- Email: [email protected]
PCR in clinical pathology
Abstracts I Journal
of Microbiological
Less attention
13
PCR in clinical pathology Cees J. Comelisse Dept.
Methods 30 (1997)
of
Pathology,
9600, Building
Leiden University
I, LI-Q,
Medical
Centre, P.O. Box
2300 RC Leiden, The Netherlands
For almost 150 years, clinical pathology largely depended on the study of morphological changes at the macroscopic-and microscopic level. The vast amount of knowledge that was gained by microscoptc examination of hematoxylin-eosin stained histological sections still forms the basis of the pathological classification of diseases. The possibilities for studying pathological changes at the molecular level were very limited until the development of monoclonal antibodies enabled the wide-spread introduction of immunohistochemistry into diagnostic pathology. Although this enabled a much more detailed study of phenotypic changes, molecular-genetic alterations were still inaccessible for diagnostic examination. Over the past decade, our insight in the molecular basis of diseases has greatly increased due to the progres, m molecular cell biology and of the Human Genome Project. Implementation of this knowledge into diagnostic pathology is now the major challenge for the pathologist at the turn of the century. It\ sensitivity and specificity makes PCR a powerful tool for diagnostic molecular pathology enabling the detection of specific molecular-genetic alterations in small tissue samples. Sampling by micro-dissection permits isolation of e.g. microscopical foci of tumour cells surrounded by large numbers of normal cells. We demonstrated the possibility to enrich cell populatrons of interest by Row cytometric cell sorting on the basis of cytoplasmic and nuclear marker expression for PCR-based molecular-genetic analysis. An overview will be given of PCR applications in molecular diagnostic pathology that are operational in our institute. These include e.g. detection of HPV in cervical carcinoma, detection of specific translocations in lymphomas and bone - and soft tissue turnours, clonality analysis of lymphomas and of multiple solid tumours, zygosity analysis on placental tissue of twin\, identification of mixed-up tissue specimens and RER analysis in colorectal cancer. Because of the paramount importance ot adequate sampling, the application of PCR on pathological cell and tissue specimens should be supervised by clinical pathologists with a solid background in molecular biology. 14 PCR application Erica Vtlla Deportmmt Modern,
in the screening
for colon carcinoma
of Internul MedicinelGastroenterology,
Via tIeI Po:zo
71. 4ilW
Modem,
University qf
Italy
The use of occult blood tests as a screening for early identification of colon cancer has given controversial results. Colonoscopy remains the gold standard; however, it cannot be used for mass screening. Sidransly’s detection of K-ras mutations on DNA extracted from stool by has suggested to use the genetic test as a non-invasive screening tool for colorectal neoplasms or colorectal adenomas. After this initial paper, almost all studies published have addressed the problem of identifying the tumour (if possible, at an early stage). In this respect, the correspondence between tindings in tumour tissue and stool test are good.
239
23-5-253
has been paid to preneoplastic
situations:
in a
prospective study in which we compared stool test with colonoscopy with, we have shown that it is possible to correctly identify subjects with polyps when these are larger than 1 cm or when there are multiple, small polyps, especially when the subject under evaluation is a relative of a CRC patient. Another group of patients in whom K-ras fragments (both wild-type or mutated) are easily identified is that of patients with inflammatory bowel disease. These results suggest that the amplification of K-ras fragments, provides
independently an useful
from
having
an associated
mutation,
information
about
the presence
of a hy-
perproliferating situation in the colon. There are only two studies performed
with another
gene (i.e.
~53): while the type of analysis required (PCR+SSCP) is more cumbersome than that for K-ras. the overall results are not better than with K-ras. In conclusion, the finding of K-ras mutation in stool DNA seems sufficiently specific and sensitive to justify a large scale trial to validate its use as a screening test. 15 “The ideal PCR test” - What have we learned? Gerda T. Noordhoek Public
Health
Laboratop
Friesland,
PO Box 21020,
8900
JA
Leeuwlarden, The Netherlands
Since the introduction of nucleic acid (NA) amplification techniques an enormous number of tests have been published for the detection of microorganisms. At this moment nearly each diagnostic laboratory has one or more NA amplification-based tests in its repertoire. In theory these molecular methods have a high sensitivity and specificity. However, the reliability of the laboratory performance is sometimes questionable. Quality control studies have shown that the interpretation of results obtained with amplification techniques should be carried out with the greatest caution. Even with commercial available test systems, false positive and false negative results may occur. The impact of NA amplification techniques is sometimes overestimated and decision making should not be based on a single test result. The laboratory management needs to point out the benetits and the pitfalls of molecular diagnostic test systems to the clinicians. Good performance is not only dependent on the amplification system and the proficiency of the technicians, but also on the moment and location of sampling, transport and storage of clinicical specimens. International available standards are required in order to estimate the detection level of a method and the interlaboratory variation. Furthermore, laboratories need guidelines and run controls to monitor their day-to-day quality performance and to check individual samples for inhibition of the enzyme reactions. At this moment we are far from what we may consider to be the “Ideal NA amplification test”. 16 Feasibility of the COBAS AMPLICOR, LCx and AMPLIFIED CT for the detection of Chlamydia trachomatis in urine R.P. Verkooyen, J.W. Mouton. WI. van der Meijden, W.H.F.
of Microbiological
Less attention
13
PCR in clinical pathology Cees J. Comelisse Dept.
