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PubMed2003

Molecular assessment of depth of myometrial invasion in stage I endometrial cancer: a model based on K-ras mutation analysis.

INTRODUCTION: Overall nearly 20% of endometrial cancer (EC) patients die of the disease and over half of these had initially presented with clinical stage I disease. There is a strong correlation between disease mortality and depth of myometrial invasion. Current assessment of depth of invasion relies on light microscopy. Tumor cells can evade detection by light microscopy if they are vastly outnumbered by myometrial cells. Molecular techniques have a great potential in the detection of such isolated cells. OBJECTIVE: The objective was to develop a model for the application of molecular techniques to advance the assessment of risk status in patients with clinical stage I EC. METHODS: The study sample included 21 stage I ECs with a documented K-ras mutation from two series of 96 and 106 ECs from the United Kingdom and Norway, respectively. K-ras was documented using heteroduplex mobility analysis and amplified created restriction site, followed by sequencing to identify the specific base substitution at codon 12 and 13 of K-ras oncogene. For each case with a K-ras mutation, a modified mutant allele-specific amplification technique was carried out on a series of tissue strips microdissected at increasing depths from the myometrium underlying tumor. The microdissected myometrium had been previously examined histologically for absence of infiltrating tumor cells on light microscopy. Presence of K-ras mutations was used to identify the tumor cells within the histologically normal myometrium. Correlations between submicroscopic myometrial tumor cell infiltration and clinicopathological factors were studied. RESULTS: Of 21 cases with K-ras mutation, 6 cases (28%) showed molecular evidence of tumor cell infiltration beyond the histological boundary. The depth of submicroscopic myometrial infiltration was found to be variable. The staging of the tumors would have changed in 3 cases (14%) if tumor cells been detected histologically. A borderline significant correlation between presence of submicroscopic myometrial invasion and depth of myometrial invasion was noted (P = 0.053). The recurrence rate and survival of patients without submicroscopic invasion were better than those with, although it did not reach statistical significance (recurrence rate P = 0.13, recurrence free survival P = 0.14, cause-specific survival P = 0.12, and total survival P = 0.2). CONCLUSIONS: Molecular assessment of depth of myometrial invasion using K-ras mutation is feasible and may add information to conventional light microscopy. Further prospective studies are required to define the clinical significance of this technology.

Adult↗
PubMed1999

SRY mutation and tumor formation on the gonads of XP pure gonadal dysgenesis patients.

We report three patients with XY pure gonadal dysgenesis. Two of these patients developed gonadoblastoma and associated dysgerminoma. Molecular analyses were undertaken to investigate the relationship between the formation of these tumors and Y chromosome aberrations. Deletion analyses were performed by polymerase chain reaction (PCR) amplification of Y chromosome-specific DNA sequences (PABY, SRY, DYS250, DYS254, and DYZ1). A cryptic deletion of the short arm of the Y chromosome that included the PABY, SRY, DYS250, and DYS254 loci was observed in one of the patients (22-years-old) with an associated tumor. In the other two patients who did not demonstrate such a deletion, the sequence of the SRY open reading frame was determined by the dideoxynucleotide method. Two nucleotide substitutions followed by a seven nucleotide deletion were observed in the 3' end of HMG (high mobility group)-box in the other patient (15-years-old) with an associated tumor. The patient (22-years-old) without an associated tumor did not have the cryptic deletion or mutation of SRY. A Y chromosome specific sequence (DYZ1) was demonstrated by PCR amplification of microdissected tumor tissues from these two patients. These results suggest that SRY may play a role in the formation of gonadal tumors, especially dysgerminoma.

Adolescent↗
PubMed1999

Update of diagnostic and prognostic markers in cutaneous malignant melanoma.

The biomolecules described in this article generally have been studied as possible diagnostic or clinically prognostic markers in the context of melanoma disease progression as measured by the gold standards of tumor thickness and development of metastasis. Most of the markers showed variations in expression phenotype only during the deeply invasive or metastatic stage of tumor progression and were thus predictive of clinical outcome only for these subgroups of patients. Some of the markers may have utility in identifying patients with deeply invasive primary tumors who are likely to develop metastasis and thus should receive earlier, more aggressive treatments. In addition, some of the markers may identify patients likely to respond better to a new type of therapy (e.g., anti-angiogenic therapy in a patient whose tumor is overexpressing VEGF or immunotherapy for a patient whose tumor is expressing high levels of MART-1). In the future, it will probably be possible to employ new techniques, such as laser-guided microdissection of tissues, to isolate individual melanocytes in order to identify the earliest stage-specific defects that contribute to an aggressive biological behavior. Identifying the subset of patients with superficially invasive melanomas who will develop metastatic disease will continue to provide a challenge.

