The development of methods for detecting Keratin 19 expression by the polymerase chain reaction in breast cancer and lymphatic tissue

James, Philip Patrick Brady (1994) The development of methods for detecting Keratin 19 expression by the polymerase chain reaction in breast cancer and lymphatic tissue. University of Southampton, Doctoral Thesis.

Abstract

A method has been devised for detecting the expression of keratin 19 (K19) in both fresh and paraffin-embedded tissue by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated by modifications of published methods. Primers and reaction conditions for RT-PCR were designed to minimise the likelihood of genomic contamination and non-specific amplification; the identity of the amplified sequence was shown by Southern hybridization.

K19 expression was demonstrated for the first time by RT-PCR in primary and secondary breast carcinomas. In addition, K19 expression was also found by RT-PCR in normal lymph nodes - this has not previously been shown by any method. Immunohistochemical analysis with a newly available monospecific anti-K19 antibody showed, also for the first time, that the cells in the normal lymph node which express K19 are the B-lymphocytes of the marginal zone, and also the follicular dendritic cells. Up till now it was generally considered that lymphatic tissue expresses only small amounts of K8 and K18.

Breast carcinomas express K19, the mRNA of which is detectable by RT-PCR. More surprisingly, this powerful and sensitive technique has shown that this simple 'embryonic' keratin is also unequivocally expressed by normal lymphatic tissue. Immunohistochemical methods have shown the cells of origin to be the marginal zone B-lymphocytes and the follicular dendritic cells.

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