Anaplastic Lymphoma Kinase FISH Research Articles

ALK immunohistochemistry positive, FISH negative NSCLC is infrequent, but associated with impaired survival following treatment with crizotinib

ObjectiveMetastasized non-small cell lung cancer (NSCLC) with an anaplastic lymphoma kinase (ALK) rearrangement is usually sensitive to a range of ALK-tyrosine kinase inhibitors. ALK-positive NSCLC have been identified in pivotal phase III trials with fluorescence in situ hybridization (ALK FISH+). These tumors are also expressing the fusion product (ALK immunohistochemistry (IHC)+). However, discrepant cases occur, including ALK IHC + FISH-. The aim of this study was to collect ALK IHC + cases and compare within this group response to crizotinib treatment of ALK FISH + cases with ALK FISH- cases. Materials and methodsIn this European prospective multicenter research study patients with Stage IV ALK IHC + NSCLC treated with crizotinib were enrolled. Tumor slides were validated centrally for ALK IHC and ALK FISH. ResultsRegistration of 3523 ALK IHC tests revealed a prevalence of 2.7% (n = 94) ALK IHC + cases. Local ALK FISH analysis resulted in 48 concordant (ALK IHC+/FISH+) and 16 discordant (ALK IHC+/FISH-) cases. Central validation revealed 37 concordant and 7 discordant cases, 5 of which had follow-up. Validation was hampered by limited amount of tissue in biopsy samples. The PFS at 1 year for ALK concordant and discordant was 58% and 20%, respectively (HR = 2.4; 95% CI: 0.78–7.3; p = 0.11). Overall survival was significantly better for concordant cases than discordant cases after central validation (HR=4.5; 95% CI= 1.2–15.9; p=0.010. ConclusionALK IHC + FISH- NSCLC is infrequent and associated with a worse outcome on personalized treatment. A suitable predictive testing strategy may be to screen first with IHC and then confirm with FISH instead of considering ALK IHC equivalent to ALK FISH according to the current guidelines.

Clinical performance of endobronchial ultrasound-guided transbronchial needle aspiration for assessing programmed death ligand-1 expression in nonsmall cell lung cancer.

Pembrolizumab was recently approved as a first line agent for metastatic NSCLC in patients with high programmed death-ligand 1 (PD-L1) expression. Since a significant portion of lung cancer is diagnosed by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS TBNA); there is a need for PD-L1 testing in these specimens. However, to date few studies have evaluated performance of cytology specimens from EBUS TBNA for PD-L1 analysis. Patients who had a diagnosis of NSCLC and in whom ancillary testing, i.e., next generation sequencing (NGS), anaplastic lymphoma kinase (ALK), and PD-L1 expression was requested between January and May 2017 were reviewed. Fifty of the 112 patients reviewed had the diagnosis of NSCLC for which ancillary testing was requested. Twelve patients (24%) had squamous cell carcinoma, twenty-seven had adenocarcinoma (54%), five had NSCLC favor adenocarcinoma (10%), two had NSCLC favor squamous cell cancer (4%), and four had NSCLC not otherwise specified (NOS) (8%). Size of the lymph nodes or lesion sampled ranged from 10 to 50 mm. Four (8%) patients had insufficient number of tumor cells in the cell block for any of the ancillary molecular testing. Forty-one (82%) patients had an adequate sample for all three ancillary tests. Satisfactory results for PD-L1 expression for all cases was 86% with 14 (32%) patients having levels of PD-L1 expression >50%. EBUS TBNA is effective and has a high proportion of satisfactory results for testing PD-L1 expression on tumor cells in addition to NGS and ALK FISH.

Equivocal ALK fluorescence in-situ hybridization (FISH) cases may benefit from ancillary ALK FISH probe testing.

