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P2.02-024 False Positivity Due to Polysomy in Fluorescence in Situ Hybridization
November 2017 nuclease protection method provides the possibility to analyze minimal amount of formalin fixed paraffin embedded (FFPE) tissue without previous extraction steps. We tested this technique and compared it to the traditional methods RNA sequencing (RNAseq) and immunohistochemistry (IHC). Method: The nuclease protection method (HTGmolecular) in combination with next-generation sequencing was used to measure gene expression of 549 immuneoncology genes in FFPE samples from NSCLC-patients. Standardized minimal tissue amounts were used for 12 samples (4 tissue circles, 4mm thick, 1 mm in diameter, from a tissue microarray). Of these tissue sections also two corresponding original tumor biopsy were analyzed. RNA sequencing data was available from a corresponding fresh frozen tissue as well as IHC annotation of the immune markers FOXP3, CDH1, CD20, CD44, CD3, CD4, CD8 and PD-L1 on the analyzed tissue cores. Result: Of the 12 core preparations, 9 samples were successfully analyzed and fulfilled the quality criteria in the first run, the three others in a second re-analysis. The mRNA expression profiles of 12 samples measured with HTG on minute FFPE samples and RNAseq from fresh frozen tissue showed most often good correlations (r¼0.41-0.87). HTG based mRNA data correlated with IHC expression for 5 of 8 genes (PD-L1 r¼0.76, CD44 r¼0.75, CDH1 r¼0.61, CD8 r¼0.60, CD4 r¼0.54). RNAseq data showed good correlations with IHC for only 3 of 8 genes (CD44 r¼0.91, PD-L1 r¼0.86, CD8 r¼0.67). Also, the HTG data of the two biopsies demonstrated very good correlations to the corresponding tissue cores and the RNAseq data (r>0.91). Finally, technical replicates of 10 of the minimal tissue core samples measured in different laboratories revealed relatively good concordance (r¼0.71-0.94). Conclusion: The applied nuclease protection technique opens the possibility to multiplex and analyze the immune profile of 549 genes in minimal diagnostic biopsies with a high success rate. This is of great value for clinical use or in NSCLC clinical studies where the amount of tissue often is a limiting factor in companion diagnostics. Keywords: immune-oncology genes, minimal amount of FFPE tissue, Nuclease protection assay
P2.02-024 False Positivity Due to Polysomy in Fluorescence in Situ Hybridization A.L. Görtz,1 S. Finn,2 L. Bubendorf,3 I. Bahce,4 B. Witte,5 E. Thunnissen6 1Pathology, Vumc Amsterdam, Amsterdam/NL, 2Dept of Histopathology, St James’s Hospital, Dublin/IE, 3Institute of Pathology, University Hospital Basel, Basel/CH, 4VU Medical Centre, Amsterdam/NL, 5 Biostatistics, Vumc Amsterdam, Amsterdam/NL, 6Pathology Department, VU University Medical Center, Amsterdam/NL Background: Pathologists may recognize the phenomenon of polyploidy in FISH, which may be misleading in interpretation of break apart fluorescence in-situ hybridization (FISH). The chance for single or split probe signals is likely to increase with the degree of polysomy. The aim of this study was to explore whether false positivity due to polyploidy occurs in practice. Method: A cohort of cases referred for study or patient care was collected from the archives. From the cases where the ALK and/or ROS1 in-situ hybridization test was repeated in our hospital the outcome of testing was compared. Additionally tumor DNA of an occasional case was tested by an orthogonal method (Ion Torrent Oncomine Focus Assay) for translocations. Result: Three cases with ALK FISH rearrangement elsewhere were diagnosed with polyploidy in the referral center. One case was reported with rearrangements in both the ALK and the ROS1 gene detected by FISH analysis. In the repeated FISH analysis the average number of colocalization signals in the tumor cell nuclei was 7.6 for ALK and 9.5 for ROS1 respectively (range 1 - 30). Moreover, the morphology of this case was a giant cell carcinoma, variant of pleomorphic carcinoma of the lung. Examination with an orthogonal method (Ion Torrent Oncomine Assay) revealed no translocations and the tumor cells were negative for ALK and ROS1 by immunohistochemistry proving the
Abstracts
S2107
original report as false positive, supported by absence of response on crizotinib. In break apart FISH the 15% threshold for positivity was obtained in cells emphasizing that in cross sections of normal nuclei occasionally split signals or 3’ probe signals may be present even in diploid nuclei. In the range of 15-20% the chance of false positive FISH is >1%.1 However, in polyploid tumors the higher number of probe signals within one nucleus comes with an increased chance of split or 3’ signals and a higher rate of false-positive results when maintaining a uniform threshold 15% irrespective of ploidy. Moreover, this may in case of ALK be an additional reason for discordancy with ALK immunohistochemistry, explaining the lack of response on targeted therapy in these patients.2 References: 1. vLaffert Lung cancer. 2015;90:465 2. vdWekken. Clin Cancer Res.epub. Conclusion: In case of polysomy there is an increased chance of false positive in break apart FISH results. An addition technique should be used to confirm a positive FISH status in tumors with highly increased gene copy number due to polysomy.