Biomarkers, Tumor↗
PubMed2003

Selection and validation of microarray candidate genes from subregions and subnuclei of the brain.

DNA array technology now allows an enormous amount of expression data to be obtained. For large-scale gene profiling enterprises, this is of course welcome. However, the scientist interested in follow-up studies of a handful of differentially expressed genes may find it hard to sift through the vast datasets to pinpoint genes with the most desirable and reliable behaviors. Here, we present the methodology we have employed to discover genes differentially expressed in the adult mouse brain. We first used Affymetrix microarrays to compare gene expression from five different brain regions: the amygdala, cerebellum, hippocampus, olfactory bulb, and periaqueductal gray. Second, we identified genes differentially expressed within three distinct amygdala subnuclei. In this case, the tissue was microdissected by laser-capture to minimize contamination from adjacent subnuclei, and extracted RNA was subjected to three rounds of linear amplification prior to hybridization to the microarrays. To select candidate genes, we developed a custom algorithm to identify those genes with the most robust changes in expression across different replicate samples. Confirmation of expression patterns with in situ hybridization uncovered further criteria to consider in the selection process.

Algorithms↗
PubMedDate not supplied

Genetic changes during the multistage pathogenesis of human papillomavirus positive and negative vulvar carcinomas.

OBJECTIVE: To identify the molecular alterations found in 30 human papillomavirus (HPV) positive (n = 15) and negative (n = 15) vulvar carcinomas (VC) and their associated preinvasive lesions (VIN [vulvar intraepithelial neoplasia]) and normal epithelium to determine a common molecular pathogenesis of HPV positive and negative VC. METHODS: Loss of heterozygosity (LOH) at seven 3p chromosomal regions (3p12, 3p14.2, 3p14.3-21.1, 3p21.3, 3p22-24, 3p24.3, 3p25), 13q14 (RB) and 17p13.1 (p53) loci, and TP53 gene mutations in microdissected archival tissues were investigated. RESULTS: Fourteen of fifteen HPV positive VC had HPV 16 DNA sequences. The fractional regional loss index (FRL), an index of total allelic loss at all chromosomal regions analyzed, was greater in the HPV negative VCs than in the HPV positive tumors (FRL = 0.55 versus 0.32; P = .048) and was also greater in the HPV negative high-grade VINs as compared with the HPV positive lesions (0.29 versus 0.02; P = .002). Overall, LOH at any 3p region was frequent (80%) in both groups of cancers and in their associated VIN lesions. Although TP53 gene mutations were present in a minority of VCs (20%), allelic losses at the TP53 locus were frequently present, especially in HPV negative VCs, as compared with the HPV positive tumors (62% versus 15%; P = .02). CONCLUSION: A greater number of molecular alterations are found in HPV negative VCs compared with HPV positive tumors. Allelic losses at 3p are common early events in vulvar carcinogenesis in HPV negative cancers detected at a high rate in the corresponding high-grade precursor lesions (VIN II/III). TP53 gene mutations with associated 17p13.1 LOH are more common in HPV negative cancers.

Carcinoma in Situ↗
PubMed2001

Pattern of chromosome 16q loss differs between an atypical proliferative lesion and an intraductal or invasive ductal carcinoma occurring subsequently in the same area of the breast.

Atypical proliferative lesions of the breast, such as atypical ductal hyperplasia and atypical papilloma, are considered to be precursors of breast carcinomas and have frequently been shown to have loss of heterozygosity (LOH) on chromosome 16q at the DNA level. We evaluated whether an atypical proliferative lesion and a carcinoma that subsequently occurred in the same area of the ipsilateral breast were of identical clonal origin in seven patients. Using DNA isolated from microdissected archival tissue of epithelial components of both the biopsy specimen of the atypical proliferative lesion and the mastectomy specimen of the carcinoma, the pattern of LOH on 16q was compared between these two lesions using polymerase chain reaction -microsatellite LOH analysis. As a control, LOH on 16q was examined in 13 cases of usual ductal hyperplasia, 10 usual papillomas, and 6 atypical ductal hyperplasias. In the seven cases, LOH on 16q was detected in three of the six atypical proliferative lesions and in five of the seven carcinomas, but the allele with LOH or a deleted region always differed between the two. LOH was detected in both atypical proliferative lesions and carcinomas in one case, only in the atypical proliferative lesion in two cases, and only in carcinomas in three cases. In the controls, LOH on 16q was absent in usual ductal hyperplasias or usual papillomas but was detected in two of six atypical ductal hyperplasias. Although atypical proliferative lesions were frequently confirmed to be of clonal nature with LOH on 16q, these lesions and carcinomas were considered to be clones, probably originated from a field with these clones.

