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Detection of ALK mRNA Transcripts in Liquid-based NSCLC ThinPrep Cytology Slides
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Abstracts
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were collected in PreservCyt Solution and mounted onto ThinPrep slides. Automated mRNA ISH was performed on a Ventana Discovery XT automated stainer using ALK, control probes (PPIB, DapB), and amplification kit (Advanced Cell Diagnostics, Hayward, CA) combined with the ChromoMap Red Kit (Ventana, Tucson, AZ). ThinPrep FISH using an ALK break-apart probe set (Abbott Molecular Vysis, Des Plaines, IL) was the reference data set. Results: Overexpression of ALK mRNA was detectable in the ThinPrep control cells. Of the pilot EBUS-TBNAs, 7 were negative and 3 were positive. Positive staining was identified as brilliant red punctuate dot signals in the nucleus and/or cytoplasm, which was visible by BF and fluorescence microscopy using fluorescein and rhodamine filter combinations. Furthermore, the red chromogenic reaction product of alkaline phosphatase was more intense when viewed via fluorescence, providing a level of sensitivity greater than BF. The ALK status in controls and patients was consistent with ThinPrep FISH results. Conclusion: Automated mRNA ISH with Alkaline Phosphatase-Fast Red reaction for the detection of NSCLC ALK mRNA in liquid-based NSCLC ThinPrep slides is feasible and delineates ALK status in cytology samples. The potential for future multiplex assays detecting mRNA could serve as a screening test for numerous molecular alterations in cytology samples.
Endobronchial Ultrasound-Guided (EBUS) Procore Sampling: Comparison to Fine Needle Aspiration for Mediastinal Lymph Nodes Steve Manos, BS, CT(ASCP), Sara Monaco, MD, David Wilson, MD, MPH, Liron Pantanowitz, MD. University of Pennsylvania Medical Center, Pittsburgh, Pennsylvania Introduction: Endobronchial ultrasound (EBUS)-guided sampling of the mediastinum has traditionally been performed by fine needle aspiration (FNA) for cytologic evaluation. The ProCore fine needle biopsy, used primarily to obtain intra-abdominal tissue core biopsies, has not been widely used for EBUS. The aim of this study was to evaluate our experience with EBUS-Procore of mediastinal nodes, and to compare its diagnostic utilization to that of conventional EBUS-FNA. Material and Methods: A retrospective review of all EBUS cases of mediastinal masses using a ProCore needle from December 2013 to March 2014 was performed. Patient demographics, anatomic site, number of procedure passes, on-site immediate evaluation, final pathology reports, and ancillary test results were recorded. All specimen slides and cell blocks were evaluated scoring lesion and contamination cellularity (0 Z acellular, 1 Z scant, 2 Z moderate, and 3 Z abundant). Results: There were 18 EBUS cases of mediastinal lymph nodes using Procore in patients (12 female, 6 male) of average age 57 years (range, 3380 years). FNA was followed by Procore biopsy in all cases, except one where only Procore was performed. Cases included benign (granulomas), atypical, and malignant (carcinoma) diagnoses. More FNAs were satisfactory (with 2 unsatisfactory cases) than Procores (with 7 unsatisfactory cases). The 2 non-diagnostic FNA cases remained unsatisfactory with subsequent Procore. More passes were performed with FNA (4/case) than Procore (2.6/case). On average, FNA provided greater cellularity of lesional material (smear score Z 2; cell block score Z 1.4) than Procore (smear score Z 1.4; cell block score Z 1.4). However, FNAs contained more bronchial contamination (smear score Z 1.6; cell block score Z 1) than Procore (smear score Z 1; cell block score Z 0.4). Both sample types contained similar bronchial and blood contamination. Ancillary studies (special stains, immunostains, FISH, molecular) were