branch stimulation and 2-dimensional surface EMG recordings showed that individual compartment territories were discrete and non-overlapping with well-defined borders. Comparisons of the three compartments revealed consistent differences in architecture, relationship to the skeleton, and function. The BFa crossed only the hip joint and appears to function as a pure hip extensor. The BFm had equal lever arm lengths to the hip and knee joints, appears to function as a hip extensor, and may contribute to knee flexion or femoral rotation. The BFp had a greater lever arm to the knee, functions as a knee flexor, and may contribute to hip extension, femoral rotation or ankle extension. Measurements of individual fascicles from the three compartments revealed a surprising range of lengths, 3.3-12.0 cm. Microdissection of gold-stained tissue showed that fascicles from all compartments were comprised of interdigitated, short fibers (range: 0.6-5.0 cm; average 2.14 cm) arranged in-series in fascicles, running parallel to the origin-insertion axis of each muscle compartment. In regions of fiber interdigitation, the fiber endings were round and tapered (taper lengths: 1-11 mm) although flat, tapering endings like ribbons were occasionally found. As hip and knee joint angles were varied over physiological ranges corresponding to minimal to maximal muscle length, fascicles of the three compartments changed length disproportionately. Long BFa fascicles maximally lengthened 10-18%, consistent with in vivo length measures during treadmill locomotion. However, the long BFp fascicles lengthened 25-45%, and the relatively short fascicles near the BFm/BFp border maximally lengthened 45-53%. How do these unexpectedly large length changes affect sarcomere lengths? Using laser diffraction to measure sarcomeres, static fascicle and sarcomere lengths were compared in muscles that went into rigor mortis after fixing the hip and knee joint angles. Sarcomeres within the short BFm/BFp and long BFp fascicles consistently lengthened proportionately less than the whole fascicle. It remains to be determined how and where the fascicle length changes are dissipated in the connective tissue between the interdigitated muscle fibers and whether such a series-compliance operates during the large excursions over which this muscle normally works.

Animals↗
PubMed1996

Frequent loss in chromosome 8p loci in liver cirrhosis accompanying hepatocellular carcinoma.

Most hepatocellular carcinoma (HCC) is preceded by liver cirrhosis, but the genetic changes involved in cirrhosis are not well understood. We therefore studied loss of heterozygosity (LOH) in cirrhotic and neoplastic foci in livers of 14 patients with HCC. The samples, microdissected from paraffin-embedded tissues, were analyzed using a polymerase-chain-reaction-based assay for dinucleotide repeat polymorphisms on 8p. Of the 14 cases, 13 showed constitutional heterozygosity for the microsatellite markers. In 7 (54%) of these 13 informative cases, LOH was detected in the primary HCC and, in these 7 doubly informative (informative and LOH-positive in primary HCC) cases, LOH was found in 16 (70%) of 23 liver cirrhotic foci. The pattern of 8p allelic loss was identical in each doubly informative tumor; however, some of the liver cirrhotic foci harbored an 8p loss identical to that seen in the primary HCC, some harbored a different 8p loss, and some did not harbor any 8p loss. The remaining 6 cases without LOH on 8p in HCC showed no 8p loss in any cirrhotic foci. Presumably HCC could develop from cirrhotic cells harboring 8p loss.

Adult↗
PubMed1984

Intralobular distribution of UDP-glucuronosyltransferase in livers from untreated, 3-methylcholanthrene- and phenobarbital-treated rats.

A 3-methylcholanthrene-inducible enzyme form of UDP-glucuronosyltransferase has been localized within the liver lobule both immunohistochemically and enzymatically in microdissected centrilobular and periportal liver tissue. Livers of untreated, 3-methylcholanthrene- and phenobarbital-treated rats have been compared. The enzyme was detected in hepatocytes throughout the liver. However both immunohistochemical determination of the enzyme level and biochemical determination of its activity towards 1-naphthol revealed a heterogeneous distribution of the enzyme. In untreated controls and 3-methylcholanthrene-treated rats both enzyme activity and histochemical staining was highest in centrilobular hepatocytes. However, after phenobarbital-treatment enzyme staining and activity was highest in periportal hepatocytes, suggesting that the differentially inducible enzyme activities may be localized in different zones of the liver lobule. The results demonstrate that the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase is preferentially expressed in centrilobular hepatocytes.

Animals↗
PubMed1988

Distribution, quantitation, and origin of immunoreactive neuropeptide Y in the human gastrointestinal tract.

