Kankatsu Yun

Diagnostic use of the sentinel node in colon cancer

PURPOSE: The aim of this study was to compare the lymphatic drainage of colon cancer with the anatomic distribution of histologic and submicroscopic lymph node metastases. METHODS: Patients attending for colectomy were eligible to enter the study. At the commencement of surgery, 40 MBq of 99mTc colloidal antimony sulfide in 2 ml of Patent Blue dye was injected subserosally around the tumor. Resection was completed in a standard fashion. After resection, specimens were imaged with a gamma camera to determine the site of sentinel lymph nodes, and then dissected, recording the position of the lymph nodes on an anatomic diagram. Recovered lymph nodes were bisected, one-half for routine histology and one-half for assessment by keratin 20 (K20) reverse transcription polymerase chain reaction. The kappa measure of agreement was used to assess concordance between sentinel nodes and histologic and submicroscopic metastases. RESULTS: Four hundred fifty-six lymph nodes were dissected from 26 tumors and evaluated using lymphoscintigraphy and lymph node mapping. Sentinel nodes were evident in 23 tumors (88 percent). The sensitivity of sentinel nodes involvement as a predictor of metastatic disease was 55 percent (95 percent confidence interval, 23–83), with a false negative (nondiagnostic) rate of 45 percent. Sentinel nodes involved the apical group in four tumors, and represented anatomic “skip” lesions in four tumors. CONCLUSIONS: Direct lymphatic drainage to the apical group does occur in colon cancer; however, sentinel node mapping of colon cancer by this technique is of little clinical value because of the poor concordance between lymph node metastases and sentinel nodes.

Prognostic significance of occult metastases in colon cancer.

AbstractPURPOSE: The purpose of this study was to determine the prognostic significance of occult lymph node metastases in colon cancer detected by cytokeratin 20 reverse transcription polymerase chain reaction. METHODS: Two hundred patients undergoing elective colonic resections were enrolled in the study. Lymph nodes from resected specimens were dissected fresh and assessed by both reverse transcription polymerase chain reaction and histopathology. Follow-up was undertaken for up to five years, and the major end point of death was recorded. Univariate survival analysis was performed by the log-rank method and the change-in-estimate method was used to construct multivariate analysis models for the effect of cytokeratin 20 reverse transcription polymerase chain reaction lymph node status on overall survival. RESULTS: A total of 2,317 lymph nodes from 200 patients were assessed by both histopathology and cytokeratin 20 reverse transcription polymerase chain reaction. Forty-eight of 141 (34 percent) histologically lymph node–negative patients had evidence of occult metastases by cytokeratin 20 reverse transcription polymerase chain reaction. An interim analysis was performed at a median of 42 (range, 23–75) months. Cytokeratin 20 reverse transcription polymerase chain reaction lymph node status was a highly significant predictor of overall survival (P < 0.0001) on univariate analysis. In addition, the number of reverse transcription polymerase chain reaction–positive lymph nodes was a significant predictor of survival in the histologically lymph node–negative group (P < 0.0001) on univariate analysis. On multivariate analysis cytokeratin 20 reverse transcription polymerase chain reaction lymph node status had independent prognostic significance for overall survival (P = 0.021; hazard ratio = 2.7) and the number of cytokeratin 20 reverse transcription polymerase chain reaction–positive lymph nodes was a significant predictor of overall survival in the histologically lymph node–negative group (P = 0.005; hazard ratio = 1.1–11.1). CONCLUSION: Cytokeratin 20 reverse transcription polymerase chain reaction has potential as a clinically useful marker for staging colorectal cancer. Further follow-up is required, but if the current trends continue, a study of the effect of adjuvant therapy in patients with occult metastases detected by cytokeratin 20 reverse transcription polymerase chain reaction is indicated.

