- Email: [email protected]
Cellular Cytology Smears Provide Sufficient Tumor DNA for Theranostic Next Generation Sequencing: A Study Defining “Molecular Adequacy”
S82
Abstracts
176 Cytological and Cytogenetic Analysis of Large Circulating Cells from Blood Derek Go, BS1, Sean O’Byrne, BS1, Rachel Conrad, MD1, Jianyu Rao, MD1, Nagesh Rao, PhD1, Edward Garon, MD1, Jonathan Goldman, MD1, Rajan Kulkarni, MD, PhD1, Elodie Sollier, PhD2, Dino Di Carlo, PhD2. 1University of California, Los Angeles, California; 2Vortex Biosciences & University of California, Los Angeles, California Introduction: Cancer cells found in circulation are believed to disseminate from tumors and form secondary metastatic sites. These cells, termed circulating tumor cells (CTCs), can provide a crucial cell source for cytology and cytogenetic analysis or cancer diagnosis, treatment monitoring, and personalized therapy identification. However, CTCs are difficult to isolate from the large number of contaminating blood cells and occur at extremely low frequencies (1-500 CTCs/mL blood). Standard modes of isolation and analysis use antibodies targeted against epithelial protein markers. However, due to intra/ inter-tumor heterogeneity, there is a need for label-free isolation and analysis of alternative characteristics that may correlate more accurately with the malignant phenotype. Our approach isolates and concentrates larger circulating cells into a small volume, enabling facile downstream cytological and fluorescence in situ hybridization (FISH) analyses compared to previous approaches. Material and Methods: We use a recently introduced microfluidic device that generates size-dependent shear gradient forces to selectively concentrate larger cells from blood into fluid vortices (Figure 1). In contrast to label-intensive methods, the isolated cells can easily be released from the device and collected in suspension for downstream analysis. Results: We have successfully isolated and detected atypical cells from patients diagnosed with adeno- and squamous cell carcinoma through conventional Papanicolau staining (Figure 2). Similar morphological features are also seen in the corresponding primary tumors. Furthermore, we have detected the anaplastic lymphoma kinase (ALK) chromosomal translocation in circulating cells isolated from patients with the same genetic abnormality in their primary tumors (Figure 3). Conclusions: The detection of circulating cells that share similar morphological and genetic signatures as the primary tumor indicates their common site of origin. This demonstrates the utility of enriching and analyzing these potentially malignant cells in a label-free fashion, which can provide a noninvasive method to monitor disease progression and help guide clinical decision-making.
177 Cellular Cytology Smears Provide Sufficient Tumor DNA for Theranostic Next Generation Sequencing: A Study Defining “Molecular Adequacy” Sarah Kerr, MD, Ferga Gleeson, MD, Michael Campion, BS, Douglas Minot, CT(ASCP), MB(ASCP), Jesse Voss, BS, CT, MB(ASCP), Michael Levy, MD, Benjamin Kipp, PhD. Mayo Clinic, Rochester, Minnesota Introduction: Demand for theranostic molecular testing is exploding. Unfortunately, fine needle aspiration/biopsies (FNAB) produce specimens (cell blocks, cores) that are often insufficient for multi-gene testing when formalin fixed. Additionally, onsite “molecular adequacy” evaluation of such specimens is difficult. The use of routine cytologic smears offers several advantages, including direct onsite adequacy evaluation, concentration of tumor cells, and higher quality DNA. This study evaluates the “molecular adequacy” of cytologic smears for a 50-gene next generation sequencing (NGS) panel. Material and Methods: Stained smears obtained from lung cancer metastasis to the adrenal gland and gastrointestinal stromal tumors (GIST) of the stomach during percutaneous or endoscopic FNAB were reviewed. A single slide was selected if it contained >300 total cells and 20% tumor cells. Slides were decoverslipped and scraped. DNA was extracted and processed for NGS evaluating 207 amplicons in 50 cancer genes. Results: Review of 102 cases produced 76 acceptable slides (56 lung, 20 GIST). Slides had low (300-1000), moderate (1000-5000), or high (>5000
Abstracts cells) cellularity in 18 (24%), 21(28%), and 37(49%) cases. Median tumor content was 80% (range 20-90%). Fluorometric DNA quantity, average depth of coverage, and % reads mapped to target varied with cellularity. Successful NGS results were obtained in 47(62%) cases, of which 39 (83%) revealed pathogenic alterations. Successful NGS cases had high cellularity in 72%; DNA concentration ranged from 3.3 - 273 ng/mL (median 26.7), average depth of coverage ranged 335 -12112 (median 3424), and the % mapped reads median was 97%. High cellularity and >5 ng/mL of DNA predicted NGS success 92% and 96% of cases, respectively. Conclusions: Cytologic smears provide adequate material for NGS, with success dependent upon cellularity. These results suggest that cytopathology professionals can provide onsite “molecular adequacy” evaluation for theranostic testing. Validation studies are required for other tumor types and locations. 