Chestclinic

CASE BASED DISCUSSIONS

A patient with complex multiple genomic ALKalterationsXin Liu,1 Shawn J Rice,1 Carlos A Jamis-Dow,2 Catherine Abendroth,3 Siraj Ali,4

Salah Almokadem,1 Chandra P Belani1

1Penn State Hershey CancerInstitute, Penn State College ofMedicine, Hershey,Pennsylvania, USA2Department of Radiology,Penn State Milton S. HersheyMedical Center, Hershey,Pennsylvania, USA3Department of Pathology,Penn State College ofMedicine, Hershey,Pennsylvania, USA4Foundation Medicine,Cambridge, Massachusetts,USA

Correspondence toDr Chandra P Belani,Department of Medicine, PennState College of Medicine, 500University Dr., Hershey, PA17033, USA;cbelani@hmc.psu.edu.

XL and SJR contributedequally.

Received 16 October 2015Revised 11 December 2015Accepted 22 December 2015

To cite: Liu X, Rice SJ,Jamis-Dow CA, et al. ThoraxPublished Online First:[please include Day MonthYear] doi:10.1136/thoraxjnl-2015-207950

INTRODUCTIONLung cancer is a disease with a heterogeneous com-plement of mutations.1 Although point mutationsand deletions are among the most common typesof mutations in lung cancer, translocations in theALK gene, which occur in approximately 5% oflung adenocarcinomas, exist predominantly in non-smokers. ALK translocations gained notorietyrecently because they are targets for the kinaseinhibitor crizotinib (Xalkori).1 Crizotinib has exhib-ited profound efficacy and has obtained FDA(United States Food and Drug Administration)approval for use in patients with non-small celllung cancer (NSCLC) with ALK translocation, asdetermined by a break-apart fluorescent in situhybridisation (FISH) assay.2 Here we report a casewhere a patient with a complicated ALK genotype,including an EML4-ALK variant 5a/b translocationand ALK tandem duplication with response to cri-zotinib treatment.

CASE PRESENTATIONA 70-year-old female patient with complaints ofprogressive dyspnoea underwent a chest CT scan,which revealed a 6 cm spiculated mass with extrinsiccompression of the trachea and the right main stembronchus. PET-CT (positron emission tomography-CT) scan confirmed the findings of the CT scan andthe mass was metabolically active, and there waspresence of metastases in the lymph nodes.Histological evaluation along with immunostainingrevealed primary lung adenocarcinoma. An MRI ofthe brain revealed nodular intraparenchymal meta-static deposits in the left cerebellar hemisphere, leftinferior cerebellar vermus and the left superior par-ietal cortex. The patient received palliative radiationto the lung mass and gamma knife treatment for thebrain metastasis. Genetic profiling performed on thebiopsied tissue using Foundation Medicines’ com-prehensive genomic profiling, reported alterationsin eight genes including premature stop codons inTP53, ARID1A, BRD4 and SETD2, single nucleotidepolymorphisms in the TERT promoter, MAP2K4,U2AF1 and an EML4-ALK translocation. Closerevaluation of the sequencing data showed a complexgenomic environment around the ALK gene, whichincluded a rare EML4-ALK variant 5a/b transloca-tion (<2% of EML4-ALK translocations in lungcancer2) and a tandem duplication of the ALK genewith breakpoints in ALK exon 19 and LOC728730intron 3 (figure 1A teal and green bar, respectively).The tissue was subsequently submitted for FISHanalysis to confirm an EML4-ALK translocation.

Approximately 65% of the cells were positive for anEML4-ALK translocation, which correlated with thesequencing results (figure 1B, C).Based on the presence of the EML4-ALK trans-

location, the patient was treated with crizotinib(Xalkori) 250 mg daily. The patient tolerated thetreatment very well; no adverse events werereported. A follow-up CT after 5 months oftherapy showed a partial response to the crizotinibtreatment indicated by a decrease in tumour sizefrom 5.9×3.2 cm, following radiation therapy, to2×1.4 cm post-treatment (figure 1D). The patientcontinues to respond to the crizotinib treatment,but brain metastases have recurred, which is knownto occur despite crizotinib treatment.3 The patientwill undergo a second round of gamma knifetherapy for the brain metastases.