Myogenesis in Wilms Tumors Is Associated with Mutations of the WT1 Gene and Activation of Bcl-2 and the Wnt Signaling Pathway

Wilms tumors with WT1 mutations [WT1(−)] have a stromal-predominant histology with varying extents of rhabdomyogenesis. These tumors also frequently have mutations in the beta-catenin gene (CTNNB1). We have investigated the molecular events that may explain the origins of rhabdomyogenesis in WT1(−) tumors. Of 35 Wilms tumors, we identified 12 with WT1 mutations, of which 9 carried CTNNB1 mutations. We compared WT1 wild-type tumors [WT1(+)] with WT1(−) tumors for histological features, localization of beta-catenin, Bcl-2 expression, and apoptosis using an in-situ end-labeling technique. WT1(+) tumors showed triphasic and blastemal- and epithelial predominant-histology. Expression of WT1, beta-catenin, and Bcl-2 recapitulated those of normal kidney epithelial development. Localization of beta-catenin was observed in the cytoplasm and cytoplasmic membrane of early glomerular epithelial structures. Bcl-2 is also expressed in condensing blastema and early glomerular epithelial structures which had little apoptosis. WT1(−) tumors, regardless of whether CTNNB1 mutations were detected or not, showed a stromal-rich phenotype with abundant expression of beta-catenin in the nucleus of the rhabdomyoblasts. Bcl-2 was expressed in rhabdomyoblasts, but not in blastemal cells undergoing apoptosis, suggesting that WT1 regulates Bcl-2 positively in the epithelial pathway, but negatively in the myogenic pathway. These data indicate that mutations in WT1 might alter the Wnt signaling pathway and Bcl-2 related-apoptosis. In WT1(−) tumors, the nuclear accumulation of beta-catenin and Bcl-2 expression are associated with rhabdomyogenesis, and dysregulation of Bcl-2 may be a mechanism by which the histogenesis (loss of blastemal component, muscle differentiation) may be explained.

Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer.

SummaryWe have developed sensitive assays for cytokeratin (K) 8, 16, 19, stromelysin 3 (ST3), MUC1 and maspin mRNAs using reverse transcription polymerase chain reaction (RT-PCR) and used these to assess lymph node status in patients undergoing surgery for breast cancer. In addition the RT-PCR assays were tested against lymph nodes from non-cancer patients to determine their specificity. Despite high sensitivity RT-PCR assays for K8, K16, K19, ST3 and maspin were not found to be useful as markers of submicroscopic disease as transcripts of these genes were detected in the great majority of control lymph nodes tested. Expression of MUC1 was also not found to be useful as it was both insensitive and non-specific. The importance of assessing potential markers against an adequately sized control population is demonstrated, as failure to do so can lead to erroneous conclusions.

The Human ASCL2 Gene Escaping Genomic Imprinting and Its Expression Pattern

The mouse achaete-scute homolog-2 gene (Ascl2 or Mash2) encodes a transcription factor playing a role in the development of the trophoblast. The Ascl2 is an imprinted gene with maternal expression and assigned to an imprinting gene cluster region (ICR) at a distal region of mouse chromosome 7. We previously isolated a phage clone carrying the human homolog, ASCL2, and mapped it to human chromosome 11p15.5, a human ICR. In the present study, we demonstrate the expression patterns of the human ASCL2 in the fetus at a stage between first and second trimesters and in the placental tissues. In addition, it has been shown that the human ASCL2 gene escapes genomic imprinting.

In situ detection of insulin-like growth factor II (IGF2) and H19 gene expression in hepatocellular carcinoma

AbstractTo assess the relationship between insulin-like growth factor II (IGF2) and H19 gene expression at the cellular level, we have examined the distribution of IGF2 and H19 mRNA by means of in situ hybridization in hepatic malignancies consisting of hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC), and metastatic liver cancer (MLC). In HCC, 15 of 27 tumors (56%) and 11 of 27 tumors (41%) demonstrated increased IGF2 and H19 gene expression, respectively. Of 16 HCCs with increased expression of either IGF2 or H19, 10 tumors coexpressed both transcripts at comparable levels. Moreover, the spatiotemporal distribution and the cellular localization of the two gene transcripts were almost identical, suggesting the presence of a reciprocal relation between IGF2 and H19. In addition, 5 HCCs showed increased IGF2 expression without concomitant H19 expression, whereas 1 HCC showed increased H19 expression without IGF2 transcripts. However, 11 HCCs showed no IGF2 or H19 expression. On the other hand, neither IGF2 transcripts nor H19 transcripts were detected in 2 CCCs or 10 MLCs studied. The data suggest that IGF2 and/or H19 gene expression may be characteristic of some HCCs.