178 Comprehensive Theranostic Summary (CTS) Reports for Thoracic Cytopathology Liron Pantanowitz, MD, Luke Wiehagen, BS, Sara Monaco, MD, Samuel Yousem, Anthony Piccoli, BS, Anil Parwani, MD,PhD. University of Pennsylvania Medical Center, Pittsburgh, Pennsylvania Introduction: Signing out cytopathology reports in the current era of personalized medicine requires incorporation of ancillary test results. For lung non-small cell carcinoma, this includes theranostic information obtained from immunohistochemistry, fluorescence in situ hybridization (FISH) and molecular tests. Frequently these results are delivered in separate reports subsequent to the cytology diagnosis, given turnaround time constraints. We aim to share our experience with using comprehensive theranostic summary (CTS) reports in thoracic cytopathology. Material and Methods: The synoptic reporting tool in our laboratory information system (CoPathPlus, Cerner) was customized to capture and report results for predictive or theranostic immunohistochemistry, FISH and/or molecular testing into one CTS report. Ancillary test results were manually entered into reports as they became available by multiple authors from relevant testing areas, and once complete, a final CTS report was signed out as an addendum. We analyzed CTS reports for lung cytopathology cases from October 2011 to March 2014 (30 month period). Clinicians were surveyed about the utility of these reports. Results: To date, we have signed out 348 lung cytopathology CTS reports. From an informatics perspective, CTS reports were moderately easy to implement and maintain. The discrete data input generates a text-based result format, ensuring reliable transmission to several downstream electronic medical records. Manual entry of data caused some redundancy in lab workflow, delay or omission of CTS reports during the early stage of implementation, and occasional transcription errors. When surveyed, clinicians receiving CTS reports found them to be useful and easier to read than complex addendum reports. Conclusions: CTS reporting for thoracic cytopathology has been successfully adopted at our institution. Such reports have been well received by most of our clinicians because of the ease in finding this important information in a concise format. CTS reports also contain relevant, structured, discrete data that can be easily searched. Implementing an automated electronic solution to support CTS reporting may help avoid workflow inefficiency and reduce errors attributed to manual tasks. 179 Validation of Protein Biomarkers in Bronchoalveolar Lavage (BAL) to Improve the Detection of Lung Cancers in Patients with Lung Nodules Hui Zhang, PhD, Susan Geddes, CT(ASCP), Frederic Askin, MD, Edward Gabrielson, MD, Qing Kay Li, MD, PhD. The Johns Hopkins Medical Institutions, Baltimore, Maryland Introduction: National Lung Cancer Screening Trial (NLST) using highly sensitive low-dose computed tomography (LDCT) has demonstrated an improved detection of lung cancers. However, NLST has also showed high
S83 repeat screening rates and high false-positive LDCT rates, causing unnecessary second-line invasive procedures and/or surgery. New strategies are needed to improve the specificity of lung cancer screenings. Bronchoalveolar lavage (BAL) is commonly used for the cytological evaluation of lung parenchymal cells in lung nodule patients and for the diagnosis of lung cancers. Ironically, BAL fluid is currently discarded after cytological examination. Our preliminary study have shown that proteins in the BAL fluid can be identified, and they may be used as potential biomarkers for the diagnosis of lung cancers. Material and Method: We collected BAL samples, from patients with benign lung disease, lung adenocarcinoma (ADC), lung squamous cell carcinoma (SqCC), and small cell lung carcinoma (SCLC), and studied protein profiles using SPEG and LC-MS/MS. We further tested the sensitivity and specificity of a subset of proteins using independently collected BAL specimens by ELISA assays Results: A total of 462 glycoproteins were identified and quatified from BAL fluids. Among them, 290 proteins were identified in the BAL of lung ADC, 376 in lung SqCC, 310 in SCLC and 318 in benign BALs. In addition to proteins found in all groups, we identified 123 unique proteins that were differentially expresssed exclusively in either benign disease, ADC, SqCC or SCLC. The levels of four proteins in cancer and benign BALs were further validated by ELISA assays, using independently collected subset of BAL specimens. Among the proteins, Napsin A and Periostin were significantly elevated in cancer BAL specimens. Conclusions: Our study demonstrates that protein biomarkers in BAL can be used to improve the specificity of lung screening tests and the early detection of lung cancers. 