DISCUSSIONCrizotinib has achieved remarkable clinical successin patients with lung cancer with ALK transloca-tions. ALK translocations can occur with variouspartners, but the most common translocation inlung cancer occurs with echinodermmicrotubule-associated protein like 4 (EML4)protein. Many EML4-ALK translocations variantshave been reported, each involve the positioning ofthe ALK kinase domain (exons 20–29) downstreamof EML4 gene at different breakpoints. Althoughthe kinase domain of ALK drives signalling, theEML4 coiled–coiled domain is thought to beimportant for homodimerisation and stability ofthe fusion protein. Preclinical models evaluatingthe most common EML4-ALK variants 1, 2 and 3a/b have indicated that these variants can show differ-ential sensitivities to crizotinib.4 A recent small(n=61) retrospective study reported no differencein objective response rate or progression-free sur-vival with crizotinib treatment between the differ-ent EML4-ALK variants.5 It is clear that furtherclinical investigations are warranted to provide amore clear view of how EML4-ALK variantresponds to ALK-inhibitors.Here we report the case of a patient with lung

adenocarcinoma who had a tumour displaying acomplex genomic organisation around the ALKgene as determined by comprehensive genomic pro-filing. FISH analysis correlated with the genomicprofiling by detecting an ALK translocation. Basedon the sequencing and FISH data, crizotinib seemedto be an ideal treatment for this patient. However,little was known regarding how the genetic com-plexity of the tumour would affect its response to

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crizotinib. It has been noted that increased ALK copy number isassociated with crizotinib resistance, therefore the magnitude andduration of the patient’s response to the treatment was unclear inspite of being positive for an ALK translocation by FISH. Prior tothis report, little was known about how a tumour with theEML4-ALK variant 5a/b translocation, which occurs in <2% oflung cancers,2 would respond to crizotinib in vivo. This variant isamong the shortest, fusing EML4 exon 2 to exon 20 of the ALKgene. Animal data suggests that this variant has the ability totransform cells and should respond to the treatment2; however,this case was complicated by the presence of tandem duplicationswithin the ALK gene.

This case highlights the applicability of next-generationsequencing (NGS) for identifying patients carrying tumourswith EML4-ALK translocations and other genetic variations inthe ALK genomic region. Although FISH is an approved stand-ard method to identify ALK alterations, NGS offers expandedfunctionality by detecting translocations in addition tosequence information from multiple other genomic regions,which could be applicable to the treatment of patients. In thepresent case, eight genetic alterations were identified in the

patient by NGS compared with only ALK rearrangement byFISH. The additional mutation information could help todirect treatment if alterations in genes known to provide resist-ance to crizotinib are detected. Additionally, tumours that arenegative for ALK translocations by FISH can actually possesscrizotinib sensitive ALK translocations. Here, we report that atumour with a complex ALK genotype, identified by NGS, wassensitive to crizotinib.

It should be noted that although the primary tumourresponded to the crizotinib, new brain metastases developedduring the course of treatment. Development of brain metasta-ses is a common occurrence despite crizotinib treatment, whichmay be a result of the drugs poor blood–brain barrier perme-ability.3 While on crizotinib, patients can develop new brainmetastases as a result of acquired drug resistance.3

Acknowledgements We would like to thank Darlene Knutson, Sara Kloft-Nelsonand Dr Patricia T. Greipp, DO, from the Mayo Cytogenetics Core, who performed theALK FISH analysis, and Eric Sanford of Foundation Medicine.

Competing interests SA is an employee and has equity interest in FoundationMedicine.

Figure 1 Molecular characterisationof the lung adenocarcinoma casepresented. (A) Sequencing datashowing the EML4-ALK translocation(teal reads in left and right panels) andtandem duplication (green reads in leftand centre panels). (B) Fluorescent insitu hybridisation analysis performed atMayo Cytogenetics Core Facility. Thewhite box indicates the area of theinlay, which shows a cell with anormal ALK allele (yellow, combinedgreen and red probes) and atranslocated ALK allele (separate greenand red probes). (C) A graph showingthe patients response to crizotinib.Zero indicates the day the patientstarted crizotinib treatment. (D) Thepatient’s response to crizotinibtherapy. A representative CT scan ofthe patient’s EML4-ALK-positivetumour prior to radiation treatment(preradiation), following radiationtreatment (postradiation) and5 months after crizotinib treatment(post-crizotinib).

2 Liu X, et al. Thorax 2016;0:1–3. doi:10.1136/thoraxjnl-2015-207950

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Patient consent Obtained.

Provenance and peer review Not commissioned; externally peer reviewed.

REFERENCES1 Govindan R, Ding L, Griffith M, et al. Genomic landscape of non-small cell lung

cancer in smokers and never-smokers. Cell 2012;150:1121–34.2 Shaw AT, Engelman JA. ALK in lung cancer: past, present, and future. J Clin Oncol

2013;31:1105–11.

3 Costa DB, Shaw AT, Ou SH, et al. Clinical Experience With Crizotinib in Patients WithAdvanced ALK-Rearranged Non-Small-Cell Lung Cancer and Brain Metastases. J ClinOncol 2015;33:1881–8.

4 Heuckmann JM, Balke-Want H, Malchers F, et al. Differential protein stability andALK inhibitor sensitivity of EML4-ALK fusion variants. Clin Cancer Res2012;18:4682–90.

5 Lei YY, Yang JJ, Zhang XC, et al. Anaplastic lymphoma kinase variants and thepercentage of ALK-positive tumor cells and the efficacy of crizotinib in advancedNSCLC. Clin Lung Cancer 2015. doi:10.1016/j.cllc.2015.09.002

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