Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Dietmaier, Wolfgang; Hartmann, Arndt; Wallinger, Sabine; Heinmöller, Ernst; Kerner, Thomas; Endl, Elmar; Jauch, Karl‐Walter; Hofstädter, Ferdinand; Rüschoff, Josef

doi:10.1016/s0002-9440(10)65254-6

Cited by 213 publications

(166 citation statements)

References 38 publications

(57 reference statements)

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“…This is additionally corroborated by the fact that we monitored distinct differences in success rates between samples obtained from different pathology departments. In accordance with our findings, Dietmaier et al (19) suggested that successful whole genome amplification for mutation analysis is critically dependent on a very quick sample procession (Ͻ30 min), and problematic when analyzing paraffin-embedded tissue samples. Thus, the use of LA-PCR is especially recommended in situations where CGH has to be routinely performed from samples derived from different tissues or from institutions with different fixation and embedding techniques.…”

Section: Discussionsupporting

confidence: 92%

“…However, as compared to the data above, success rates (44% versus 17% for LA-PCR and DOP-PCR, respectively) were relatively low. This might have been caused by the high amounts of degrading enzymes in the liver as well as enhanced DNA-degradation by formalin fixation (18,19). Stoecklein et al (9) suggested the sample age of formalin-fixed, paraffin-embedded tissues as a decisive factor limiting successful amplification, especially by DOP-PCR.…”

Section: Discussionmentioning

confidence: 99%

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Whole genome amplification for CGH analysis: Linker‐adapter PCR as the method of choice for difficult and limited samples

Pirker¹,

Raidl²,

Steiner³

et al. 2004

Cytometry Pt A

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BackgroundComparative genomic hybridization (CGH) is a powerful method to investigate chromosomal imbalances in tumor cells. However, DNA quantity and quality can be limiting factors for successful CGH analysis. The aim of this study was to investigate the applicability of degenerate oligonucleotide‐primed PCR (DOP‐PCR) and a recently developed linker‐adapter‐mediated PCR (LA‐PCR) for whole genome amplification for use in CGH, especially for difficult source material.MethodsWe comparatively analyzed DNA of variable quality derived from different cell/tissue types. Additionally, dilution experiments down to the DNA content of a single cell were performed. FISH and/or classical cytogenetic analyses were used as controls.ResultsIn the case of high quality DNA samples, both methods were equally suitable for CGH. When analyzing very small amounts of these DNA samples (equivalent to one or a few human diploid cells), DOP‐PCR‐CGH, but not LA‐PCR‐CGH, frequently produced false‐positive signals (e.g., gains in 1p and 16p, and losses in chromosome 4q). In case of formalin‐fixed paraffin‐embedded tissues, success rates by LA‐PCR‐CGH were significantly higher as compared to DOP‐PCR‐CGH. DNA of minor quality frequently could be analyzed correctly by LA‐PCR‐CGH, but was prone to give false‐positive and/or false‐negative results by DOP‐PCR‐CGH.ConclusionsLA‐PCR is superior to DOP‐PCR for amplification of DNA for CGH analysis, especially in the case of very limited or partly degraded source material. © 2004 Wiley‐Liss, Inc.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Whole genome amplification for CGH analysis: Linker‐adapter PCR as the method of choice for difficult and limited samples

Pirker¹,

Raidl²,

Steiner³

et al. 2004

Cytometry Pt A

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“…For whole genome amplification (WGA), genomic DNA was PCR-amplified using random 15-mer primers. 33 Procedures of WGA has been validated as previously described. 34 Methods of PCR and sequencing targeted for KRAS codons 12 and 13, and BRAF codon 600, have been previously described.…”