180 Validation of the Novel Triple Marker (Combination of TTF, Napsin-A and p40) in the Subclassification of Non-Small Cell Lung Carcinomas (NSCLC) Using Fine Needle Aspiration (FNA) Cytological Materials Rajni Sharma, MS, PhD, Grzegorz Gurda, MD, PhD, Susan Geddes, CT(ASCP), Edward Gabrielson, MD, Frederic Askin, MD, Qing Kay Li, MD, PhD. The Johns Hopkins Medical Institutions, Baltimore, Maryland Introduction: Personalized treatment of lung cancers requires the accurate subclassification of NSCLC into adenocarcinoma (ADC), squamous cell carcinoma (SqCC) and other subtypes. In most cases, NSCLC can be subclassified using routine H&E sections. In poorly differentiated tumors or on small FNA specimens, it may be difficult. The use of immunohistochemical (IHC) markers plays an important role in this circumstance. However, the commonly used panels need to be performed on multiple sections, and cause exhaustion of FNA specimen needed for molecular tests. Our recent study using TMAs has shown that the novel triple marker (combination of TTF, Napsin-A and p40) has the ability to accurately subclassify NSCLCs (Human Pathol 2014;45:926-934). In this study, we further validate the triple marker for the subclassification of NSCLC using cytological materials. Material and Methods: From the pathology archives, FNA cases with cell block preparations were collected, including 71 cases of SqCC, 41 cases of ADC (including both primary pulmonary and non-pulmonary ADCs), and 6 cases of small cell lung carcinoma. Immunostains of tumor cells with the triple marker were scored semi-quantitatively and compared. The performance of the triple marker was also compared with individual markers. Results: In SqCCs, 68 of 71 (96%) cases were appropriately immunoreactive for the triple marker, and the sensitivity and specificity were 95.77% and 100%, respectively. In primary pulmonary ADCs, the triple marker showed a superior performance than individual marker alone with the immunoreactivity in 93.5% of cases, and the sensitivity and specificity were 93.5% and 100%, respectively. In non-pulmonary ADCs, the triple marker showed 0% of immunoreactivity. Conclusions: Our study provides further evidence that the triple marker is a useful combination of immunomarkers for the subclassification of NSCLC using minimal cytological material. More importantly, it also provides optimal conservation of tumor tissue for further molecular testing of lung cancers.
Abstracts
176 Cytological and Cytogenetic Analysis of Large Circulating Cells from Blood Derek Go, BS1, Sean O’Byrne, BS1, Rachel Conrad, MD1, Jianyu Rao, MD1, Nagesh Rao, PhD1, Edward Garon, MD1, Jonathan Goldman, MD1, Rajan Kulkarni, MD, PhD1, Elodie Sollier, PhD2, Dino Di Carlo, PhD2. 1University of California, Los Angeles, California; 2Vortex Biosciences & University of California, Los Angeles, California Introduction: Cancer cells found in circulation are believed to disseminate from tumors and form secondary metastatic sites. These cells, termed circulating tumor cells (CTCs), can provide a crucial cell source for cytology and cytogenetic analysis or cancer diagnosis, treatment monitoring, and personalized therapy identification. However, CTCs are difficult to isolate from the large number of contaminating blood cells and occur at extremely low frequencies (1-500 CTCs/mL blood). Standard modes of isolation and analysis use antibodies targeted against epithelial protein markers. However, due to intra/ inter-tumor heterogeneity, there is a need for label-free isolation and analysis of alternative characteristics that may correlate more accurately with the malignant phenotype. Our approach isolates and concentrates larger circulating cells into a small volume, enabling facile downstream cytological and fluorescence in situ hybridization (FISH) analyses compared to previous approaches. Material and Methods: We use a recently introduced microfluidic device that generates size-dependent shear gradient forces to selectively concentrate larger cells from blood into fluid vortices (Figure 1). In contrast to label-intensive methods, the isolated cells can easily be released from the device and collected in suspension for downstream analysis. Results: We have successfully isolated and detected atypical cells from patients diagnosed with adeno- and squamous cell carcinoma through conventional Papanicolau staining (Figure 2). Similar morphological features are also seen in the corresponding primary tumors. Furthermore, we have detected the anaplastic lymphoma kinase (ALK) chromosomal translocation in circulating cells isolated from patients with the same genetic abnormality in their primary tumors (Figure 3). Conclusions: The detection of circulating cells that share similar morphological and genetic signatures as the primary tumor indicates their common site of origin. This demonstrates the utility of enriching and analyzing these potentially malignant cells in a label-free fashion, which can provide a noninvasive method to monitor disease progression and help guide clinical decision-making.