Methods 30 (1997)
of
Pathology,
9600, Building
Leiden University
I, LI-Q,
Medical
Centre, P.O. Box
2300 RC Leiden, The Netherlands
For almost 150 years, clinical pathology largely depended on the study of morphological changes at the macroscopic-and microscopic level. The vast amount of knowledge that was gained by microscoptc examination of hematoxylin-eosin stained histological sections still forms the basis of the pathological classification of diseases. The possibilities for studying pathological changes at the molecular level were very limited until the development of monoclonal antibodies enabled the wide-spread introduction of immunohistochemistry into diagnostic pathology. Although this enabled a much more detailed study of phenotypic changes, molecular-genetic alterations were still inaccessible for diagnostic examination. Over the past decade, our insight in the molecular basis of diseases has greatly increased due to the progres, m molecular cell biology and of the Human Genome Project. Implementation of this knowledge into diagnostic pathology is now the major challenge for the pathologist at the turn of the century. It\ sensitivity and specificity makes PCR a powerful tool for diagnostic molecular pathology enabling the detection of specific molecular-genetic alterations in small tissue samples. Sampling by micro-dissection permits isolation of e.g. microscopical foci of tumour cells surrounded by large numbers of normal cells. We demonstrated the possibility to enrich cell populatrons of interest by Row cytometric cell sorting on the basis of cytoplasmic and nuclear marker expression for PCR-based molecular-genetic analysis. An overview will be given of PCR applications in molecular diagnostic pathology that are operational in our institute. These include e.g. detection of HPV in cervical carcinoma, detection of specific translocations in lymphomas and bone - and soft tissue turnours, clonality analysis of lymphomas and of multiple solid tumours, zygosity analysis on placental tissue of twin\, identification of mixed-up tissue specimens and RER analysis in colorectal cancer. Because of the paramount importance ot adequate sampling, the application of PCR on pathological cell and tissue specimens should be supervised by clinical pathologists with a solid background in molecular biology. 14 PCR application Erica Vtlla Deportmmt Modern,
in the screening
for colon carcinoma
of Internul MedicinelGastroenterology,
Via tIeI Po:zo
71. 4ilW
Modem,
University qf
Italy
The use of occult blood tests as a screening for early identification of colon cancer has given controversial results. Colonoscopy remains the gold standard; however, it cannot be used for mass screening. Sidransly’s detection of K-ras mutations on DNA extracted from stool by has suggested to use the genetic test as a non-invasive screening tool for colorectal neoplasms or colorectal adenomas. After this initial paper, almost all studies published have addressed the problem of identifying the tumour (if possible, at an early stage). In this respect, the correspondence between tindings in tumour tissue and stool test are good.
239
23-5-253
has been paid to preneoplastic
situations:
in a
prospective study in which we compared stool test with colonoscopy with, we have shown that it is possible to correctly identify subjects with polyps when these are larger than 1 cm or when there are multiple, small polyps, especially when the subject under evaluation is a relative of a CRC patient. Another group of patients in whom K-ras fragments (both wild-type or mutated) are easily identified is that of patients with inflammatory bowel disease. These results suggest that the amplification of K-ras fragments, provides
independently an useful
from
having
an associated
mutation,
information
about
the presence
of a hy-
perproliferating situation in the colon. There are only two studies performed
with another
gene (i.e.
~53): while the type of analysis required (PCR+SSCP) is more cumbersome than that for K-ras. the overall results are not better than with K-ras. In conclusion, the finding of K-ras mutation in stool DNA seems sufficiently specific and sensitive to justify a large scale trial to validate its use as a screening test. 15 “The ideal PCR test” - What have we learned? Gerda T. Noordhoek Public
Health
Laboratop
Friesland,
PO Box 21020,
8900
JA
Leeuwlarden, The Netherlands
Since the introduction of nucleic acid (NA) amplification techniques an enormous number of tests have been published for the detection of microorganisms. At this moment nearly each diagnostic laboratory has one or more NA amplification-based tests in its repertoire. In theory these molecular methods have a high sensitivity and specificity. However, the reliability of the laboratory performance is sometimes questionable. Quality control studies have shown that the interpretation of results obtained with amplification techniques should be carried out with the greatest caution. Even with commercial available test systems, false positive and false negative results may occur. The impact of NA amplification techniques is sometimes overestimated and decision making should not be based on a single test result. The laboratory management needs to point out the benetits and the pitfalls of molecular diagnostic test systems to the clinicians. Good performance is not only dependent on the amplification system and the proficiency of the technicians, but also on the moment and location of sampling, transport and storage of clinicical specimens. International available standards are required in order to estimate the detection level of a method and the interlaboratory variation. Furthermore, laboratories need guidelines and run controls to monitor their day-to-day quality performance and to check individual samples for inhibition of the enzyme reactions. At this moment we are far from what we may consider to be the “Ideal NA amplification test”. 16 Feasibility of the COBAS AMPLICOR, LCx and AMPLIFIED CT for the detection of Chlamydia trachomatis in urine R.P. Verkooyen, J.W. Mouton. WI. van der Meijden, W.H.F.