P2.02-025 Histological Difference of Tumor-Infiltrate Lymphocytes in Non-Small Cell Lung Cancer N. Kobayashi,1 S. Kikuchi,1 Y. Goto,1 Y. Sato,1 S. Sakashita,2 M. Noguchi2 1Department of Surgery (Thoracic Surgery), University of Tsukuba, Tsukuba Ibaraki/JP, 2Pathology, University of Tsukuba, Tsukuba-Shi/JP Background: Lymphocytes play important roles in cancer immunity. Tumor-infiltrate lymphocytes (TILs) are seen in non-small cell lung cancer (NSCLC) and generally classified according to their localization (epithelial area and stromal area). The distribution and the number of TILs are quite different. Cancer cells have an ability to evade from cancer immunity, and the several mechanisms of the ability have been reported; decreased expression of tumor antigen, inhibition of immune response, induction of immunosuppressive cells, and secretion of immunosuppressive cytokines. We hypothesized that the mechanisms of evasion from cancer immunity would influence TIL representation. In this study, we investigated the differences of TILs in histological differentiation, since we considered that histological difference could affect cancer immunity. Method: We retrospectively investigated surgical specimens between 2009 and 2015. Consecutive 20 cases with minimally invasive adenocarcinoma (MIA), lepidic adenocarcinoma (Ad lepidic), acinar or papillary adenocarcinoma (Ad aci/pap), solid adenocarcinoma (Ad solid) and squamous cell carcinoma (Sq) were selected (total 100 cases). We checked all fields of the tumors in the slice with maximum tumor-diameter microscopically at 100-fold magnification. TILs in the field were judged as positive when more than 10 lymphocytes flocking in tumor epithelial area or stromal area were observed. TILs of the tumors were assessed as the rate of the TIL positive fields in all, and separately evaluated in epithelial area and stromal area. Then, analysis of variance was used to assess the histological differences of TILs. Significant difference was considered as p-value was less than 0.05. Result: The average rates of TIL positive fields in epithelial area of MIA, Ad lepidic, Ad aci/pap, Ad solid and Sq were 11.2 ± 20.4%, 15.8 ± 20.4%, 26.9 ± 20.9%, 52.4 ± 30.0% and 27.8± 28.8%, respectively. The rate of Ad solid was significantly higher than those of MIA, Ad lepidic and Ad aci/pap, and the rate of Sq was also significantly higher than those of MIA and Ad lepidic. The average rates of TIL positive fields in stromal area of MIA, Ad lepidic, Ad aci/ pap, Ad solid and Sq were 41.9 ± 26.1%, 51.2 ± 28.3%, 57.6 ± 23.2%, 67.7 ± 25.4% and 67.8 ± 30.0%, respectively. The rate of MIA was significantly lower than Ad solid and Sq. Conclusion: TILs were significantly different representation depending on the histology. Especially in adenocarcinoma, the TILs differed according to the grade of differentiation. These results might show that highly differentiated lung adenocarcinoma has low expression of tumor antigen. Keywords: Tumor-infiltrate lymphocyte, histology
P2.02-024 False Positivity Due to Polysomy in Fluorescence in Situ Hybridization A.L. Görtz,1 S. Finn,2 L. Bubendorf,3 I. Bahce,4 B. Witte,5 E. Thunnissen6 1Pathology, Vumc Amsterdam, Amsterdam/NL, 2Dept of Histopathology, St James’s Hospital, Dublin/IE, 3Institute of Pathology, University Hospital Basel, Basel/CH, 4VU Medical Centre, Amsterdam/NL, 5 Biostatistics, Vumc Amsterdam, Amsterdam/NL, 6Pathology Department, VU University Medical Center, Amsterdam/NL Background: Pathologists may recognize the phenomenon of polyploidy in FISH, which may be misleading in interpretation of break apart fluorescence in-situ hybridization (FISH). The chance for single or split probe signals is likely to increase with the degree of polysomy. The aim of this study was to explore whether false positivity due to polyploidy occurs in practice. Method: A cohort of cases referred for study or patient care was collected from the archives. From the cases where the ALK and/or ROS1 in-situ hybridization test was repeated in our hospital the outcome of testing was compared. Additionally tumor DNA of an occasional case was tested by an orthogonal method (Ion Torrent Oncomine Focus Assay) for translocations. Result: Three cases with ALK FISH rearrangement elsewhere were diagnosed with polyploidy in the referral center. One case was reported with rearrangements in both the ALK and the ROS1 gene detected by FISH analysis. In the repeated FISH analysis the average number of colocalization signals in the tumor cell nuclei was 7.6 for ALK and 9.5 for ROS1 respectively (range 1 - 30). Moreover, the morphology of this case was a giant cell carcinoma, variant of pleomorphic carcinoma of the lung. Examination with an orthogonal method (Ion Torrent Oncomine Assay) revealed no translocations and the tumor cells were negative for ALK and ROS1 by immunohistochemistry proving the