equally successful using cell blocks from both specimen types. Conclusions: Our preliminary experience shows that EBUS-guided Procore sampling of mediastinal lymph nodes is feasible. Although FNA samples appear to provide more diagnostic cellular material in this pilot series, fewer subsequent Procore passes were required to achieve satisfactory specimens. Procore specimens also contained less obscuring bronchial contamination. Evaluation of more cases and more experience with this new technique is necessary to determine if the diagnostic yield using Procore during EBUS can be improved. 182 Detection of ALK mRNA Transcripts in Liquid-based NSCLC ThinPrep Cytology Slides Jordan Reynolds, MD1, Eugen Minca, MD, PhD1, Sherrie Sanda, PhD1, Christopher Lanigan, MS1, Daniel Kim, MS2, Sui Bui, MS2, Nan Su, PhD2, Xiao-Jun Ma, PhD2, Yuling Luo, PhD2, Kaixin Wang, PhD1, Zhen Wang, MD, PhD1. 1Cleveland Clinic, Cleveland, Ohio; 2Advanced Cell Diagnostics, Hayward, California Background: ALK gene rearrangements have been identified as oncogenic drivers in patients with non-small cell lung cancer (NSCLC). ALK status confers qualification for crizotinib therapy, currently performed by fluorescence in situ hybridization (FISH) or immunohistochemistry. Some patients with advanced NSCLC only have an FNA material for testing. We tested the utility of an automated bright field (BF) ALK mRNA in situ hybridization (ISH) assay combined with alkaline phosphatase-fast red reaction for detection of ALK mRNA transcripts in liquid-based NSCLC ThinPrep slides. Design: Endobronchial ultrasound guided transbronchial needle aspiration biopsies (EBUS-TBNA) and Control cells (SU-DHL-1/HL-60 ratio 1:1)
183 Detection of BRAF Mutation in Metastatic Melanoma Utilizing Celltransferred Cytological Smears Shaoxiong Chen, MD, Melissa Randolph, BS, CT(ASCP), Tracy Watkins, BS, CT (ASCP)cm, Holly McCullough, Harvey Cramer, MD, Kristin Post, MPH, Joyashree Sen, Liang Cheng, MD, Howard Wu, MD. Indiana University Health, Indianapolis, Indiana Introduction: Patients with advanced-stage melanoma containing BRAF mutations are candidates for BRAF-inhibitor therapy. Although fine-needle aspiration (FNA) is frequently used to diagnose metastatic melanoma, cell blocks prepared from these aspirates sometimes lack sufficient tumor cells for BRAF testing. In this study, we use cell-transferred cytological smears (CTC) as an alternative source for BRAF molecular testing. Material and Methods: A search of our laboratory information system was performed from 2011 through 2013 to identify surgical pathology cases of primary melanomas in which BRAF mutation analysis had been performed. Then, cases of metastatic melanoma from these same patients that were subsequently diagnosed by FNA were identified. One representative ethanol-fixed direct smear and one air-dried direct smear from each FNA case were selected for processing. The areas on the direct smears containing tumor cells were circled by a pathologist and submitted for mutation analysis using the cell transfer technique. Results: Mutation analysis was successfully performed in 28 of 30 FNA cases (93%), using the CTC technique. In 24 cases (7 BRAF-positive, 17 BRAF-negative), there was 100% agreement for the BRAF mutation analysis between the FNA using CTC and the formalin fixed paraffin embedded tissue. Interestingly, there were 4 FNA cases that showed BRAF mutations that had not been detected in the patients’ original surgical biopsy. Conclusions: When FNA cell blocks lack adequate material, the cell transfer technique can be used as a reliable, alternative method for detecting BRAF mutations. Additional mutational events may occur during acquisition of the metastatic phenotype that might explain why BRAF mutations were unexpectedly identified in 4 FNAs of metastatic melanoma whose primaries were BRAF-negative.