A radioimmunoassay for measurement of immunoreactive neuropeptide Y has been developed using antiserum from a rabbit (221) immunized with porcine neuropeptide Y. Antibody 221 has been characterized for both sensitivity and specificity. To determine the distribution of neuropeptide Y in the human gastrointestinal tract, fresh tissue specimens were separated by microdissection into the muscularis externa and the mucosa-submucosa. To examine the origin of neuropeptide Y in human colon, specimens of aganglionic and ganglionic colon were obtained from patients with Hirschsprung's disease. Immunoreactive neuropeptide Y in human gut was present in highest concentrations in the muscularis externa of the stomach and in lowest concentrations in the muscularis externa of the ileum and descending colon. Neuropeptide Y in the stomach was present in higher concentrations in the muscularis externa than in the mucosa-submucosa, but in the descending colon there were lower concentrations of neuropeptide Y in the muscularis externa than in the mucosa-submucosa. In Hirschsprung's disease, concentrations of neuropeptide Y were increased in aganglionic colon in both the muscularis externa and the mucosa-submucosa, compared to corresponding layers from proximal ganglionic colon. Extracts of the gastric muscularis externa and the colonic mucosa-submucosa were separated by C18 reverse-phase high-performance liquid chromatography. One major immunoreactive species was identified by radioimmunoassay which eluted in a position similar to synthetic human neuropeptide Y. These results demonstrated both regional and layer differences in concentrations of neuropeptide Y in human gut. Increased concentrations of neuropeptide Y in aganglionic colon from Hirschsprung's disease most likely result from enlargement of neuropeptide Y-containing extrinsic nerve fibers in both the mucosa-submucosa and the muscularis externa.

Animals↗
PubMed1983

Effects of pinealectomy on neurohypophysial hormones in the SFO and plasma of dehydrated rats exposed to 12 hours of light.

Magnocellular neurons in the supraoptic and paraventricular nuclei synthesize and release vasopressin and oxytocin in response to dehydration. Pinealectomy has been observed to decrease the distribution in the supraoptic nuclei of thiamine diphosphate-phosphohydrolase, an enzyme specific for the Golgi apparatus that correlates positively with neurosecretory activity. Based upon these studies we postulated that pinealectomy would alter the concentration of neurohypohysial hormones in plasma elevated by 48 hr of water deprivation. In addition, we investigated the possibility that pinealectomy would affect vasopressin concentration in another circumventricular organ, the subfornical organ (SFO) and in a adjacent fiber tract of the limbic system, the hippocampal commissure-fornix (HC-F). Adult, male, Sprague-Dawley rats exposed to a 12 hr light/dark cycle were either unoperated (controls; C), sham-operated (Sham; S) or pinealectomized (PX) three weeks prior to testing. Food and water consumption and urinary excretion of Na and K were measured for 7 days. On the fifth day, half of the animals in each treatment group (C, S, PX) were deprived of water for 48 hr. Animals were decapitated on day 8. Vasopressin and oxytocin in plasma were extracted using bentonite and acetone-ether, respectively, then quantified by radioimmunoassay. The SFO and HC-F were microdissected from each brain. Like tissues from 4 rats were pooled, homogenized in 0.1 N HCl, and centrifuged. The supernatant was neutralized and vasopressin was quantified by radioimmunoassay. Dehydration resulted in antidiuresis, increased urine concentrations of Na and K, a decreased ratio of Na:K in urine, and reduced food consumption of similar magnitudes in all groups (C, S, PX; p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Somatic D-loop mitochondrial DNA mutations are frequent in uterine serous carcinoma.

The mitochondria plays a role in apoptosis. Its genome is also more susceptible to mutations because of high levels of reactive oxygen species and limited repair mechanisms. The D-loop of mitochondrial DNA (mtDNA) contains essential transcription and replication elements, and mutations in this region might alter the rate of DNA replication. We examined genetic alterations in the D-loop region of mtDNA in uterine serous carcinoma (USC) samples and their paired normal adjacent endometrium. DNA was extracted after laser-capture microdissection of paraffin-embedded tissues from eight patients with USC. The entire D-loop genome was amplified using nine pairs of overlapping primers. Denatured polymerase chain reaction (PCR) products were subjected to single-strand conformation polymorphism (SSCP) analysis. Somatic mtDNA alterations were detected in five tumours (63%). Our study indicates that mtDNA D-loop sequence alterations occur at a high frequency in USC suggesting that mtDNA mutations may play a role in the development of USC.

Aged↗
PubMed2005

Deletions of chromosomes 9 and 8p in histologically normal urothelium of patients with bladder cancer.