Abstracts
S2107
original report as false positive, supported by absence of response on crizotinib. In break apart FISH the 15% threshold for positivity was obtained in cells emphasizing that in cross sections of normal nuclei occasionally split signals or 3’ probe signals may be present even in diploid nuclei. In the range of 15-20% the chance of false positive FISH is >1%.1 However, in polyploid tumors the higher number of probe signals within one nucleus comes with an increased chance of split or 3’ signals and a higher rate of false-positive results when maintaining a uniform threshold 15% irrespective of ploidy. Moreover, this may in case of ALK be an additional reason for discordancy with ALK immunohistochemistry, explaining the lack of response on targeted therapy in these patients.2 References: 1. vLaffert Lung cancer. 2015;90:465 2. vdWekken. Clin Cancer Res.epub. Conclusion: In case of polysomy there is an increased chance of false positive in break apart FISH results. An addition technique should be used to confirm a positive FISH status in tumors with highly increased gene copy number due to polysomy.
P2.02-025 Histological Difference of Tumor-Infiltrate Lymphocytes in Non-Small Cell Lung Cancer N. Kobayashi,1 S. Kikuchi,1 Y. Goto,1 Y. Sato,1 S. Sakashita,2 M. Noguchi2 1Department of Surgery (Thoracic Surgery), University of Tsukuba, Tsukuba Ibaraki/JP, 2Pathology, University of Tsukuba, Tsukuba-Shi/JP Background: Lymphocytes play important roles in cancer immunity. Tumor-infiltrate lymphocytes (TILs) are seen in non-small cell lung cancer (NSCLC) and generally classified according to their localization (epithelial area and stromal area). The distribution and the number of TILs are quite different. Cancer cells have an ability to evade from cancer immunity, and the several mechanisms of the ability have been reported; decreased expression of tumor antigen, inhibition of immune response, induction of immunosuppressive cells, and secretion of immunosuppressive cytokines. We hypothesized that the mechanisms of evasion from cancer immunity would influence TIL representation. In this study, we investigated the differences of TILs in histological differentiation, since we considered that histological difference could affect cancer immunity. Method: We retrospectively investigated surgical specimens between 2009 and 2015. Consecutive 20 cases with minimally invasive adenocarcinoma (MIA), lepidic adenocarcinoma (Ad lepidic), acinar or papillary adenocarcinoma (Ad aci/pap), solid adenocarcinoma (Ad solid) and squamous cell carcinoma (Sq) were selected (total 100 cases). We checked all fields of the tumors in the slice with maximum tumor-diameter microscopically at 100-fold magnification. TILs in the field were judged as positive when more than 10 lymphocytes flocking in tumor epithelial area or stromal area were observed. TILs of the tumors were assessed as the rate of the TIL positive fields in all, and separately evaluated in epithelial area and stromal area. Then, analysis of variance was used to assess the histological differences of TILs. Significant difference was considered as p-value was less than 0.05. Result: The average rates of TIL positive fields in epithelial area of MIA, Ad lepidic, Ad aci/pap, Ad solid and Sq were 11.2 ± 20.4%, 15.8 ± 20.4%, 26.9 ± 20.9%, 52.4 ± 30.0% and 27.8± 28.8%, respectively. The rate of Ad solid was significantly higher than those of MIA, Ad lepidic and Ad aci/pap, and the rate of Sq was also significantly higher than those of MIA and Ad lepidic. The average rates of TIL positive fields in stromal area of MIA, Ad lepidic, Ad aci/ pap, Ad solid and Sq were 41.9 ± 26.1%, 51.2 ± 28.3%, 57.6 ± 23.2%, 67.7 ± 25.4% and 67.8 ± 30.0%, respectively. The rate of MIA was significantly lower than Ad solid and Sq. Conclusion: TILs were significantly different representation depending on the histology. Especially in adenocarcinoma, the TILs differed according to the grade of differentiation. These results might show that highly differentiated lung adenocarcinoma has low expression of tumor antigen. Keywords: Tumor-infiltrate lymphocyte, histology