Section: Tissue Specimens and Histopathologic Evaluationsmentioning

confidence: 99%

Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component

et al. 2006

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Signet ring cell carcinoma and mucinous carcinoma are distinct subtypes of colorectal adenocarcinoma. The morphologic and molecular spectra of colorectal carcinomas with various signet ring cell components and colorectal carcinomas with various mucinous components, compared to non-mucinous adenocarcinomas, have not been examined. The study groups consisted of 39 carcinomas with various signet ring cell components ('the signet group'), 167 carcinomas with various mucinous components ('the mucinous group'), and 457 nonmucinous adenocarcinoma. We visually estimated the amounts of signet ring cell and mucinous components in tumors, and subclassified the signet and mucinous groups according to the amount of each component (r19, 20-49, and Z50%). We sequenced BRAF and KRAS, analyzed for microsatellite instability (MSI) and 18q loss of heterozygosity (LOH), and performed immunohistochemistry for TP53, cyclooxygenase-2 (COX2), MLH1, O-6-methylguanine DNA methyltransferase (MGMT), p16 (CDKN2A), and fatty acid synthase (FASN). Signet ring cell carcinoma (Z50% signet ring cell tumors) and r49% signet ring cell tumors showed similar molecular features. Except for MSI and MGMT, Z50% mucinous tumors and r49% mucinous tumors also showed similar molecular features. BRAF mutations, MSI, and MLH1 loss were more frequent in both the signet and mucinous groups than nonmucinous carcinoma. More frequent KRAS mutations and less frequent p16 loss and TP53 positivity were observed in the mucinous group than nonmucinous carcinoma. 18q LOH and COX2 overexpression were less common in the signet group than nonmucinous carcinoma. FASN levels were highest in the mucinous group, followed by nonmucinous carcinoma, and lowest in the signet group. In conclusion, a minor (r49%) signet ring cell or mucinous component in colorectal carcinoma suggests molecular features similar to Z50% signet ring cell or mucinous carcinoma, respectively. Signet ring cell carcinoma and mucinous carcinoma are related subtypes of colorectal adenocarcinoma, but have molecular features distinct from each other. Keywords: BRAF; colon cancer; COX2; fatty acid synthase; MSI; mucinous; signet ring cell Signet ring cell colorectal carcinoma and mucinous colorectal carcinoma are subtypes of colorectal adenocarcinoma with prominent mucin secretion. A unique pathologic feature of signet ring cell carcinoma is the presence of signet ring cells, which are single tumor cells with intracytoplasmic mucin displacing their nuclei aside. In contrast, mucinous colorectal carcinoma is characterized by abundant extracellular mucin produced by tumor cells. By definition, a 50% or greater signet ring cell component is required for the designation of signet ring cell colorectal carcinoma. Mucinous colorectal carcinoma has also 50% or more mucinous components. Signet ring cell colorectal carcinoma has been associated with poor clinical outcomes. [1][2][3][4][5][6] A number of studies have examined the molecular features of signet ring cell colorectal carcinoma and mucinous

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“…37 Previous studies by us and others showed that WGA did not significantly affect KRAS mutation detection or microsatellite analysis. 37,38 …”

Section: Genomic Dna Extraction and Whole Genome Amplificationmentioning

confidence: 99%

WRN promoter methylation possibly connects mucinous differentiation, microsatellite instability and CpG island methylator phenotype in colorectal cancer

et al. 2008

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Werner syndrome is a premature aging syndrome characterized by early onset of cancer and abnormal cellular metabolism of glycosaminoglycan. The WRN helicase plays an important role in the maintenance of telomere function. WRN promoter methylation and gene silencing are common in colorectal cancer with the CpG island methylator phenotype (CIMP), which is associated with microsatellite instability (MSI) and mucinous tumors. However, no study has examined the relationship between mucinous differentiation, WRN methylation, CIMP and MSI in colorectal cancer. Utilizing 903 population-based colorectal cancers and real-time PCR (MethyLight), we quantified DNA methylation in WRN and eight other promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) known to be specific for CIMP. Supporting WRN as a good CIMP marker, WRN methylation was correlated well with CIMP-high diagnosis (Z6/8 methylated promoters), demonstrating 89% sensitivity and 81% specificity. WRN methylation was associated with the presence of any mucinous component and Z50% mucinous component (Po0.0001). Because both MSI and CIMP were associated with mucinous tumors and WRN methylation, we stratified tumors into 9 MSI/CIMP subtypes, to examine whether the relationship between WRN methylation and mucin still persisted. In each MSI/CIMP subtype, tumors with mucinous component were persistently more common in WRN-methylated tumors than WRN-unmethylated tumors (P ¼ 0.004). No relations of WRN methylation with other variables (age, sex, tumor location, poor differentiation, signet ring cells, lymphocytic reactions, KRAS, BRAF, p53, p21 or 18q loss of heterozygosity) persisted after tumors were stratified by CIMP status. In conclusion, WRN methylation is associated with mucinous differentiation independent of CIMP and MSI status. Our data suggest a possible role of WRN methylation in mucinous differentiation, and may provide explanation to the enigmatic association between mucin and MSI/CIMP. Modern Pathology (2008) 21, 150-158;

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Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Cited by 213 publications

References 38 publications

Whole genome amplification for CGH analysis: Linker‐adapter PCR as the method of choice for difficult and limited samples

Whole genome amplification for CGH analysis: Linker‐adapter PCR as the method of choice for difficult and limited samples

Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component

WRN promoter methylation possibly connects mucinous differentiation, microsatellite instability and CpG island methylator phenotype in colorectal cancer

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