OBJECTIVES: Multifocality and frequent recurrence of urothelial carcinoma of the urinary bladder indicate a disease of the entire "urothelial field", but little is known about chromosomal alterations in histologically normal urothelium. Histopathologically normal urothelium from patients with and without concurrent urothelial carcinoma was analyzed for loss of heterozygosity (LOH) on chromosomes 8p, 9 and 17p, regions that are known to play an important role in urothelial carcinogenesis. MATERIALS AND METHODS: Histologically inconspicuous urothelial mucosa samples (n = 160) from cystectomy specimens of patients with urothelial carcinoma (n = 15, max. diagnosis: CIS, pT1-4; all tumors grade 3) were studied. Control samples (n = 50) were obtained from patients with benign prostatic hyperplasia (n = 30) and from cystectomies performed for invasive non-urothelial carcinoma (n = 20). Tissue samples were laser-microdissected and DNA was isolated using standard protocols. LOH analyses were performed using 16 polymorphic markers on chromosomes 8p, 9p/q and 17p. RESULTS: All investigated samples were informative for at least one microsatellite marker on each chromosome/chromosomal arm. Deletions were found on chromosome 9 and/or 8p in 15/160 (9.4%) of normal urothelial samples from urothelial cancer patients. These alterations were only found in 5/15 patients with urothelial carcinoma. There were no deletions on chromosome 17p. The marker D9S1113 on chromosome 9q33-34 was most frequently affected (11/15 samples, 73%). No chromosomal deletions were found in any of the 50 urothelial control samples. CONCLUSION: Specific genetic alterations frequently associated with bladder cancer are detectable in histologically normal urothelium of patients with bladder cancer, supporting the field effect hypothesis in urothelial carcinogenesis. However, intramucosal spread of tumor cells that escape light microscopic detection remains a possibility, since normal urothelium and concurrent carcinomas showed matching deletions. Chromosomal deletions in normal bladder mucosa are not a common finding and argue against a frequent genomic alteration of the entire urothelial field in bladder carcinogenesis.

Aged↗
PubMed2005

K-ras mutation influences macroscopic features of gastric carcinoma.

INTRODUCTION: Gastric carcinoma is classified morphologically as type 1 to 4. Type 1 is defined as a polypoid tumor; types 2 and 3 are defined as ulcerated tumors with polypoid growth or gastric wall infiltration, respectively, and type 4 tumors are defined as flat. This morphological classification is important because biological characteristics differ between the four morphological types, but little is known about genetic differences between them. MATERIALS AND METHODS: One hundred eight gastric tumors were classified macroscopically as type 1 to 4. Tumoral DNA was microdissected from paraffin-embedded tissue sections. PCR amplification of exon 1 of a K-ras containing codons 12 and 13 was performed. K-ras amplicons were dot-blotted onto nylon filters and hybridized with radiolabeled oligomer primers. RESULTS: A K-ras mutation was found in 20 of 108 gastric cancers. A significant relationship of K-ras mutation with polypoid cancer was found. The frequency of K-ras mutation was 6/14 (43%), 8/29 (28%), 2/11 (18%), and 4/54 (7%) in type 1 to 4 tumors, respectively. K-ras mutation was correlated with well-differentiated tumors. Of various types of K-ras mutations, 12 Asp often was seen in type 1 and 2 gastric cancers (well-demarcated, elevated tumors), while 12 Val and 12 Ser were often seen in type 3 and 4 cases (infiltrating carcinomas). CONCLUSION: K-ras mutations occur prominently in type 1 and type 2 gastric cancers.

Adenocarcinoma↗
PubMed2004

Marked allelic imbalance on chromosome 5q31 does not explain alpha-catenin expression in epithelial ovarian cancer.