177 Cellular Cytology Smears Provide Sufficient Tumor DNA for Theranostic Next Generation Sequencing: A Study Defining “Molecular Adequacy” Sarah Kerr, MD, Ferga Gleeson, MD, Michael Campion, BS, Douglas Minot, CT(ASCP), MB(ASCP), Jesse Voss, BS, CT, MB(ASCP), Michael Levy, MD, Benjamin Kipp, PhD. Mayo Clinic, Rochester, Minnesota Introduction: Demand for theranostic molecular testing is exploding. Unfortunately, fine needle aspiration/biopsies (FNAB) produce specimens (cell blocks, cores) that are often insufficient for multi-gene testing when formalin fixed. Additionally, onsite “molecular adequacy” evaluation of such specimens is difficult. The use of routine cytologic smears offers several advantages, including direct onsite adequacy evaluation, concentration of tumor cells, and higher quality DNA. This study evaluates the “molecular adequacy” of cytologic smears for a 50-gene next generation sequencing (NGS) panel. Material and Methods: Stained smears obtained from lung cancer metastasis to the adrenal gland and gastrointestinal stromal tumors (GIST) of the stomach during percutaneous or endoscopic FNAB were reviewed. A single slide was selected if it contained >300 total cells and 20% tumor cells. Slides were decoverslipped and scraped. DNA was extracted and processed for NGS evaluating 207 amplicons in 50 cancer genes. Results: Review of 102 cases produced 76 acceptable slides (56 lung, 20 GIST). Slides had low (300-1000), moderate (1000-5000), or high (>5000
Abstracts cells) cellularity in 18 (24%), 21(28%), and 37(49%) cases. Median tumor content was 80% (range 20-90%). Fluorometric DNA quantity, average depth of coverage, and % reads mapped to target varied with cellularity. Successful NGS results were obtained in 47(62%) cases, of which 39 (83%) revealed pathogenic alterations. Successful NGS cases had high cellularity in 72%; DNA concentration ranged from 3.3 - 273 ng/mL (median 26.7), average depth of coverage ranged 335 -12112 (median 3424), and the % mapped reads median was 97%. High cellularity and >5 ng/mL of DNA predicted NGS success 92% and 96% of cases, respectively. Conclusions: Cytologic smears provide adequate material for NGS, with success dependent upon cellularity. These results suggest that cytopathology professionals can provide onsite “molecular adequacy” evaluation for theranostic testing. Validation studies are required for other tumor types and locations. 178 Comprehensive Theranostic Summary (CTS) Reports for Thoracic Cytopathology Liron Pantanowitz, MD, Luke Wiehagen, BS, Sara Monaco, MD, Samuel Yousem, Anthony Piccoli, BS, Anil Parwani, MD,PhD. University of Pennsylvania Medical Center, Pittsburgh, Pennsylvania Introduction: Signing out cytopathology reports in the current era of personalized medicine requires incorporation of ancillary test results. For lung non-small cell carcinoma, this includes theranostic information obtained from immunohistochemistry, fluorescence in situ hybridization (FISH) and molecular tests. Frequently these results are delivered in separate reports subsequent to the cytology diagnosis, given turnaround time constraints. We aim to share our experience with using comprehensive theranostic summary (CTS) reports in thoracic cytopathology. Material and Methods: The synoptic reporting tool in our laboratory information system (CoPathPlus, Cerner) was customized to capture and report results for predictive or theranostic immunohistochemistry, FISH and/or molecular testing into one CTS report. Ancillary test results were manually entered into reports as they became available by multiple authors from relevant testing areas, and once complete, a final CTS report was signed out as an addendum. We analyzed CTS reports for lung cytopathology cases from October 2011 to March 2014 (30 month period). Clinicians were surveyed about the utility of these reports. Results: To date, we have signed out 348 lung cytopathology CTS reports. From an informatics perspective, CTS reports were moderately easy to implement and maintain. The discrete data input generates a text-based result format, ensuring reliable transmission to several downstream electronic medical records. Manual entry of data caused some redundancy in lab workflow, delay or omission of CTS reports during the early stage of implementation, and occasional transcription errors. When surveyed, clinicians receiving CTS reports found them to be useful and easier to read than complex addendum reports. Conclusions: CTS reporting for thoracic cytopathology has been successfully adopted at our institution. Such reports have been well received by most of our clinicians because of the ease in finding this important information in a concise format. CTS reports also contain relevant, structured, discrete data that can be easily searched. Implementing an automated electronic solution to support CTS reporting may help avoid workflow inefficiency and reduce errors attributed to manual tasks. 179 Validation of Protein Biomarkers in Bronchoalveolar Lavage (BAL) to Improve the Detection of Lung Cancers in Patients with Lung Nodules Hui Zhang, PhD, Susan Geddes, CT(ASCP), Frederic Askin, MD, Edward Gabrielson, MD, Qing Kay Li, MD, PhD. The Johns Hopkins Medical Institutions, Baltimore, Maryland Introduction: National Lung Cancer Screening Trial (NLST) using highly sensitive low-dose computed tomography (LDCT) has demonstrated an improved detection of lung cancers. However, NLST has also showed high