Accurate assessment of anaplastic lymphoma kinase (ALK) gene rearrangement in non-small-cell lung cancers (NSCLCs) is critical to identify patients who are likely to respond to crizotinib. The aim of this study was to evaluate the ALK/EML4 TriCheck FISH probe in a series of NSCLCs enriched for tumours with equivocal ALK status. ALK FISH was prospectively performed on 45 NSCLCs with the ALK/EML4 TriCheck probe (ZytoVision) and the Vysis ALK break-apart probe (Abbott Molecular). ALK immunohistochemistry was performed with 5A4 and D5F3 antibodies. Fourteen cases had equivocal ALK status, based on borderline or focal FISH positivity, an atypical FISH pattern, or discrepancy between ALK FISH and immunohistochemistry. Four of the 14 equivocal cases showed discordance between the two FISH probes. All other cases were concordant. The TriCheck probe showed that, of 31 unequivocal cases, 15 were ALK-rearranged, and 60% of these had EML4 as the translocation partner. Within the group of 14 equivocal cases, 12 showed rearrangement with the Tricheck probe; only one of these showed EML4 rearrangement. Of the six equivocal cases that received crizotinib, four showed clinical benefit. The ALK/EML4 TriCheck FISH probe may be useful for the detection of ALK rearrangements, especially in borderline or atypical cases, where an additional unique ALK FISH probe may provide further confirmation of rearrangement.

Abstract P3-06-39: Anaplastic lymphoma kinase (ALK) protein overexpression is not a feature of inflammatory breast cancer

Abstract Recent studies suggest that anaplastic lymphoma kinase (ALK) could serve as a therapeutic target for inflammatory breast cancer (IBC) and that strategies targeting ALK should be considered for evaluation in clinical trials. Reverse phase protein arays revealed activation of the ALK receptor tyrosine kinase and biochemically-linked downstream signalling molecules in pre-clinical models of IBC. ALK genetic abnormalities have been reported in 80% (20/25) of IBC patients with a high prevalence of ALK alterations in basal-like IBC (Robertson F. et al., 2013). However, ALK protein expression has not been studied in human IBC samples. Immunohistochemical detection of the ALK protein is increasingly being used as a screening tool to test samples for ALK rearrangements. The biological premise is that the genetic abnormality of the ALK gene leads to overexpression of the ALK protein and therefore overactivity of the ALK tyrosine kinase; since this kinase is the target of crizotinib, it makes sense to assess the drug target directly. Formalin fixed paraffin embedded tissue samples from pre-treatment surgical biopsies of 79 consecutive IBC patients were immunohistochemically (IHC) tested for ALK protein over-expression using a validated IHC test (NCL-ALK, clone 5A4) with a sensitivity of 93% and a specificity of 100% when the Vysis ALK-FISH break apart test is used as a gold standard. Stained tissue sections were evaluated using the IHC histoscore proposed by Thunnissen E. et al., 2012, assessing both the proportion of tumor cells showing cytoplasmic staining and the staining intensity. Cytoplasmic staining of appendiceal ganglion cells was used as an on-slide external positive control; weak to moderate cytoplasmic staining of macrophages was used as an internal positive control. In 75 of the 79 tissue samples none of the tumor cells showed any ALK immunoreactivity. One tumor showed moderate cytoplasmic staining in a few cells (<1%, score 2+); ALK (2p23) FISH showed no rearrangement. This tumor was hormone receptor negative and HER2 negative. Three tumors showed minimal cytoplasmic staining in a few cells (<1%, score 1+). These results demonstrate that ALK protein overexpression is not a feature of IBC. Although genetic abnormalities of ALK without protein overexpression are not excluded, our results show that ALK IHC cannot be used to identify IBC patients eligible for enrollment in clinical trials evaluating ALK targeted therapeutics. Citation Format: Cecile Colpaert, Melike Marsan, Peter Vermeulen, Luc Dirix, Steven Van Laere, Inflammatory Breast Cancer International Consortium. Anaplastic lymphoma kinase (ALK) protein overexpression is not a feature of inflammatory breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-39.