OBJECTIVE: Human alpha-catenin gene (CTNNA1) on chromosome 5q31 is aberrantly expressed in various types of cancer including epithelial ovarian tumors. Allelic imbalance on this region has also been described in several malignant diseases. In the present work, the role of CTNNA1 as a candidate tumor suppressor gene was studied by comparing protein expression with allelic imbalance in human epithelial ovarian tumors. METHODS: Alpha-catenin protein expression was determined from two areas of 41 tumors, and tissues from these areas were microdissected. After DNA extraction, AI analysis was carried out with eight microsatellite markers. RESULTS: Altogether, 93% of the tumors (38 of 41) showed allelic imbalance at one or more loci. Two distinct common regions of allelic imbalance were identified, one comprising markers D5S2002 and D5S1995 and the other markers D5S393 and D5S476. Loss of the CTNNA1 gene did not appear to be involved in down-regulation of alpha-catenin in ovarian tumors, since allelic imbalance with a variety of markers, including CTNNA1 associated marker D5S476, was found in tumor samples independently of alpha-catenin expression. Furthermore, allelic imbalance analyses of two different samples from the same tumor revealed genetic heterogeneity. CONCLUSIONS: High allelic imbalance frequency indicates that chromosomal region 5q31 is functionally important in epithelial ovarian cancer. Allelic imbalance occurs at two distinct regions of which one includes the CTNNA1 gene. However, this gene is likely to be inactivated by mechanisms other than allelic imbalance. In addition, genetic heterogeneity observed in these tumors demonstrates the multiclonal nature of epithelial ovarian tumors.

Adenocarcinoma↗
PubMed2005

Clinical applications of microarray technology: creatine kinase B is an up-regulated gene in epithelial ovarian cancer and shows promise as a serum marker.

OBJECTIVE: (1) To identify and (2) validate genes that are up-regulated in ovarian cancer, and (3) to investigate whether the activity of a candidate gene, creatine kinase B (CKB) is elevated in pre-operative sera from ovarian cancer patients compared to patients with benign pelvic masses and normal controls. METHODS: MICROMAX cDNA microarray system and RNA derived from pooled ovarian cancer cell lines and normal ovary surface epithelial cells (HOSE) were used to identify differentially expressed genes. Using RNA from both cell lines and from tissue obtained through laser capture microdissection (LCM), we performed quantitative PCR in order to validate up-regulation of one of these genes, creatine kinase B (CKB). Using a commercially available enzyme assay, CKB activity was measured in pre-operative serum samples obtained from 45 ovarian cancer patients, 49 patients with a benign pelvic mass, as well as 37 normal controls. Statistical analysis was preformed using an unpaired Student's t test. RESULTS: Microarray technology revealed that CKB gene expression had a cancer to HOSE ratio of 18. RNA levels of CKB, measured by real-time PCR, were elevated a mean (and standard error) of 36-fold (8.4) in cancer cell lines compared with HOSE cells and 22.75-fold (10.45) in microdissected ovarian cancer epithelial cells compared with normal ovarian epithelial cells. In serum, the mean (+/-standard error) of CKB enzyme activity in cancer cases was 24.7 U/L units (+/- 5.1) compared to 9.6 U/L (+/- 1.6) for benign mass cases (P = 0.0088) and to 8.5 U/L (1.7) for normal controls (P = 0.0096). CONCLUSIONS: Microarray technology offers a method to identify tumor biomarkers with potential clinical usefulness. Our data indicated that CKB gene expression is up-regulated in ovarian cancer cells in vitro and in vivo and that CKB enzyme activity is significantly elevated in sera from ovarian cancer patients, including those with stage I disease. These findings suggest a potential role for CKB as a marker for early diagnosis.

Adult↗
PubMed2002

Loss of heterozygosity on chromosome 5p13-12 predicts adverse prognosis in advanced bladder cancer independent of tumor stage and grade.

PURPOSE: The individual prognosis in patients with bladder cancer is only partially predicted by tumor stage and grade. Various molecular markers have been shown to correlate with disease progression and prognosis but few add some predictive capacity beyond that offered by standard clinical and pathological parameters. MATERIALS AND METHODS: A total of 191 urothelial carcinomas from 157 patients were analyzed. Paired normal and tumor cells were microdissected by manual and microbeam-microdissection of membrane mounted native tissue, and analyzed for loss of heterozygosity (LOH) at a critical region of LOH on chromosome 5p13-12 that is involved in bladder cancer progression. LOH data were correlated with progression-free and tumor specific survival by multivariate analysis. RESULTS: Informativity of the assay was 60.7%. Log rank analysis of 100 evaluable patients showed a progression-free survival benefit without LOH at 5p13-12 of more than 3 years (p <0.01). Mean followup was 36 months. Multivariate analysis revealed that this benefit was not predicted by tumor stage and grade alone in advanced disease (stages T3/4 and/or N+/M+, stages III/IV). CONCLUSIONS: LOH at the critical region on chromosome 5p13-12 is a molecular marker of adverse prognosis in advanced bladder carcinoma independent of tumor stage and grade.

Carcinoma, Transitional Cell↗
PubMed1998

Carcinosarcoma of the breast: molecular-biological study for analysis of histogenesis.

