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Validation of Stained Cytology Smears as a Sample Type for a 50-Gene Solid Tumor Targeted Next Generation Sequencing Panel
Journal of the American Society of Cytopathology (2015) 4, S1eS87
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.jascyto.org/
ABSTRACT SECTION-ANNUAL MEETING 2015 PP01
PP02
Validation of Stained Cytology Smears as a Sample Type for a 50-Gene Solid Tumor Targeted Next Generation Sequencing Panel
Aptima HR-HPV Testing From Diff-Quick Stained FNA Smears of Oropharyngeal Squamous Cell Carcinoma
Douglas Minot, CT, MB(ASCP), Jesse Voss, BS, CT, MB(ASCP), Benjamin Kipp, PhD, Sarah Kerr, MD Mayo Clinic, Rochester, Minnesota
Min Han, MD, PhD, Cory Bernadt, MD, PhD, Benjamin Murray, BS, Jalal Jalaly, MBBS, Telly Garcia, CT(ASCP), Laura Adhikari, MD Washington University/Barnes-Jewish Hospital, St. Louis, Missouri
Introduction: Next generation sequencing (NGS) is used to detect clinically actionable mutations in solid tumors, but tests are usually validated for formalin fixed, paraffin embedded tissue (FFPE). Stained cytology smears (SCS) are an attractive alternative sample due to increasing use of minimally invasive procedures and direct onsite evaluation of sample adequacy. We describe a clinical validation designed to meet regulatory requirements for NGS using SCS. Materials and Methods: A SCS with >5000 total cells and >20% tumor nuclei was obtained from 14 cases with a concurrent, previously tested (NGS or single gene testing) FFPE needle biopsy. Tumor (RL-95-2, P12Ichikawa, Molt-4, and HT-29) and HapMap cell lines (NA12878, NA19240) were also prepared as SCS. Three tumor cell lines were processed by 3 methods: Rapid Romanowsky, Papanicolau stained smear or liquid based preparation. After coverslip removal, DNA was extracted by column and quantitated by fluorometry. 50 gene NGS panel testing was performed using a previously validated FFPE method. Results: Mean DNA concentration was 17 ng/uL (range 3 to 197 ng/mL). Sequencing accuracy per base for HapMap samples ranged from 99.1 to 99.7%. All expected variants (40) were detected in the SCS. Mean detected variant frequency (VF) was consistently higher for SCS vs paired FFPE (69.17% [range 50 -87] vs 51% [range 23-78]; pZ0.0120), see Table 1. One additional mutation was detected in a SCS that was not detected in paired FFPE. Conclusion: SCS can be validated as a sample type for NGS in a way that meets regulatory requirements. Consistently higher VFs are obtained from SCS vs FFPE, confirming enrichment of tumor cells in SCS. As a result, SCS may be preferred over FFPE needle biopsy for NGS due to enhanced tumor percentage and ease of direct onsite adequacy assessment. Use of SCS could spare paired FFPE tissue for other necessary tests.
Introduction: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is a biologically unique form of carcinoma that is important to identify for prognosis and treatment. Diff-Quick (DQ) stained smears from fine needle aspirations (FNAs) are amenable for rapid on-site adequacy assessment and have proven useful for molecular testing. The objective of this study was to evaluate the performance of the Aptima HPV assay using DQ stained smears from FNAs of HPV-related oropharyngeal SCC. Materials and Methods: Patients with a diagnosis of oropharyngeal SCC who also had an FNA demonstrating metastatic SCC were identified. Using a mounting media-based cell transfer technique, approximately 200 tumor cells were selected and harvested from DQ stained FNA slides. The selected cells were tested for high risk HPV using the Aptima HPV assay, an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger RNA from high-risk types of HPV. These results were compared to the p16 immunohistochemical staining of the corresponding surgical pathology specimens. Results: Twenty of 22 (90.9%) FNAs of p16 positive oropharyngeal SCC were positive for high-risk HPV by the Aptima assay and 11/11 (100%) FNAs of p16 negative head and neck SCC were negative for high-risk HPV by the Aptima assay. One of the two false negatives was noted to have technical failure during cell transfer. Conclusions: DQ-stained FNA smears can be used by the Aptima HPV assay to accurately detect high-risk HPV the in -oropharyngeal SCCs with a sensitivity of 90.9% and a specificity of 100%. This provides an alternative to p16 immunohistochemical staining of FNA cell block material, which may not be available on all specimens. PP03 Benefit of Computerized Adverse Entry Documentation for NonGynecological Cytopathology Performance Improvement Jacqueline Cuda, BS, SCT(ASCP), Lindsey Seigh, BS, Kathleen Cieply, BS, Sara Monaco, MD, Samuel Yousem, Anthony Piccoli, BS, Liron Pantanowitz, MD University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania Introduction: In cytopathology it is important to document and track errors as part of an ongoing quality assurance program. At our institution we