Anaplastic Lymphoma Kinase Immunocytochemistry on Cell-Transferred Cytologic Smears of Lung Adenocarcinoma

Background: Anaplastic lymphoma kinase (ALK) immunohistochemical staining on formalin-fixed paraffin-embedded tissue or cell blocks (CB) has been reported as an effective alternative to fluorescence hybridization in situ (FISH) for the detection of ALK gene rearrangement. However, CB frequently lack adequate cellularity even when the direct smears are cellular. This study aims to assess the utility of ALK immunocytochemical (ICC) staining on direct smears using the cell transfer (CT) technique for the detection of ALK rearrangement. Methods: Fine-needle aspiration (FNA) cases of lung adenocarcinoma in which the ALK status had been determined by FISH on CB or a concurrent biopsy were identified. ICC staining for ALK was performed on alcohol-fixed Papanicolaou-stained direct smears using the CT technique. ALK immunoreactivity was evaluated using a modified semiquantitative scale. Results were compared with those of FISH. Results: A total of 47 FNA specimens were included. Five of 7 FISH-positive cases showed positive ALK ICC staining (71.4%), and 39 of 40 FISH-negative cases were negative on ALK ICC staining (97.5%). The overall correlation between ALK ICC and FISH was 93.6%. Conclusion: ICC performed on FNA smears using the CT technique is an alternative method for the assessment of ALK rearrangement, especially when CB lack adequate cellularity.

ALK Rearrangements Are Mutually Exclusive with Mutations in EGFR or KRAS: An Analysis of 1,683 Patients with Non–Small Cell Lung Cancer

Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of non-small cell lung cancer (NSCLC). Recently, several case reports and small series have reported that ALK rearrangements can overlap with other oncogenic drivers in NSCLC in crizotinib-naïve and crizotinib-resistant cancers. We reviewed clinical genotyping data from 1,683 patients with NSCLC and investigated the prevalence of concomitant EGFR or KRAS mutations among patients with ALK-positive NSCLC. We also examined biopsy specimens from 34 patients with ALK-positive NSCLC after the development of resistance to crizotinib. Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. Four patients with KRAS mutations were found to have abnormal ALK FISH patterns, most commonly involving isolated 5' green probes. Sufficient tissue was available for confirmatory ALK immunohistochemistry in 3 cases, all of which were negative for ALK expression. Among patients with ALK-positive NSCLC who acquired resistance to crizotinib, repeat biopsy specimens were ALK FISH positive in 29 of 29 (100%) cases. Secondary mutations in the ALK kinase domain and ALK gene amplification were observed in 7 of 34 (20.6%) and 3 of 29 (10.3%) cases, respectively. No EGFR or KRAS mutations were identified among any of the 25 crizotinib-resistant, ALK-positive patients with sufficient tissue for testing. Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK-positive cancers with acquired resistance to crizotinib.

A retrospective analysis of the prevalence of EGFR or KRAS mutations in patients (pts) with crizotinib-naïve and crizotinib-resistant, ALK-positive non-small cell lung cancer (NSCLC).

8083 Background: Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of NSCLC. Recently, several studies have reported that ALK+ pts occasionally harbor concomitant mutations in other oncogenic drivers. Methods: We retrospectively analyzed tumor genotyping data from 1,683 pts with NSCLC seen at 3 U.S. centers from 2009 – 2012 to determine rates of overlapping alterations in EGFR, KRAS and ALK. Mutations in EGFR and KRAS were mainly identified using the SNaPshot multiplexed assay (>95% of cases). ALK FISH was performed in all cases. To determine if this prevalence is impacted by crizotinib, we also updated our earlier analysis (Katayama et al., Sci Transl Med, 2012) of a series of repeat biopsy specimens from 34 crizotinib-resistant, ALK+ pts. Resistant specimens were examined using ALK FISH, SNaPshot, and direct sequencing of the ALK tyrosine kinase domain (TKD). Results: Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. 4 pts with KRAS mutations also had abnormal ALK FISH patterns, involving isolated 5’ green probes (3/4 cases) and an isolated 3’ red probe that was unusually small (1/4 cases). Sufficient tissue was available for confirmatory ALK immunohistochemistry (clone 5A4, Novacastra, UK) in 3 of these cases, all of which were negative for ALK expression. Among pts with ALK+ NSCLC and acquired crizotinib resistance, repeat biopsy specimens remained ALK fusion positive in 28/28 (100%) cases. Secondary mutations in the ALK TKD (1151Tins, L1196M, G1202R, S1206Y, and G1269A) were identified in 7/34 (20.6%) cases. L1196M was the most common secondary mutation (3/34, 8.8% cases). ALK gene amplification was present in 3/28 (10.71%) pts. No EGFR or KRAS mutations were identified in 23 crizotinib-resistant, ALK+ pts with sufficient tissue for testing. Conclusions: Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK+ pts with acquired resistance to crizotinib.