The histogenesis of carcinosarcoma of the breast is controversial. In the current case, the demarcation between the carcinomatous and sarcomatous components was distinct in all microscopic fields. Immunohistochemical analysis was negative for epithelial membrane antigen (EMA) and keratin in the sarcomatous component and was negative for desmin in the carcinomatous component, suggesting that this tumor could be derived from the two different stem cells. To determine the histogenesis of this tumor, both carcinomatous and sarcomatous lesions were microdissected from formalin-fixed tissues and DNAs were prepared by proteinase K digestion. PCR amplification of the human androgen receptor (HUMARA) short tandem repeat (STR), after Hpa II digestion of the genomic DNA, indicated that the patterns of X-chromosome inactivation were identical in both components. Moreover, both components contained the identical TGT --> TTT transversion in codon 275 of the p53 gene. These observations strongly support the hypothesis that this tumor is derived from a single totipotent stem cell.

Breast Neoplasms↗
PubMed1999

Molecular abnormalities associated with secretory carcinomas of the breast.

Secretory carcinomas (SCAs) represent a unique histological variant of invasive breast carcinomas, occurring predominantly in patients younger than 30 years of age. Data from limited series have shown SCAs to have a favorable prognosis in patients younger than 20 years of age, whereas the clinical course tends to parallel the more common in filtrating ductal carcinomas (IDCs) in patients older than 20 years. There are no reports on the molecular abnormalities associated with this unusual tumor. Microdissected archival formalin-fixed tissue from 10 SCAs collected from 2 institutions were used to determine the frequencies of allelic loss at 13 chromosomal regions with 19 microsatellite markers, using multiplex polymerase chain reaction (PCR)-based techniques. The results of loss of heterozygosity (LOH) and microsatellite alterations (MAs) analyses were compared with 20 cases of IDCs. P53 gene mutation analysis was also performed on the 10 SCAs using single-strand conformation polymorphism (SSCP) analysis, followed by sequencing of abnormal bands. LOH at multiple regions of chromosome 3p were the most common abnormality in both SCAs (55%) and IDCs (50%), followed by LOH at 17q21 (BRCA1 locus), 13q14 (retinoblastoma gene locus), and 8p21-23. No significant differences were seen in the frequencies of LOH at any chromosomal region except for 17p13 (p53 gene locus), where allelic losses were absent in SCAs, but evident in 46% of IDCs (P < .05). The 2 histological entities were similar in the fractional regional loss (FRL) index (0.26 v 0.24), fractional allelic loss (FAL) index (0.23 v 0.27), as well as in the frequency of MAs (0.015 v 0.005), P > .05. P53 gene missense mutation (G:C::A:T) was detected in 1 of 10(10%) SCAs. Based on the considerable similarities in the molecular abnormalities associated with both tumors, the formation of secondary lumina in both the in situ and the invasive components, as well as suggestions from limited series that the clinical behavior in adult patients parallels that of IDCs, SCA most likely reflects a secretory variant of IDCs.

Adult↗
PubMed2003

p16 Hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis.

Abnormality of the p16 expression is involved in the pathogenesis of hepatocellular carcinoma (HCC), and hypermethylation of p16 gene is known as a major p16 inactivation mechanism. Cirrhotic nodule (CN) is now regarded as a preneoplastic lesion that is frequently associated with microscopic foci of HCC through dysplastic nodules (DNs). This observation clearly supports a multistep hepatocarcinogenesis from CNs through DNs. We thus examined the methylation status of p16 gene in HCCs surrounded by DNs and CNs to define the significance of p16 hypermethylation in the early stage of hepatocarcinogenesis. We tested 24 hepatitis B virus (HBV)-associated CNs, 37 DNs, and 18 HCCs within DNs that were microdissected from paraffin-embedded tissue sections. Frequency of p16 hypermethylation was significantly high in HCCs within DNs (15/18. 83.3%) and it increased from CNs (15/24. 62.5%) through DNs (26/37, 70.3%). Interestingly, 11 out of 12 (91.7%) HCC associated with methylation-positive DNs revealed hypermethylation of p16, and 18 out of 23 (78.2%) DNs associated with methylation-positive CNs showed p16 hypermethylation. These data suggest that p16 hypermethylation in the early stages, CNs and DNs may predispose to HCC. In addition, p16 methylation status of five cell lines with or without HBV infection was examined to test whether the high frequency of hypermethylation is related to HBV infection. HBV-infected cell lines were exclusively methylation-positive. These data suggest that high frequency of hypermethylation may be associated with hepatitis B virus infection.

DNA Methylation↗