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P1.02-046 ALK IHC is Highly Sensitive to Fixation Parameters
January 2017
Results: Seven cases (7.3%) showed discordant results with ALK FISH-positive and IHC negative. Among these cases the mean number of FISH positive rearranged nuclei was 40.2% (range 20-54%). All cases showed coexistent split signals pattern positivity with mean percentage 19.1% (range 10-32%.), 5’ deletions pattern positivity with mean percentage 21.7% (range 12-34%) and one case also had gene amplifications pattern positivity with percentage of 64%. The polysomy was observed in all cases with mean percentage of 45.7% (range of 16-72%). Five patients with discordant ALK FISH and IHC results received crizotinib; of them, four progressed and one has stable disease. Conclusion: In most of discordant cases, a coexistent complex pattern of rearrangements (deleted, inverted and amplified/polysomic patterns) of ALK positive cells in FISH analysis was observed; these complex rearranged cases were not detectable by IHC, probably due to the lack of protein expression. Considering that crizotinib inhibits the ALK protein and not specifically ALK rearrangements, it could be speculated that NSCLCs without IHC ALK expression could be not sensitive to crizotinib. The ALK FISH+/IHC negative discordant group showed a lower percentage of nuclei positive for ALK rearrangement (19.1% in split signals and 21.7% in 5’ deletions) as compared with 64% of gene amplification in one case and with polysomy (45.7%) observed in all cases. These preliminary data highlight the role of IHC analysis and of the percentage of the genetic pattern of ALK rearranged cells in FISH analysis to select patients for anti ALK treatment. Further investigation is suggested about the correlation of complex mutational findings and clinical outcome. Keywords: ALK rearrangements, advanced NSCLC, IHC/ FISH discordance
P1.02-046 ALK IHC is Highly Sensitive to Fixation Parameters Topic: Driver Genes in NSCLC, Resistance, and Other Isabell Loftin,1 Rachel Miller,1 Patricia Thorne-Nuzzo,1 Abigail Mcelhinny,2 Penny Towne,1 Shalini Singh,1 June Clements1 1 Ventana Medical Systems Inc., A Member of the Roche Group, Tucson/AZ/United States of America, 2Personal Genome Diagnostics, Baltimore/MD/United States of America Background: An ALK genetic translocation event occurs in w2-7% of non-small cell lung carcinoma patients (NSCLC), resulting in the constitutive expression of an active chimeric ALK protein, which leads to tumor proliferation. Ventana has developed a fully automated
Abstracts
S515
immunohistochemistry (IHC) assay using the VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody (ALK (D5F3)) to detect the ALK protein in formalin fixed paraffin embedded tissue. ALK IHC testing is becoming more widespread in NSCLC; however, information concerning the impact of pre-analytical conditions on the ALK antigen is limited. We used human cell lines expressing ALK, generated as xenografts to evaluate the effect of Fixation Type, Time, and Delay to Fixation (Ischemia) on the ALK antigen as measured by the ALK (D5F3) antibody staining intensity. Methods: NCI-H2228 human NSCLC cell lines were used to generate xenograft tumors in SCID mice. In order to assess ischemia (delay to fixation) H2228 xenografts were used to model ischemia in 10% NBF. The impact of fixation on staining performance of the ALK (D5F3) primary antibody was assessed using the H2228 xenografts to model fixation type and fixation time for 10% NBF, Zinc Formalin, 95% Alcohol, Alcoholic Formalin Acid, B5, and Prefer for 1, 6, 12, 24 and 72 hours. Each of the stained slides were evaluated by a pathologist using a qualitative assessment and scored on a 0-3+ intensity scale (0 ¼ no staining detected, 1+¼ weak staining detected, 2+¼ moderate staining detected, 3+ ¼ strong staining detected). Results: Fixation in all time points in B5, Prefer, and AFA, as well as ethanol, severely compromised staining intensity of ALK (by decreasing staining intensity greater than 0.5 points). Ischemia greater than 6 hours also decreased staining intensity. In contrast, EGFR (5B7) and TTF1 (SP141) antigens were robust across a wide range of fixation times and types, as well as ischemia times. Conclusion: The ALK antigen is highly sensitive to fixative time, type and ischemia. The data indicate that the detection of ALK by IHC is impacted by fixation conditions. Strikingly, antigens detected by other lung antibodies (EGFR, TTF1) are more robust in comparison across a range of conditions. Standardized pre-analytical conditions are critical to achieve appropriate staining for ALK IHC and to mitigate the risk of false negative results. The recommendations for pre-analytical conditions for ALK are to fix at least 6 hours in 10% neutral buffered formalin. Keywords: ALK, IHC, Fixation
P1.02-047 Effect of Dasatinib on EMT-MediatedMechanism of Resistance against EGFR Inhibitors in Lung Cancer Cells Topic: Driver Genes in NSCLC, Resistance, and Other Yuichi Sesumi, Kenichi Suda, Hiroshi Mizuuchi, Yoshihisa Kobayashi, Masaya Nishino, Masato Chiba,