Adult↗
PubMed2026

Ribo-ITP enables identification of translons from limited input samples.

In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can act as regulatory elements or encode functional micropeptides. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we address this input limitation using Ribo-ITP on difficult-to-collect samples such as microdissected hippocampal tissues and single preimplantation embryos to identify thousands of translons. To test the translational capacity of the identified translons, we engineer a translon-dependent GFP reporter system and detect expression of translons initiating at ATG and near-cognate start codons in mouse embryonic stem cells (mESCs). We identify distinct expression patterns of translons using a comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types. Further, using a machine learning model, we predict that specific upstream translons in synaptically enriched mRNAs regulate translation efficiency of the annotated coding region. Taken together, we present a proof-of-concept study to identify non-canonical translation events from low input samples which can be applied to cell and tissue types inaccessible to conventional methods.

Animals↗
PubMed1997

A complex nine base pair deletion in RET exon 11 common in sporadic medullary thyroid carcinoma.

Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.

Base Sequence↗
PubMed1998

Allelic deletion at 11q23.3-q25 is an early event in cervical neoplasia.

Twenty per cent of cervical intraepithelial neoplasias-III (CIN-III) progress to invasive cancer. Human papillomavirus (HPV) infection alone does not determine progression. CIN-III lesions were collected from 161 women. Each tissue was microdissected into a maximum of 32 contiguous units and assayed at multiple microsatellite loci on chromosome 11q, a region frequently deleted in invasive cervical and other cancers. Eight of 108 informative cases (7%) had 11q23.3-q25 deletions; focally intra-lesional in six (one with focal loss of alternate alleles), and pan-lesional in two cases. Hence, 11q deletion can occur early in cervical neoplasia, and possibly predisposes to invasion.

Adolescent↗
PubMed1999

Evidence for a polyclonal etiology of palmar fibromatosis.

X chromosome inactivation patterns at the androgen receptor locus were evaluated to determine clonality in microdissected lesional tissue and in leukocytes from 2 women with Dupuytren's disease. The tissue from both patients generated a polyclonal pattern of X chromosome inactivation of the human androgen receptor gene. This finding supports a polyclonal reactive process as the underlying etiology for palmar fibromatosis.

Dosage Compensation, Genetic↗
PubMed1990

Regulation of the fibroblast growth factor receptor in early Xenopus embryos.

Recent evidence suggests that fibroblast growth factor (FGF) is a primary mesoderm inducer in Xenopus development. We have isolated a full-length cDNA clone for the Xenopus FGF receptor. Like other FGF receptors, the Xenopus homolog is a membrane-spanning protein with a split intracellular tyrosine kinase domain. The Xenopus FGF receptor mRNA is present as a maternal message whose levels are constant through early development. There is no specific regional localization of the transcript by analysis of FGF receptor mRNA levels in microdissected embryonic tissue. In isolated animal-pole blastomeres, FGF receptor mRNA declines over 16 hr in culture and this loss can be prevented by incubation with FGF or activin. Despite the presence of the FGF receptor mRNA in the oocyte, oocytes in culture do not respond to added FGF. However, injection of exogenous Xenopus FGF receptor transcripts into oocytes does generate a functional response to FGF. Our data suggest that posttranscriptional response to FGF. Our data suggest that posttranscriptional mechanisms regulate the FGF receptor in the oocyte and early embryo and further suggest that mesoderm-inducing factors influence receptor mRNA levels during the time of early tissue formation.

Amino Acid Sequence↗
PubMed2002

Down-regulation of Cdc6, a cell cycle regulatory gene, in prostate cancer.

CDC6 plays a critical role in regulation of the onset of DNA replication in eukaryotic cells. We have found that Cdc6 expression is down-regulated in prostate cancer as detected by semiquantitative reverse transcriptase-PCR of prostate cell lines and laser-captured microdissected prostate tissues. This result was substantiated by immunohistochemical analysis of paraffin-embedded tissue sections and immunoblot analysis of benign (BPH-1) and adenocarcinomatous prostatic cells. Furthermore, a 100-fold reduction in the transcription efficiency of the Cdc6 promoter-luciferase construct was noted in the metastatic PC3 cells compared with that in BPH-1 cells. Concentration of the E2F and Oct1 transcription factors that have putative binding sites in the Cdc6 promoter was substantially low in PC3 cells compared with BPH cells. Mutagenesis of the two E2F binding sites on the Cdc6 promoter resulted in increased promoter activity in PC3 cells owing to elimination of the negative regulation by pRb.E2F complex but not to the level of that obtained in BPH cells. We conclude that an altered interaction of transcription factors may be responsible for the down-regulation of Cdc6 transcription in PC3 cells. Our study suggests a potential use of the lack of CDC6 expression as an index of prostate cancer development.