EML4-ALK mutation in Austrian patients with NSCLC: A multicenter study.

e19093 Background: EML4 (echinoderm microtubule-associated protein-like 4) - ALK (anaplastic lymphoma kinase) fusion-type tyrosine kinase, an oncoprotein found in a subgroup of non-small-cell lung cancer (NSCLC) predicts the response to ALK inhibitors. In general, AML4-ALK mutation is found in 2 to 7% of Caucasian patients with NSCLC and occurs more often in never and former smokers, adenocarcinomas, and younger, male patients. However, the frequency of EML4-ALK mutation in Austrian patients with NSCLC is unknown. The aim of the study was to evaluate the prevalence of EML4-ALK mutation in Austrian patients with NSCLC. Methods: From September 2011 to October 2012 tumour tissue from bronchoscopy, CT- and ultrasound guided biopsies and surgical specimen with histological type of adenocarcinoma and NSCLC NOS (Not Otherwise Specified) excluding squamous cell carcinoma, large cell carcinoma and neuroendocrine carcinoma were analysed for EML4-ALK mutations from 4 hospitals in Austria with high expertise in the management of lung cancer. Mutation detection was performed with a two-step procedure. First an immunhistochemical staining was done (ALK confirm/Ventana) and further on positive cases were tested by ALK FISH (dual colour breakapart FISH/Abbott Vysis). Results: In total 639 patients were analysed. EML4-ALK positive immunohistochemical staining was found in 35 patients (5,48%). 14 of these patients (2,19%) showed positive ALK FISH analysis. Conclusions: Frequency of EML-ALK mutations in Austrian patients with NSCLC was similar to other Caucasian peers.

Abstract 4466: Integrative study of dysregulated genes in translocated ALK-positive non-small cell lung cancer and personalized targeted therapy resistance.

Abstract Background: Recent studies revealed that ALK (anaplastic lymphoma kinase) 2p23 translocation, most commonly fused with echinoderm microtubule-associated protein-like 4 (EML4), is a novel driver oncogene in about 5% of non-small cell lung cancer (NSCLC). ALK 2p23 fusion (ALK+) can be diagnosed by dual color break-part ALK FISH assay to inform personalized targeted therapy using the ALK inhibitor crizotinib. However, the underlying mechanisms of ALK oncogenic signaling and more importantly ALK inhibitor crizotinib resistance are not well understood. Materials and Methods: To understand the mechanism of oncogenic ALK signaling, an ALK+ NSCLC microarray dataset was retrieved from NCBI-GEO data repository (GSE31210) and analyzed using data-mining bioinformatics approaches. This dataset includes the gene expression profiles of 11 ALK+ NSCLC and 20 normal lung tissue samples. Statistical analysis was carried out to identify altered genes in ALK+ NSCLC and Ingenuity Pathway Analysis (IPA) software was deployed to uncover their network interactions. To study the resistance mechanism of the ALK+ H3122 human NSCLC cell line against the specific ALK inhibitor TAE684, real-time polymerase chain reaction (qRT-PCR), cell viability assay and immunoblotting were performed using standard techniques. Results: We identified a set of 1,656 genes that were significantly altered in ALK+ NSCLC, consisting of 662 upregulated and 994 downregulated. The principal component analysis (PCA) based on the top 51 of these dysregulated genes evidently separated ALK+ and normal lung tissue samples, implicating that these genes could represent potential genomic signature of ALK+ NSCLC. The IPA analysis revealed 35 canonical pathways linked to ALK signaling, including beta-adrenergic and angiopoietin signaling pathways. The results of qRT-PCR, cell viability assay and immunoblotting showed that in translocated ALK+ H3122 human NSCLC cell line, HGF (hepatocyte growth factor) overexpression could activate ALK downstream signaling via activation of MET receptor kinase pathway, thus potentially bypassing the oncogenic ALK signaling under ALK inhibitor TAE684 treatment. Hence, alternative direct activation of ALK downstream signaling molecules via MET/HGF axis activation may potentially contribute to ALK inhibitor drug resistance. Conclusion: Using bioinformatics data-mining, we have identified dysregulated genes and pathways in ALK+ NSCLC, many of which are novel biomarkers not previously reported. We also verified the relevance and potential key role of MET/HGF signaling axis in ALK inhibitor resistance. Taken together, our results highlighted the cross-talk between ALK and MET signaling in NSCLC and shed new insights into possible pitfalls of potential specific ALK targeting inhibitor drugs. Citation Format: Ivy Shi, Lihong Yin, Patrick C. Ma. Integrative study of dysregulated genes in translocated ALK-positive non-small cell lung cancer and personalized targeted therapy resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4466. doi:10.1158/1538-7445.AM2013-4466