Results: Seven cases (7.3%) showed discordant results with ALK FISH-positive and IHC negative. Among these cases the mean number of FISH positive rearranged nuclei was 40.2% (range 20-54%). All cases showed coexistent split signals pattern positivity with mean percentage 19.1% (range 10-32%.), 5’ deletions pattern positivity with mean percentage 21.7% (range 12-34%) and one case also had gene amplifications pattern positivity with percentage of 64%. The polysomy was observed in all cases with mean percentage of 45.7% (range of 16-72%). Five patients with discordant ALK FISH and IHC results received crizotinib; of them, four progressed and one has stable disease. Conclusion: In most of discordant cases, a coexistent complex pattern of rearrangements (deleted, inverted and amplified/polysomic patterns) of ALK positive cells in FISH analysis was observed; these complex rearranged cases were not detectable by IHC, probably due to the lack of protein expression. Considering that crizotinib inhibits the ALK protein and not specifically ALK rearrangements, it could be speculated that NSCLCs without IHC ALK expression could be not sensitive to crizotinib. The ALK FISH+/IHC negative discordant group showed a lower percentage of nuclei positive for ALK rearrangement (19.1% in split signals and 21.7% in 5’ deletions) as compared with 64% of gene amplification in one case and with polysomy (45.7%) observed in all cases. These preliminary data highlight the role of IHC analysis and of the percentage of the genetic pattern of ALK rearranged cells in FISH analysis to select patients for anti ALK treatment. Further investigation is suggested about the correlation of complex mutational findings and clinical outcome. Keywords: ALK rearrangements, advanced NSCLC, IHC/ FISH discordance
P1.02-046 ALK IHC is Highly Sensitive to Fixation Parameters Topic: Driver Genes in NSCLC, Resistance, and Other Isabell Loftin,1 Rachel Miller,1 Patricia Thorne-Nuzzo,1 Abigail Mcelhinny,2 Penny Towne,1 Shalini Singh,1 June Clements1 1 Ventana Medical Systems Inc., A Member of the Roche Group, Tucson/AZ/United States of America, 2Personal Genome Diagnostics, Baltimore/MD/United States of America Background: An ALK genetic translocation event occurs in w2-7% of non-small cell lung carcinoma patients (NSCLC), resulting in the constitutive expression of an active chimeric ALK protein, which leads to tumor proliferation. Ventana has developed a fully automated
Abstracts
S515
immunohistochemistry (IHC) assay using the VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody (ALK (D5F3)) to detect the ALK protein in formalin fixed paraffin embedded tissue. ALK IHC testing is becoming more widespread in NSCLC; however, information concerning the impact of pre-analytical conditions on the ALK antigen is limited. We used human cell lines expressing ALK, generated as xenografts to evaluate the effect of Fixation Type, Time, and Delay to Fixation (Ischemia) on the ALK antigen as measured by the ALK (D5F3) antibody staining intensity. Methods: NCI-H2228 human NSCLC cell lines were used to generate xenograft tumors in SCID mice. In order to assess ischemia (delay to fixation) H2228 xenografts were used to model ischemia in 10% NBF. The impact of fixation on staining performance of the ALK (D5F3) primary antibody was assessed using the H2228 xenografts to model fixation type and fixation time for 10% NBF, Zinc Formalin, 95% Alcohol, Alcoholic Formalin Acid, B5, and Prefer for 1, 6, 12, 24 and 72 hours. Each of the stained slides were evaluated by a pathologist using a qualitative assessment and scored on a 0-3+ intensity scale (0 ¼ no staining detected, 1+¼ weak staining detected, 2+¼ moderate staining detected, 3+ ¼ strong staining detected). Results: Fixation in all time points in B5, Prefer, and AFA, as well as ethanol, severely compromised staining intensity of ALK (by decreasing staining intensity greater than 0.5 points). Ischemia greater than 6 hours also decreased staining intensity. In contrast, EGFR (5B7) and TTF1 (SP141) antigens were robust across a wide range of fixation times and types, as well as ischemia times. Conclusion: The ALK antigen is highly sensitive to fixative time, type and ischemia. The data indicate that the detection of ALK by IHC is impacted by fixation conditions. Strikingly, antigens detected by other lung antibodies (EGFR, TTF1) are more robust in comparison across a range of conditions. Standardized pre-analytical conditions are critical to achieve appropriate staining for ALK IHC and to mitigate the risk of false negative results. The recommendations for pre-analytical conditions for ALK are to fix at least 6 hours in 10% neutral buffered formalin. Keywords: ALK, IHC, Fixation
P1.02-047 Effect of Dasatinib on EMT-MediatedMechanism of Resistance against EGFR Inhibitors in Lung Cancer Cells Topic: Driver Genes in NSCLC, Resistance, and Other Yuichi Sesumi, Kenichi Suda, Hiroshi Mizuuchi, Yoshihisa Kobayashi, Masaya Nishino, Masato Chiba,