S83 repeat screening rates and high false-positive LDCT rates, causing unnecessary second-line invasive procedures and/or surgery. New strategies are needed to improve the specificity of lung cancer screenings. Bronchoalveolar lavage (BAL) is commonly used for the cytological evaluation of lung parenchymal cells in lung nodule patients and for the diagnosis of lung cancers. Ironically, BAL fluid is currently discarded after cytological examination. Our preliminary study have shown that proteins in the BAL fluid can be identified, and they may be used as potential biomarkers for the diagnosis of lung cancers. Material and Method: We collected BAL samples, from patients with benign lung disease, lung adenocarcinoma (ADC), lung squamous cell carcinoma (SqCC), and small cell lung carcinoma (SCLC), and studied protein profiles using SPEG and LC-MS/MS. We further tested the sensitivity and specificity of a subset of proteins using independently collected BAL specimens by ELISA assays Results: A total of 462 glycoproteins were identified and quatified from BAL fluids. Among them, 290 proteins were identified in the BAL of lung ADC, 376 in lung SqCC, 310 in SCLC and 318 in benign BALs. In addition to proteins found in all groups, we identified 123 unique proteins that were differentially expresssed exclusively in either benign disease, ADC, SqCC or SCLC. The levels of four proteins in cancer and benign BALs were further validated by ELISA assays, using independently collected subset of BAL specimens. Among the proteins, Napsin A and Periostin were significantly elevated in cancer BAL specimens. Conclusions: Our study demonstrates that protein biomarkers in BAL can be used to improve the specificity of lung screening tests and the early detection of lung cancers. 180 Validation of the Novel Triple Marker (Combination of TTF, Napsin-A and p40) in the Subclassification of Non-Small Cell Lung Carcinomas (NSCLC) Using Fine Needle Aspiration (FNA) Cytological Materials Rajni Sharma, MS, PhD, Grzegorz Gurda, MD, PhD, Susan Geddes, CT(ASCP), Edward Gabrielson, MD, Frederic Askin, MD, Qing Kay Li, MD, PhD. The Johns Hopkins Medical Institutions, Baltimore, Maryland Introduction: Personalized treatment of lung cancers requires the accurate subclassification of NSCLC into adenocarcinoma (ADC), squamous cell carcinoma (SqCC) and other subtypes. In most cases, NSCLC can be subclassified using routine H&E sections. In poorly differentiated tumors or on small FNA specimens, it may be difficult. The use of immunohistochemical (IHC) markers plays an important role in this circumstance. However, the commonly used panels need to be performed on multiple sections, and cause exhaustion of FNA specimen needed for molecular tests. Our recent study using TMAs has shown that the novel triple marker (combination of TTF, Napsin-A and p40) has the ability to accurately subclassify NSCLCs (Human Pathol 2014;45:926-934). In this study, we further validate the triple marker for the subclassification of NSCLC using cytological materials. Material and Methods: From the pathology archives, FNA cases with cell block preparations were collected, including 71 cases of SqCC, 41 cases of ADC (including both primary pulmonary and non-pulmonary ADCs), and 6 cases of small cell lung carcinoma. Immunostains of tumor cells with the triple marker were scored semi-quantitatively and compared. The performance of the triple marker was also compared with individual markers. Results: In SqCCs, 68 of 71 (96%) cases were appropriately immunoreactive for the triple marker, and the sensitivity and specificity were 95.77% and 100%, respectively. In primary pulmonary ADCs, the triple marker showed a superior performance than individual marker alone with the immunoreactivity in 93.5% of cases, and the sensitivity and specificity were 93.5% and 100%, respectively. In non-pulmonary ADCs, the triple marker showed 0% of immunoreactivity. Conclusions: Our study provides further evidence that the triple marker is a useful combination of immunomarkers for the subclassification of NSCLC using minimal cytological material. More importantly, it also provides optimal conservation of tumor tissue for further molecular testing of lung cancers.