Abstract 3853: Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma.

Abstract Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma that includes two entities ALK+ and ALK- ALCL based on chromosomal translocations of the anaplastic lymphoma kinase (ALK). ALK- ALCL have very poor clinical outcome with conventional chemotherapy. Lack of representative cell lines and/or models has compromised the development of successful therapy. Toward this end, we report the generation and characterization of a group of novel ALCL tumor grafts. We implanted subcutaneously (s.c.) tumor tissue fragments, fresh or viable cryopreserved, from 11 cases of primary systemic ALCL (3 ALK+ and 8 ALK-) in six- to eight-week old severe immunocombined immunodeficiency NOD Cg-Prkdcscid/Il2rgtm1wjl/SzJ (NSG) mice. A single leukaemic ALCL sample was injected i.v. or s.c. Six cases led to successful lymphoma growth within 6-12 weeks as subcutaneous solid spheroid masses constituted by a uniform population of large pleomorphic cells CD30-positive. All tumor grafts were re-implanted in recipient animals, in same cases up to 21 consecutive implants with an analogous time growth rate. Two cases, in the same way of the original human neoplasia, were early associated with concomitant enlargement of multiple organs. All tumor graft lines eventually showed disseminate disease with involvement of distant lymph nodes and of spleen (with homig in peri-arteriolar spaces), liver (initially observed within the sinusoidal spaces) and kidney. The morphological and immunohistochemical (IHC) evaluation (expression of CD30, cytotoxic proteins and ALK) of the serially propagated ALCL tumor grafts demonstrated a high concordance between primary and derived animal tumors which showed highly stable profile along different passages from the early generation (T1) until the final ones (up to T16). The clonal relationship with primary and corresponding tumor grafts was confirmed by gene TCR rearrangement analyses and by ALK FISH studies. In addition we interrogated the model using gene expression profiling and single nucleotide polymorphism which established a close relationship among primary and derived tumors. Massive parallel sequencing on mRNA transcripts defines the presence of two novel chimeras involving NF1-MAZ and TRAF1-ALK1, which confirmes the evidence of ALK signaling and pinpoint a role of RAS in ALK- ALCL. The tumor grafts displayed, also, therapeutic responses with conventional therapy (CHOP) which reflected that seen in the donor patients: initial partial responses followed invariably by clinical relapses. To overcome this refractory we assayed the sensitivity of two tumorgraft lines, generated from ALK+ ALCL, to a new selective ALK inhibitor (CEP28122) that led a rapid and stable response. This study demonstrated the key role of these libraries of tumor grafts, especially in developing and testing new therapeutic regimens in refractory ALCL patients. Citation Format: Fabrizio Tabbo’, Antonella Barreca, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Francesco Abate, Michela Boi, Maria Todaro, Cristina Abele, Domenico Novero, Alberto Zamo’, Marco Chilosi, Maurilio Ponzoni, Mangeng Cheng, Bruce Ruggeri, Pierpaolo Piccaluga, Stefano Pileri, Enrico Tiacci, Brunangelo Falini, Lenny Shultz, Roberto Piva, Enzo Medico, Francesco Bertoni, Raul Rabadan, Giorgio Inghirami. Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3853. doi:10.1158/1538-7445.AM2013-3853