TISSUE/BONE 184 The Cytomorphologic Approach and Utilization of Fine needle Aspiration for the Diagnosis in Epithelioid Pattern Sarcomas Bilge Baskir Elcin, MD, Sule Canberk, MD, Atay Uludokumaci, MD, Deniz Unluer, MD, Rana Ramazanoglu, MD, Sergulen Dervisoglu, MD,
Abstracts
181
were collected in PreservCyt Solution and mounted onto ThinPrep slides. Automated mRNA ISH was performed on a Ventana Discovery XT automated stainer using ALK, control probes (PPIB, DapB), and amplification kit (Advanced Cell Diagnostics, Hayward, CA) combined with the ChromoMap Red Kit (Ventana, Tucson, AZ). ThinPrep FISH using an ALK break-apart probe set (Abbott Molecular Vysis, Des Plaines, IL) was the reference data set. Results: Overexpression of ALK mRNA was detectable in the ThinPrep control cells. Of the pilot EBUS-TBNAs, 7 were negative and 3 were positive. Positive staining was identified as brilliant red punctuate dot signals in the nucleus and/or cytoplasm, which was visible by BF and fluorescence microscopy using fluorescein and rhodamine filter combinations. Furthermore, the red chromogenic reaction product of alkaline phosphatase was more intense when viewed via fluorescence, providing a level of sensitivity greater than BF. The ALK status in controls and patients was consistent with ThinPrep FISH results. Conclusion: Automated mRNA ISH with Alkaline Phosphatase-Fast Red reaction for the detection of NSCLC ALK mRNA in liquid-based NSCLC ThinPrep slides is feasible and delineates ALK status in cytology samples. The potential for future multiplex assays detecting mRNA could serve as a screening test for numerous molecular alterations in cytology samples.
Endobronchial Ultrasound-Guided (EBUS) Procore Sampling: Comparison to Fine Needle Aspiration for Mediastinal Lymph Nodes Steve Manos, BS, CT(ASCP), Sara Monaco, MD, David Wilson, MD, MPH, Liron Pantanowitz, MD. University of Pennsylvania Medical Center, Pittsburgh, Pennsylvania Introduction: Endobronchial ultrasound (EBUS)-guided sampling of the mediastinum has traditionally been performed by fine needle aspiration (FNA) for cytologic evaluation. The ProCore fine needle biopsy, used primarily to obtain intra-abdominal tissue core biopsies, has not been widely used for EBUS. The aim of this study was to evaluate our experience with EBUS-Procore of mediastinal nodes, and to compare its diagnostic utilization to that of conventional EBUS-FNA. Material and Methods: A retrospective review of all EBUS cases of mediastinal masses using a ProCore needle from December 2013 to March 2014 was performed. Patient demographics, anatomic site, number of procedure passes, on-site immediate evaluation, final pathology reports, and ancillary test results were recorded. All specimen slides and cell blocks were evaluated scoring lesion and contamination cellularity (0 Z acellular, 1 Z scant, 2 Z moderate, and 3 Z abundant). Results: There were 18 EBUS cases of mediastinal lymph nodes using Procore in patients (12 female, 6 male) of average age 57 years (range, 3380 years). FNA was followed by Procore biopsy in all cases, except one where only Procore was performed. Cases included benign (granulomas), atypical, and malignant (carcinoma) diagnoses. More FNAs were satisfactory (with 2 unsatisfactory cases) than Procores (with 7 unsatisfactory cases). The 2 non-diagnostic FNA cases remained unsatisfactory with subsequent Procore. More passes were performed with FNA (4/case) than Procore (2.6/case). On average, FNA provided greater cellularity of lesional material (smear score Z 2; cell block score Z 1.4) than Procore (smear score Z 1.4; cell block score Z 1.4). However, FNAs contained more bronchial contamination (smear score Z 1.6; cell block score Z 1) than Procore (smear score Z 1; cell block score Z 0.4). Both sample types contained similar bronchial and blood contamination. Ancillary studies (special stains, immunostains, FISH, molecular) were equally successful using cell blocks from both specimen types. Conclusions: Our preliminary experience shows that EBUS-guided Procore sampling of mediastinal lymph nodes is feasible. Although FNA samples appear to provide more diagnostic cellular material in this pilot series, fewer subsequent Procore passes were required to achieve satisfactory specimens. Procore specimens also contained less obscuring bronchial contamination. Evaluation of more cases and more experience with this new technique is necessary to determine if the diagnostic yield using Procore during EBUS can be improved. 