Use of archival fine-needle aspirates for the allelotyping of tumors

David M Euhus, Anirban Maitra, Ignacio I. Wistuba, Raheela Ashfaq, Arland Alberts, David Gibbons, Adi F. Gazdar

Research output: Contribution to journalArticle

Abstract

BACKGROUND. Loss of heterozygosity (LOH) analysis (allelotyping) based on polymorphic microsatellite DNA is one of the most powerful molecular tools currently available for studying carcinogenesis. However, allelotyping studies that require archival paraffin embedded tissues are often hampered by technical difficulties related to microdissection and poor DNA quality. METHODS. The authors compared allelotyping results from 12 paraffin embedded breast carcinoma cases with those from matching alcohol fixed fine-needle aspiration (FNA) cytology slides obtained for routine diagnostic purposes, using 30 polymorphic microsatellite markers at chromosomes 3p, 4p, 4q, 5q, 6p, 8p, 9p, 11q, 17p, and 17q. Cells from the alcohol fixed FNA slides were dissected and processed in three different ways, and DNA dilution experiments were performed to determine the minimum number of cells required for accurate allelotyping. RESULTS. LOH results were identical for paraffin embedded and alcohol fixed tumors for 97% of 114 polymerase chain reactions (PCR) when 1000-2000 cells were dissected from each FNA slide and DNA from 100 cells was used for each multiplex PCR. However, with lower cell numbers, the discordance rate increased and artifactual LOH was observed. Intratumor allelotype heterogeneity could not be documented. CONCLUSIONS. The use of alcohol fixed cytology preparations improves the ease of PCR-based allelotyping and greatly expands the range of archival materials available for study. The allelotyping is accurate and reproducible when DNA from ≥25 cells is used in the initial multiplex PCR.

Original languageEnglish (US)
Pages (from-to)372-379
Number of pages8
JournalCancer
Volume87
Issue number6
DOIs
StatePublished - Dec 25 1999
Externally publishedYes

Fingerprint

Needles
Loss of Heterozygosity
Fine Needle Biopsy
Alcohols
Paraffin
DNA
Multiplex Polymerase Chain Reaction
Neoplasms
Microsatellite Repeats
Cell Biology
Cell Count
Polymerase Chain Reaction
Microdissection
Carcinogenesis
Chromosomes
Breast Neoplasms

Keywords

  • Allelotyping
  • Artifactual loss of heterozygosity
  • Breast
  • Cytology
  • Microsatellite DNA
  • Tumor heterogeneity

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Euhus, D. M., Maitra, A., Wistuba, I. I., Ashfaq, R., Alberts, A., Gibbons, D., & Gazdar, A. F. (1999). Use of archival fine-needle aspirates for the allelotyping of tumors. Cancer, 87(6), 372-379. https://doi.org/10.1002/(SICI)1097-0142(19991225)87:6<372::AID-CNCR8>3.0.CO;2-5

Use of archival fine-needle aspirates for the allelotyping of tumors. / Euhus, David M; Maitra, Anirban; Wistuba, Ignacio I.; Ashfaq, Raheela; Alberts, Arland; Gibbons, David; Gazdar, Adi F.

In: Cancer, Vol. 87, No. 6, 25.12.1999, p. 372-379.

Research output: Contribution to journalArticle

Euhus, DM, Maitra, A, Wistuba, II, Ashfaq, R, Alberts, A, Gibbons, D & Gazdar, AF 1999, 'Use of archival fine-needle aspirates for the allelotyping of tumors', Cancer, vol. 87, no. 6, pp. 372-379. https://doi.org/10.1002/(SICI)1097-0142(19991225)87:6<372::AID-CNCR8>3.0.CO;2-5
Euhus, David M ; Maitra, Anirban ; Wistuba, Ignacio I. ; Ashfaq, Raheela ; Alberts, Arland ; Gibbons, David ; Gazdar, Adi F. / Use of archival fine-needle aspirates for the allelotyping of tumors. In: Cancer. 1999 ; Vol. 87, No. 6. pp. 372-379.
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AU - Gibbons, David

AU - Gazdar, Adi F.

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N2 - BACKGROUND. Loss of heterozygosity (LOH) analysis (allelotyping) based on polymorphic microsatellite DNA is one of the most powerful molecular tools currently available for studying carcinogenesis. However, allelotyping studies that require archival paraffin embedded tissues are often hampered by technical difficulties related to microdissection and poor DNA quality. METHODS. The authors compared allelotyping results from 12 paraffin embedded breast carcinoma cases with those from matching alcohol fixed fine-needle aspiration (FNA) cytology slides obtained for routine diagnostic purposes, using 30 polymorphic microsatellite markers at chromosomes 3p, 4p, 4q, 5q, 6p, 8p, 9p, 11q, 17p, and 17q. Cells from the alcohol fixed FNA slides were dissected and processed in three different ways, and DNA dilution experiments were performed to determine the minimum number of cells required for accurate allelotyping. RESULTS. LOH results were identical for paraffin embedded and alcohol fixed tumors for 97% of 114 polymerase chain reactions (PCR) when 1000-2000 cells were dissected from each FNA slide and DNA from 100 cells was used for each multiplex PCR. However, with lower cell numbers, the discordance rate increased and artifactual LOH was observed. Intratumor allelotype heterogeneity could not be documented. CONCLUSIONS. The use of alcohol fixed cytology preparations improves the ease of PCR-based allelotyping and greatly expands the range of archival materials available for study. The allelotyping is accurate and reproducible when DNA from ≥25 cells is used in the initial multiplex PCR.

AB - BACKGROUND. Loss of heterozygosity (LOH) analysis (allelotyping) based on polymorphic microsatellite DNA is one of the most powerful molecular tools currently available for studying carcinogenesis. However, allelotyping studies that require archival paraffin embedded tissues are often hampered by technical difficulties related to microdissection and poor DNA quality. METHODS. The authors compared allelotyping results from 12 paraffin embedded breast carcinoma cases with those from matching alcohol fixed fine-needle aspiration (FNA) cytology slides obtained for routine diagnostic purposes, using 30 polymorphic microsatellite markers at chromosomes 3p, 4p, 4q, 5q, 6p, 8p, 9p, 11q, 17p, and 17q. Cells from the alcohol fixed FNA slides were dissected and processed in three different ways, and DNA dilution experiments were performed to determine the minimum number of cells required for accurate allelotyping. RESULTS. LOH results were identical for paraffin embedded and alcohol fixed tumors for 97% of 114 polymerase chain reactions (PCR) when 1000-2000 cells were dissected from each FNA slide and DNA from 100 cells was used for each multiplex PCR. However, with lower cell numbers, the discordance rate increased and artifactual LOH was observed. Intratumor allelotype heterogeneity could not be documented. CONCLUSIONS. The use of alcohol fixed cytology preparations improves the ease of PCR-based allelotyping and greatly expands the range of archival materials available for study. The allelotyping is accurate and reproducible when DNA from ≥25 cells is used in the initial multiplex PCR.

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