Food and Drug Administration Approval of Laboratory Tests

The United States Food and Drug Administration (FDA) recently granted accelerated approval to crizotinib (Xalkori; Pfizer, New York, New York) to treat patients with advanced-stage non–small cell lung cancer (NSCLC) that is positive for anaplastic lymphoma kinase (ALK) fusion genes. Concurrently, the FDA granted accelerated approval for a companion diagnostic test, the Xysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Inc, Chicago, Illinois), designed to detect rearrangements of the ALK gene, which are found in about 5% of patients with NSCLC. Crizotinib comes with ‘‘a hefty price tag’’— $9600 per month, or $115 000 per year; however, ‘‘[w]e are going to be treating patients more effectively and avoiding treatments that don’t work.’’ 1 ‘‘This is the first targeted agent to be developed with an accompanying diagnostic test, said Mace Rothenberg, MD, senior vice president of Pfizer’s Oncology Business Unit.’’ 1 ‘‘‘The Abbott ALK FISH test was developed in conjunction with Pfizer and the clinical trials for crizotinib,’ said Kathryn B. Becker, PhD, director of Abbott Molecular Oncology. ‘It was configured to work specifically in NSCLC patients . . .’’’ 1 (emphasis added). The launch of crizotinib and the companion Abbott ALK FISH test has made FDA-approved laboratory tests a hot topic among pathologists. Food and Drug Administration approval of laboratory tests is new, and raises many questions. How did FDA approval of laboratory tests come about? What laboratory tests, if any, should require FDA approval? What is FDA approval meant to accomplish? What does this change mean to the pathologist? To the patient? Is FDA approval desirable? Political? Inevitable?

Abstract 1715: High ALK gene copy number as a predictor of response to crizotinib in non-small cell lung cancer cell lines

Abstract Introduction: The anaplastic lymphoma kinase (ALK) gene translocation is a rare event, described in approximately 4-6% of lung adenocarcinoma. ALK translocation is a robust predictive factor for ALK inhibitors sensitivity. However, increased ALK gene copy number seems to be a frequent event, described in 13-17% of Non-small cell lung cancer (NSCLC). The goal of this study was to explore the predictive value of ALK High copy number for 2 ALK inhibitors, crizotinib and TAE684 in NSCLC cell lines. Methods: 27 NSCLC cell lines were screened for ALK copy number by FISH after cytospin. ALK high copy number (HCN) was defined as the presence of ≤ 5 copies of ALK. FISH with CEP2 was performed to determine the ploïdy status in cell lines with HCN. As crizotinib is a dual c-MET and ALK inhibitor, FISH c-MET was performed in selected cell lines for in vitro studies. C-MET scoring was based on Cappuzzo criteria. In vitro sensitivity was evaluated through WST1 assays and clonogenic tests. Kelly, an ALK mutated neuroblastic cell line, was added as a positive control. Cells were seeded in triplicates in 96-well plates 18h before the treatment with TAE 684 or crizotinib with concentrations varying from 50 to 4000nM and 100 to 10000nM respectively. The half maximal inhibitory concentration of the two drugs (IC50) was estimated after 72h of treatment with 4 and 5 parameters logistic regression models. In clonogenic tests, crizotinib was tested with three schedules of concentrations: low dose (50nM), intermediate (200nM) and high (1000nM). Results: Among NSCLC cell lines, 6 (22%) displayed more than 5 copies of ALK, 19 (70%) presented a gain of 3 or 4 ALK copy number, only one cell line exhibited normal ALK copies and one harbored EML4-ALK translocation. FISH with CEP2 revealed a polysomy of chromosome 2 in cases with ALK HCN. C-MET amplification was observed in only three cell lines. Based on ALK FISH status, 9 NSCLC cell lines were selected to undergo in vitro assays, 6 exhibiting ALK HCN (H2030, H661, A427, H3255, BEN, H1299) and 3 exhibiting low ALK copy number (LCN) (H1975, H1651, H1650). Kelly and A549 cell lines were added as sensitive and resistant controls respectively. In ALK HCN cell lines, two (H661 and A427) were as sensitive as Kelly for crizotinib through clonogenic and WST1 assays. In addition, ALK HCN cell lines exhibited lower IC50 for anti-ALK drugs than cell lines with ALK LCN (median IC50 with crizotinib values: 1750nM [300-2800nM] in ALK HCN cell lines versus 4500nM [800-8000nM] in ALK LCN cell lines, p=0.35). The trend of crizotinib activity was not due to c-MET inhibition. Conclusion: The in vitro assays suggest that ALK HCN may be a predictive marker for sensitivity to crizotinib. The predictive value of ALK HCN would be investigated in in vivo models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1715. doi:1538-7445.AM2012-1715