2213-2945 http://dx.doi.org/10.1016/j.jasc.2015.09.002
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.jascyto.org/
ABSTRACT SECTION-ANNUAL MEETING 2015 PP01
PP02
Validation of Stained Cytology Smears as a Sample Type for a 50-Gene Solid Tumor Targeted Next Generation Sequencing Panel
Aptima HR-HPV Testing From Diff-Quick Stained FNA Smears of Oropharyngeal Squamous Cell Carcinoma
Douglas Minot, CT, MB(ASCP), Jesse Voss, BS, CT, MB(ASCP), Benjamin Kipp, PhD, Sarah Kerr, MD Mayo Clinic, Rochester, Minnesota
Min Han, MD, PhD, Cory Bernadt, MD, PhD, Benjamin Murray, BS, Jalal Jalaly, MBBS, Telly Garcia, CT(ASCP), Laura Adhikari, MD Washington University/Barnes-Jewish Hospital, St. Louis, Missouri
Introduction: Next generation sequencing (NGS) is used to detect clinically actionable mutations in solid tumors, but tests are usually validated for formalin fixed, paraffin embedded tissue (FFPE). Stained cytology smears (SCS) are an attractive alternative sample due to increasing use of minimally invasive procedures and direct onsite evaluation of sample adequacy. We describe a clinical validation designed to meet regulatory requirements for NGS using SCS. Materials and Methods: A SCS with >5000 total cells and >20% tumor nuclei was obtained from 14 cases with a concurrent, previously tested (NGS or single gene testing) FFPE needle biopsy. Tumor (RL-95-2, P12Ichikawa, Molt-4, and HT-29) and HapMap cell lines (NA12878, NA19240) were also prepared as SCS. Three tumor cell lines were processed by 3 methods: Rapid Romanowsky, Papanicolau stained smear or liquid based preparation. After coverslip removal, DNA was extracted by column and quantitated by fluorometry. 50 gene NGS panel testing was performed using a previously validated FFPE method. Results: Mean DNA concentration was 17 ng/uL (range 3 to 197 ng/mL). Sequencing accuracy per base for HapMap samples ranged from 99.1 to 99.7%. All expected variants (40) were detected in the SCS. Mean detected variant frequency (VF) was consistently higher for SCS vs paired FFPE (69.17% [range 50 -87] vs 51% [range 23-78]; pZ0.0120), see Table 1. One additional mutation was detected in a SCS that was not detected in paired FFPE. Conclusion: SCS can be validated as a sample type for NGS in a way that meets regulatory requirements. Consistently higher VFs are obtained from SCS vs FFPE, confirming enrichment of tumor cells in SCS. As a result, SCS may be preferred over FFPE needle biopsy for NGS due to enhanced tumor percentage and ease of direct onsite adequacy assessment. Use of SCS could spare paired FFPE tissue for other necessary tests.
Introduction: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is a biologically unique form of carcinoma that is important to identify for prognosis and treatment. Diff-Quick (DQ) stained smears from fine needle aspirations (FNAs) are amenable for rapid on-site adequacy assessment and have proven useful for molecular testing. The objective of this study was to evaluate the performance of the Aptima HPV assay using DQ stained smears from FNAs of HPV-related oropharyngeal SCC. Materials and Methods: Patients with a diagnosis of oropharyngeal SCC who also had an FNA demonstrating metastatic SCC were identified. Using a mounting media-based cell transfer technique, approximately 200 tumor cells were selected and harvested from DQ stained FNA slides. The selected cells were tested for high risk HPV using the Aptima HPV assay, an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger RNA from high-risk types of HPV. These results were compared to the p16 immunohistochemical staining of the corresponding surgical pathology specimens. Results: Twenty of 22 (90.9%) FNAs of p16 positive oropharyngeal SCC were positive for high-risk HPV by the Aptima assay and 11/11 (100%) FNAs of p16 negative head and neck SCC were negative for high-risk HPV by the Aptima assay. One of the two false negatives was noted to have technical failure during cell transfer. Conclusions: DQ-stained FNA smears can be used by the Aptima HPV assay to accurately detect high-risk HPV the in -oropharyngeal SCCs with a sensitivity of 90.9% and a specificity of 100%. This provides an alternative to p16 immunohistochemical staining of FNA cell block material, which may not be available on all specimens. PP03 Benefit of Computerized Adverse Entry Documentation for NonGynecological Cytopathology Performance Improvement Jacqueline Cuda, BS, SCT(ASCP), Lindsey Seigh, BS, Kathleen Cieply, BS, Sara Monaco, MD, Samuel Yousem, Anthony Piccoli, BS, Liron Pantanowitz, MD University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania Introduction: In cytopathology it is important to document and track errors as part of an ongoing quality assurance program. At our institution we
2213-2945 http://dx.doi.org/10.1016/j.jasc.2015.09.002