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Quantitative evaluation of metastases in axillary lymph nodes of breast cancer.

Inokuchi M, Ninomiya I, Tsugawa K, Terada I, Miwa K - Br. J. Cancer (2003)

Bottom Line: Amplifying cytokeratin 19 (CK19) mRNA transcripts using real-time TaqMan PCR made it possible to quantify axillary metastatic burden.Cytokeratin 19 mRNA expression values of both histologically and immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001), and those of histologically negative, but immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001).Furthermore, metastatic rates of sentinel nodes were higher than the rates of nonsentinel lymph nodes as measured by all three methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterologic Surgery, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan.

ABSTRACT
We have established a highly sensitive and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect axillary lymph node metastases of breast cancer. Amplifying cytokeratin 19 (CK19) mRNA transcripts using real-time TaqMan PCR made it possible to quantify axillary metastatic burden. Metastases in 358 axillary lymph nodes obtained from 23 breast cancers of 22 patients were investigated by conventional haematoxylin and eosin (H&E) staining, immunohistochemical staining and quantitative RT-PCR assay. The detection rates of axillary lymph node metastasis using H&E staining, immunohistochemistry and RT-PCR assay were 4.5, 5.9 and 13.1%, respectively. RT-PCR assay was the most sensitive of these three methods for detecting lymph node metastases. Cytokeratin 19 mRNA expression values of both histologically and immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001), and those of histologically negative, but immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001). Furthermore, metastatic rates of sentinel nodes were higher than the rates of nonsentinel lymph nodes as measured by all three methods. These results indicate that quantitative RT-PCR assay is a sensitive and reliable method for detecting lymph node metastasis. Furthermore, quantification of metastases in sentinel lymph nodes by quantitative RT-PCR assay may be useful to assess the entire axillary burden of breast cancer patients.

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Related in: MedlinePlus

Amplification of CK19 mRNA by quantitative PCR assay. (A) Amplification plots of CK19 mRNA for serial dilution of MCF-7 total RNA. ΔRn is defined as the cycle-to-cycle change in the reporter fluorescence signal normalised to a passive reference fluorescence signal. The initial amount of total RNA is displayed: •, 102 ng; ▴, 10 ng; □, 1 ng; ♦, 10−1 ng; , 10−2 ng; ▪, 10−3 ng and ○, 10−4 ng. Ct is calculated as the cycle at which fluorescence signal passes a fixed threshold line. (B) Standard curve of the CK19 RT–PCR assay. Ct is plotted against the starting quantity of MCF-7 total RNA.
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fig3: Amplification of CK19 mRNA by quantitative PCR assay. (A) Amplification plots of CK19 mRNA for serial dilution of MCF-7 total RNA. ΔRn is defined as the cycle-to-cycle change in the reporter fluorescence signal normalised to a passive reference fluorescence signal. The initial amount of total RNA is displayed: •, 102 ng; ▴, 10 ng; □, 1 ng; ♦, 10−1 ng; , 10−2 ng; ▪, 10−3 ng and ○, 10−4 ng. Ct is calculated as the cycle at which fluorescence signal passes a fixed threshold line. (B) Standard curve of the CK19 RT–PCR assay. Ct is plotted against the starting quantity of MCF-7 total RNA.

Mentions: In order to estimate the sensitivity of RT–PCR assays, serial dilution experiments were carried out. A volume of 100 ng of RNA derived from MCF-7 was diluted from 10−1 to 10−6 and subjected to RT–PCR assay. In Figure 3AFigure 3


Quantitative evaluation of metastases in axillary lymph nodes of breast cancer.

Inokuchi M, Ninomiya I, Tsugawa K, Terada I, Miwa K - Br. J. Cancer (2003)

Amplification of CK19 mRNA by quantitative PCR assay. (A) Amplification plots of CK19 mRNA for serial dilution of MCF-7 total RNA. ΔRn is defined as the cycle-to-cycle change in the reporter fluorescence signal normalised to a passive reference fluorescence signal. The initial amount of total RNA is displayed: •, 102 ng; ▴, 10 ng; □, 1 ng; ♦, 10−1 ng; , 10−2 ng; ▪, 10−3 ng and ○, 10−4 ng. Ct is calculated as the cycle at which fluorescence signal passes a fixed threshold line. (B) Standard curve of the CK19 RT–PCR assay. Ct is plotted against the starting quantity of MCF-7 total RNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2394408&req=5

fig3: Amplification of CK19 mRNA by quantitative PCR assay. (A) Amplification plots of CK19 mRNA for serial dilution of MCF-7 total RNA. ΔRn is defined as the cycle-to-cycle change in the reporter fluorescence signal normalised to a passive reference fluorescence signal. The initial amount of total RNA is displayed: •, 102 ng; ▴, 10 ng; □, 1 ng; ♦, 10−1 ng; , 10−2 ng; ▪, 10−3 ng and ○, 10−4 ng. Ct is calculated as the cycle at which fluorescence signal passes a fixed threshold line. (B) Standard curve of the CK19 RT–PCR assay. Ct is plotted against the starting quantity of MCF-7 total RNA.
Mentions: In order to estimate the sensitivity of RT–PCR assays, serial dilution experiments were carried out. A volume of 100 ng of RNA derived from MCF-7 was diluted from 10−1 to 10−6 and subjected to RT–PCR assay. In Figure 3AFigure 3

Bottom Line: Amplifying cytokeratin 19 (CK19) mRNA transcripts using real-time TaqMan PCR made it possible to quantify axillary metastatic burden.Cytokeratin 19 mRNA expression values of both histologically and immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001), and those of histologically negative, but immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001).Furthermore, metastatic rates of sentinel nodes were higher than the rates of nonsentinel lymph nodes as measured by all three methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterologic Surgery, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan.

ABSTRACT
We have established a highly sensitive and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect axillary lymph node metastases of breast cancer. Amplifying cytokeratin 19 (CK19) mRNA transcripts using real-time TaqMan PCR made it possible to quantify axillary metastatic burden. Metastases in 358 axillary lymph nodes obtained from 23 breast cancers of 22 patients were investigated by conventional haematoxylin and eosin (H&E) staining, immunohistochemical staining and quantitative RT-PCR assay. The detection rates of axillary lymph node metastasis using H&E staining, immunohistochemistry and RT-PCR assay were 4.5, 5.9 and 13.1%, respectively. RT-PCR assay was the most sensitive of these three methods for detecting lymph node metastases. Cytokeratin 19 mRNA expression values of both histologically and immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001), and those of histologically negative, but immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001). Furthermore, metastatic rates of sentinel nodes were higher than the rates of nonsentinel lymph nodes as measured by all three methods. These results indicate that quantitative RT-PCR assay is a sensitive and reliable method for detecting lymph node metastasis. Furthermore, quantification of metastases in sentinel lymph nodes by quantitative RT-PCR assay may be useful to assess the entire axillary burden of breast cancer patients.

Show MeSH
Related in: MedlinePlus