Crizotinib and Testing for ALK

Crizotinib was recently approved by the US FDA for the treatment of advanced non-small cell lung cancer (NSCLC) harboring the ALK (anaplastic lymphoma kinase) gene rearrangement. To ensure identification of patients most likely to benefit, the FDA approved crizotinib concurrently with a companion diagnostic test-the Vysis ALK Break Apart FISH Probe Kit. This kit was used in 1 of the 2 pivotal trials leading to the FDA approval of crizotinib and has become the gold standard for detecting ALK rearrangement in NSCLC. Although ALK FISH is clinically validated, the assay can be technically challenging and costly. Therefore, other diagnostic modalities are being explored, including immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction. This article provides an overview of the diagnostic assays available for detecting ALK rearrangement. Each assay, including ALK FISH, has its strengths and weaknesses. Recent work with commercially available ALK antibodies suggests that IHC-based tests may represent a reliable and cost-effective screening strategy; however, large multicenter studies comparing IHC with FISH are needed to validate ALK IHC. While ALK FISH remains the current standard for diagnosing ALK positivity, large-scale screening of patients with newly diagnosed advanced NSCLC, as recommended by NCCN, may require development and validation of alternative screening strategies, such as combination IHC and FISH.

Comparative analyses of overall survival of anaplastic lymphoma kinase (ALK)–positive advanced non-small cell lung cancer (NSCLC) patients who did not receive ALK inhibitors.

7515 Background: There have been limited data on the overall survival (OS) of ALK gene rearranged (ALK-positive) NSCLC patients who did not receive ALK inhibitors. We investigated the OS of advanced ALK-positive NSCLC patients who were managed in the pre-ALK inhibitors era. Methods: This is a retrospective case-case study of ALK-positive patients vs. ALK wildtype (WT) comparators who were treated in Seoul National University Hospital (SNUH), Seoul, Korea. A total of 1,100 stage IIIB/IV patients with non-squamous histology were collected in the NSCLC database of SNUH between 2003 and 2009. To enrich for ALK-positive cases, we performed ALK FISH on 257 cases that were either EGFR WT or non-responders to prior EGFR tyrosine kinase inhibitor (TKI) therapy. We compared survival outcomes between 3 groups of patients: 1) ALK-positive, 2) EGFR mutation positive (EGFR mut+), and 3) ALK WT/EGFR WT. For each comparison, 1 ALK-positive patient was matched to 2 EGFR mut + and 2 ALK WT/EGFR WT patients based on: age at diagnosis, sex, and stage. Progression-free survival (PFS) of 1st-line chemotherapy and EGFR TKIs were also analyzed. Results: Twenty two cases were ALK-positive by FISH and did not receive ALK inhibitors during the follow-up period. The median OS of ALK-positive (n=22), EGFR mut+ (n=44), and ALK WT/EGFR WT (n=44) patients was 10.4, 28.0, and 14.5 months, respectively (P-value; vs. EGFR mut+: 0.012, vs. ALK WT/EGFR WT: 0.384). The PFS of 1st-line platinum-based chemotherapy for the 3 groups was not different. However, the PFS of EGFR TKIs was shorter in ALK-positive patients, compared with the 2 other groups (P-value; vs. EGFR mut+: < 0.001, vs. ALK WT/EGFR WT: 0.048) Conclusions: In the pre-ALK inhibitor era, ALK-positive patients experienced the shortest survival. Although their responses to platinum-based chemotherapy were not different from comparator groups, they were even more resistant to EGFR TKI treatment than were ALK WT/EGFR WT patients.