Analysis of IGF2 gene imprinting in breast and colorectal cancer by allele specific-PCR

The insulin‐like growth factor II (IGF2) gene is imprinted with the paternal allele expressed and the maternal one silent. Loss of imprinting (LOI) of IGF2 has been suggested to play a role in the development of tumours, but the reported incidence of IGF2 LOI in tumours shows considerable variation, which may stem from different methodologies employed. In particular, partial digestion of reverse transcriptase‐polymerase chain reaction (RT‐PCR) products by restriction enzymes can lead to inaccurate measurements. To overcome the problem of partial enzymatic digestion, a novel method termed allele specific‐polymerase chain reaction (AS‐PCR) has recently been reported, which provides a significant advance over enzymatic digestion. A second problem with measurements of biallelic IGF2 transcription is that the co‐amplification of contaminating genomic DNA during the RT‐PCR step can lead to an overestimation of the frequency of biallelic IGF2 expression. To investigate the extent of this problem, total RNA from breast and colorectal cancer was analysed using two methods. The first method involved a first‐round PCR using cDNA generated with primers spanning exons 8 and 9 (exon connection), followed by a second round of AS‐PCR using primers from within exon 9. The second method used only AS‐PCR with primers from within exon 9. The result was that the exon‐connection approach was more accurate, thereby highlighting a significant problem in imprinting analyses where genomic DNA contamination cannot be completely ruled out. Copyright

Rapid identification of an A1555G mutation in human mitochondrial DNA implicated in aminoglycoside-induced ototoxicity

AbstractThis article describes a multiplex allele-specific PCR (AS-PCR) approach for detection of an A to G mutation occurring in the human mitochondrial 12s RNA gene at nucleotide 1555. Possession of this mutation has been shown to be associated with irreversible hearing loss following administration of aminoglycoside antibiotics, and in some families is associated with profound sensorineural deafness in the absence of aminoglycoside antibiotics. We screened 206 unrelated individuals from the province of Otago, New Zealand, and found one who possessed the mitochondrial 1555 A to G mutation (0.48%; 95% confidence interval, 0.01–2.75).

Insulin-like growth factor 2 and insulin-like growth factor binding protein 2 expression in hepatoblastoma.

The expression of insulin-like growth factor 2 (IGF2) and insulin-like growth factor binding protein 2 (IGFBP2) in 11 cases of hepatoblastoma was studied by means of in situ mRNA hybridization using digoxigenin (DIG)-labeled riboprobes. The results showed that both IGF2 and IGFBP2 transcripts are present in hepatoblastoma and that their expression is inversely correlated with the degree of tumor cell differentiation. The data suggested that IGF2 and IGFBP2 gene expression could be regarded as a marker for assessment of the degree of differentiation in hepatoblastoma.

Keratin 20 is a specific marker of submicroscopic lymph node metastases in colorectal cancer: validation by K-RAS mutations

Lymph node status has major prognostic importance in colorectal cancer and greater precision in the diagnosis of lymph node metastases should provide better prognostic and therapeutic guidance. Keratin 20 (K20) gene expression has been used as a marker of lymph node metastases, but the evidence for this remains circumstantial. This study has therefore sought to determine K20 specificity and to correlate K20 expression with mutant K‐RAS expression, in order to provide direct evidence that K20 expression in lymph nodes of colorectal cancer patients genuinely reflects metastatic disease. Specificity of K20 expression was established against a range of tissue types and 289 lymph nodes from 41 non‐cancer control patients. K20 expression was restricted to gastrointestinal epithelia and was only present in one of the 289 control lymph nodes, giving a calculated specificity of 97.6% (95% confidence limits: 87.1–99.9%). Forty‐two tumour samples were analysed for the presence of K‐RAS codon 12 gene mutations using a RT‐PCR mutant allele‐specific amplification (MASA) technique. Thirteen tumours (31%) had codon 12 mutations detected by MASA and these were further analysed to determine the exact nature of the mutation. MASA was then used to screen the lymph nodes from these patients for the presence of the tumour‐specific K‐RAS transcript and the results were compared with K20 RT‐PCR and histopathology from the same samples. Whilst K‐RAS MASA was not as sensitive as K20 RT‐PCR, there was substantial agreement between the assays. There were no K20‐negative lymph nodes which were found to be K‐RAS MASA‐positive, whereas seven nodes in four patients were K20‐positive and K‐RAS‐negative, in keeping with the differences in assay sensitivity. These results further validate K20 as a marker by providing greater certainty that what is being detected represents occult metastatic disease. Copyright