Adenocarcinoma↗
PubMed2001

Staphylococcus aureus in inflammatory bowel disease.

BACKGROUND: The potential role of superantigens in inflammatory bowel disease (IBD), particularly Crohn disease, has been broached in studies of the functions of T cell receptors. Staphylococcal cells have been found in intestinal lymph follicles of IBDs. To clarify a role of staphylococcal superantigens in IBD, we attempted to determine whether Staphylococcus aureus could be detected in intestinal mucosa, including surgical specimens and lymph follicles of initial cases. METHODS: One-hundred-and-six colonic and ileal specimens were obtained from 38 Crohn disease, 25 ulcerative colitis and 36 non-IBD patients through therapeutic surgery or endoscopic biopsy. In Crohn disease, 23 surgical specimens and 11 biopsy specimens from initial cases were included. DNA was extracted with phenol-chloroform after homogenization and proteinase K treatment in 73 mucosal specimens. Using an inverted microscope, lymph follicle tissue was microdissected from the remaining 33, mostly biopsy, specimens. DNA was then extracted by freeze-thawing. A coagulase gene characteristic of S. aureus was sought. A nested polymerase chain reaction was performed utilizing primers that amplify a region of the coagulase gene. Polymerase chain reaction products were analyzed with polyacrylamide gel electrophoresis. RESULTS: Only one surgically resected colonic specimen, from a 42-year-old male ulcerative colitis patient, registered positive staphylocoagulase amplification. CONCLUSIONS: Staphylococcal superantigens are not involved in either the early lesions or the established lesions of Crohn disease. However, S. aureus infection occasionally may occur during the course of IBD.

Adult↗
PubMed2002

The hematopoietic system-specific minor histocompatibility antigen HA-1 shows aberrant expression in epithelial cancer cells.

Allogeneic stem cell transplantation (SCT) can induce curative graft-versus-tumor reactions in patients with hematological malignancies and solid tumors. The graft-versus-tumor reaction after human histocompatibility leukocyte antigen (HLA)-identical SCT is mediated by alloimmune donor T cells specific for polymorphic minor histocompatibility antigens (mHags). Among these, the mHag HA-1 was found to be restricted to the hematopoietic system. Here, we report on the HA-1 ribonucleic acid expression by microdissected carcinoma tissues and by single disseminated tumor cells isolated from patients with various epithelial tumors. The HA-1 peptide is molecularly defined, as it forms an immunogenic peptide ligand with HLA-A2 on the cell membrane of carcinoma cell lines. HA-1-specific cytotoxic T cells lyse epithelial tumor cell lines in vitro, whereas normal epithelial cells are not recognized. Thus, HA-1-specific immunotherapy combined with HLA-identical allogeneic SCT may now be feasible for patients with HA-1(+) carcinomas.

Hematopoietic Stem Cell Transplantation↗
PubMed2002

Rapid subcellular redistribution of Bax precedes caspase-3 and endonuclease activation during excitotoxic neuronal apoptosis in rat brain.

Neuronal apoptosis is induced prominently in the newborn rodent brain by glutamate receptor excitotoxicity and related insults, including trauma and hypoxia-ischemia. However, the molecular mechanisms of this neurodegeneration are unclear. We tested the hypothesis that changes in the subcellular distribution of the proapoptotic protein Bax precede the activation of downstream apoptosis-effector mechanisms such as caspase-3 cleavage and endonuclease activation during the progression of excitotoxic neuronal apoptosis in the striatum of newborn rat. Kainic acid (4 nmol) was injected into striatum of anesthetized 7-day-old rats, and the animals were killed at 2, 6, 12, and 24 h postinsult. Controls were age-matched, vehicle-injected, or naive rats. Counts of ultrastructurally confirmed striatal neuron apoptosis in brain sections were highest at 24 h. Striatal tissue was microdissected and fractionated into cytosolic, mitochondrial-, and nuclear-enriched compartments. Immunoblots showed that Bax translocates from the cytosol fraction to the mitochondrial fraction, with maximal translocation by 2 h in the absence of changes in mitochondrial accumulation. Cleaved caspase-3 levels increase progressively in both cytosolic and mitochondrial fractions between 6 and 24 h. Cleaved caspase-3 accumulates in apoptotic striatal neurons as shown by immunolocalization. Internucleosomal fragmentation of DNA coincides with caspase-3 cleavage. We conclude that rapid translocation of Bax to mitochondria precedes caspase-3 and endonuclease activation during excitotoxic neuronal apoptosis in newborn rat brain and that initiation of this death cascade occurs within 2 h after glutamate receptor activation.

