P2.09-35 Proposal to Revise VENTANA ALK Scoring Interpretation Guide for Non-Small Cell Lung Carcinoma: Interpretation of ALK Heterogeneity

Abstract

A small number of non-small cell lung cancer (NSCLC) cases showed heterogeneity of anaplastic lymphoma kinase (ALK) by VENTANA immunohistochemistry (IHC) in clinical practice. According to the ALK Scoring Interpretation Guide for VENTANA anti-ALK (D5F3), the presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) is called positive for ALK. However, we know little about ALK heterogeneous cases. Multiple detection platforms are used to analyze molecular variability and pathological features in anticipation of clinical treatment decisions for such cases. A total of 2228 NSCLC cases with successful ALK IHC (VENTANA, D5F3, Roche) detection in Guangdong Provincial People`s Hospital were recruited between January 2012 and April 2018. Positive and negative system control and a negative agent control were established for each case. ALK IHC positivity was defined as the presence of strong granular cytoplasmic staining in 100% tumor cells; ALK IHC heterogeneity as the presence of strong granular cytoplasmic staining in 1-99% tumor cells; ALK IHC negativity as no tumor cells show strong granular cytoplasmic staining. Fluorescence in-situ hybridization (FISH) (Vysis ALK Break Apart FISH Probe Kit, Abbott) and next-generation sequencing (NGS) (OseqTMLung Cancer Gene Detection, BGI, China) was performed for cases showed ALK (D5F3) IHC heterogeneity. ALK (D5F3) double-blind review analysis showed 201 (9.0%) ALK-positive cases, 10 (0.4%) ALK-heterogeneous cases, and 2017 (90.5%) ALK-negative cases. The heterogeneity cases included 2 large cell neuroendocrine carcinomas, 1 lymphoid epithelioid carcinoma, and 7 squamous cell carcinomas. The percentages of tumor cells with strong granular cytoplasmic staining were 1% to 30%. The ALK FISH break apart signal of these ten cases were 0% to 12%, indicating ALK FISH negativity. Nine ALK-heterogeneous cases were successfully detected by NGS, and no ALKgene variations (including gene fusion, copy number variation, insertion/deletion or single nucleotide variation) were found. Immunohistochemical staining showed that some ALK-heterogeneous cases showed neuroendocrine differentiation.TableComparison of IHC, FISH and NGS results of the ALK-heterogeneous cases.Case No.GenderAgeBiopsy/surgeryDiagnosisTumor cell contentALK IHCALK FISHALK FISHALK NGSALK NGS8thTNMStrong positive staining tumor cellsBreak apart signalInterpretation resultALK fusionALK INDEL/SNV/CNV1M69Wedge resectionLarge cell neuroendocrine carcinoma70%2%0%NegativeNegativeNegativepT1bN0M02M55LobectomyLarge cell neuroendocrine carcinoma85%20%4%NegativeNegativeNegativepT2bN0M03F33LobectomyLymphoid epithelioid carcinoma60%5%0%NegativeNegativeNegativepT3N0M04M68LobectomySquamous cell carcinoma70%5%6%NegativeNegativeNegativepT3N0M05M69LobectomySquamous cell carcinoma70%5%12%NegativeNegativeNegativepT2aN0M06M52LobectomySquamous cell carcinoma80%1%0%NegativeNegativeNegativepT2aN1M07M73LobectomySquamous cell carcinoma90%5%2%NegativeNegativeNegativepT3N0M08M70LobectomySquamous cell carcinoma80%5%4%NegativeNegativeNegativepT2bN1M09M69BiopsySquamous cell carcinoma85%15%0%NegativeNegativeNegativesT3N1M010M60BiopsySquamous cell carcinoma85%30%0%NegativeNANAcT2bN3M0Abbreviations: INDEL, insertion and deletion; SNV, single nucleotide variation; CNV, copy number variation; NA, NGS was not performed due to insufficient tumor tissues. Open table in a new tab Abbreviations: INDEL, insertion and deletion; SNV, single nucleotide variation; CNV, copy number variation; NA, NGS was not performed due to insufficient tumor tissues. Multi-platform detection of ALK-heterogeneous cases did not show evidence of ALKgene variation, and the effect of ALK-TKI treatment was unknown. therefore, the VENTANA ALK scoring interpretation guide for non-small cell lung carcinoma should be revised. It is recommended that ALK-heterogeneous cases be defined as ALK (D5F3) uncertain equivocal cases. The molecular pathology report should clearly state that the clinical significance is unclear, and it is recommended to conduct further testing by FISH or NGS.