182 Detection of ALK mRNA Transcripts in Liquid-based NSCLC ThinPrep Cytology Slides Jordan Reynolds, MD1, Eugen Minca, MD, PhD1, Sherrie Sanda, PhD1, Christopher Lanigan, MS1, Daniel Kim, MS2, Sui Bui, MS2, Nan Su, PhD2, Xiao-Jun Ma, PhD2, Yuling Luo, PhD2, Kaixin Wang, PhD1, Zhen Wang, MD, PhD1. 1Cleveland Clinic, Cleveland, Ohio; 2Advanced Cell Diagnostics, Hayward, California Background: ALK gene rearrangements have been identified as oncogenic drivers in patients with non-small cell lung cancer (NSCLC). ALK status confers qualification for crizotinib therapy, currently performed by fluorescence in situ hybridization (FISH) or immunohistochemistry. Some patients with advanced NSCLC only have an FNA material for testing. We tested the utility of an automated bright field (BF) ALK mRNA in situ hybridization (ISH) assay combined with alkaline phosphatase-fast red reaction for detection of ALK mRNA transcripts in liquid-based NSCLC ThinPrep slides. Design: Endobronchial ultrasound guided transbronchial needle aspiration biopsies (EBUS-TBNA) and Control cells (SU-DHL-1/HL-60 ratio 1:1)
183 Detection of BRAF Mutation in Metastatic Melanoma Utilizing Celltransferred Cytological Smears Shaoxiong Chen, MD, Melissa Randolph, BS, CT(ASCP), Tracy Watkins, BS, CT (ASCP)cm, Holly McCullough, Harvey Cramer, MD, Kristin Post, MPH, Joyashree Sen, Liang Cheng, MD, Howard Wu, MD. Indiana University Health, Indianapolis, Indiana Introduction: Patients with advanced-stage melanoma containing BRAF mutations are candidates for BRAF-inhibitor therapy. Although fine-needle aspiration (FNA) is frequently used to diagnose metastatic melanoma, cell blocks prepared from these aspirates sometimes lack sufficient tumor cells for BRAF testing. In this study, we use cell-transferred cytological smears (CTC) as an alternative source for BRAF molecular testing. Material and Methods: A search of our laboratory information system was performed from 2011 through 2013 to identify surgical pathology cases of primary melanomas in which BRAF mutation analysis had been performed. Then, cases of metastatic melanoma from these same patients that were subsequently diagnosed by FNA were identified. One representative ethanol-fixed direct smear and one air-dried direct smear from each FNA case were selected for processing. The areas on the direct smears containing tumor cells were circled by a pathologist and submitted for mutation analysis using the cell transfer technique. Results: Mutation analysis was successfully performed in 28 of 30 FNA cases (93%), using the CTC technique. In 24 cases (7 BRAF-positive, 17 BRAF-negative), there was 100% agreement for the BRAF mutation analysis between the FNA using CTC and the formalin fixed paraffin embedded tissue. Interestingly, there were 4 FNA cases that showed BRAF mutations that had not been detected in the patients’ original surgical biopsy. Conclusions: When FNA cell blocks lack adequate material, the cell transfer technique can be used as a reliable, alternative method for detecting BRAF mutations. Additional mutational events may occur during acquisition of the metastatic phenotype that might explain why BRAF mutations were unexpectedly identified in 4 FNAs of metastatic melanoma whose primaries were BRAF-negative.
TISSUE/BONE 184 The Cytomorphologic Approach and Utilization of Fine needle Aspiration for the Diagnosis in Epithelioid Pattern Sarcomas Bilge Baskir Elcin, MD, Sule Canberk, MD, Atay Uludokumaci, MD, Deniz Unluer, MD, Rana Ramazanoglu, MD, Sergulen Dervisoglu, MD,