Animals↗
PubMed2006

From bio-molecular and technology innovations to clinical practice: focus on ovarian cancer.

Ovarian cancer (OC) still represents the most lethal of gynecological malignancies with the chance for death in 5 years exceeding the chance for life. In recent years, the development of knowledge in molecular biology of OC coupled with the new technologies offers enormous opportunity to learn about aetiology of OC, and also give us a powerful tool for early diagnosis, prognosis and treatment of this disease. In particular, small cancer specimens from patients have become extremely informative thanks to techniques such as laser capture microdissection (LCM), tissue lysate arrays (TLAs), reverse trascriptase polymerase chain reaction (RT-PCR), and mass spectrometry. All of this coupled with advancements in bioinformatics have allowed the explosion of genomics, transcriptomics and proteomics. This paper focusses on the influence that advancement in the "-omics" bio-technology will reserve in OC diagnosis, prognostic characterization, and treatment.

Biotechnology↗
PubMed1994

Mutational spectrum in the p53 gene in bladder tumors from the endemic area of black foot disease in Taiwan.

An elevated risk of bladder cancer has been reported in the endemic region of 'black foot disease' on the southwest coast of Taiwan and may be related to high arsenic levels in artesian well water. Thirteen urothelial tumors from this endemic region were examined for mutations in exons 5-8 of the p53 gene to identify the effects of possible exogenous factors at the DNA level. DNA was extracted from archival tissue after microdissection of tumors and analyzed by PCR-SSCP (polymerase chain reaction-based single strand conformation polymorphism), followed by direct sequencing. Eight cases (62%) showed mutations and 9 of the 10 point mutations observed were transitions. The type and position of the mutations were not significantly different when compared with the spectra of p53 mutations previously reported for transitional cell carcinomas (TCCs). However, two of the mutations were CGC-->CAC base changes at codon 175, a mutational hotspot for many tumor types but previously unreported in TCCs except in cases associated with inflammatory agents. Three of the tumors examined were found to contain double mutations, a relatively rare mutagenic event in human cancers. Our results suggest that the agents responsible for the high risk of bladder cancer in the black foot disease region may operate through an inflammation-based mechanism which increases the amount of DNA damage per mutagenic event.

Adult↗
PubMed1996

An aflatoxin-associated mutational hotspot at codon 249 in the p53 tumor suppressor gene occurs in hepatocellular carcinomas from Mexico.

The p53 tumor suppressor gene is commonly mutated in human hepatocellular carcinoma (HCC). The most frequent mutation in HCC in populations exposed to a high dietary intake of aflatoxin B1 (AFB1) is an AGGarg-->AGTser missense mutation in codon 249 of the p53 gene. We analyzed HCCs from Monterrey, Mexico, for the codon 249ser hotspot mutation. We also analyzed the serum AFB1-albumin adduct levels of the donors and family members to measure the current AFB1 exposure in this population. Moreover, the presence of hepatitis B and/or C viral infection (HBV or HCV) was analyzed serologically in the patients. Tumor cells were microdissected from tissue sections and exon 7 p53 sequences were amplified by polymerase chain reaction from genomic DNA and sequenced directly. The serological tests for anti-p53 antibodies, HBV or HCV were done by ELISA. Immunohistochemical analysis of p53 protein was done using a polyclonal rabbit antiserum (CM-1). Eight of 21 cases were positive by p53 immunohistochemistry. Of the 16 cases sequenced for exon 7 of p53 three codon 249 AGGarg-->AGTser mutations were found. Serum antibodies recognizing p53 protein were found in one of 18 patients. Positive serology for HBV and/or HCV was found in 12 of 20 cases. The serum AFB1-albumin adduct levels in this population ranged from 0.54 to 4.64 pmol aflatoxin/mg albumin. These results indicate that dietary AFB1 and hepatitis viruses are etiological agents in the molecular pathogenesis of HCC in this geographic region of Mexico.

Adult↗