Clinical Trial Brief Report

Anaplastic Lymphoma Kinase Mutation (ALKF1174C) in Small Cell Carcinoma of the Prostateand Molecular Response to AlectinibBenedito A. Carneiro1, Sahithi Pamarthy2,3, Ami N. Shah4, Vinay Sagar2,3,Kenji Unno2,3, HuiYing Han2,3, Ximing J. Yang5, Rubens B. Costa4, Rebecca J. Nagy6,Richard B. Lanman6, Timothy M. Kuzel7, Jeffrey S. Ross8,9, Laurie Gay8, Julia A. Elvin8,Siraj M. Ali8, Massimo Cristofanilli2, Young K. Chae2,4, Francis J. Giles10, andSarki A. Abdulkadir2,3

Abstract

Purpose: Small cell carcinoma of the prostate (SCCP) is anaggressive disease that can arise de novo or by transdiffer-entiation from prostate adenocarcinoma. Alterations in ana-plastic lymphoma kinase (ALK) gene are involved in neuro-blastoma, lung cancer, and other malignancies, but its role inSCCP has not been documented. We describe a patient withrefractory de novo SCCP with ALK F1174C–activating muta-tion who obtained clinical benefit from treatment with ALKinhibitor.

Experimental Design:Next-generation sequencing (NGS) wasused to analyze primary and circulating tumor DNA (ctDNA).Prostate cancer databaseswere queried for alterations inALK gene,mRNA, and its impact in clinical outcomes. In vitro prostate cellline/organoid models were generated by lentiviral-mediatedexpression of ALK and ALK F1174C and assessed for response toALK inhibitors crizotinib and alectinib.

Results:NGS analysis of the primary tumor and ctDNA of a 39-year-old patient with refractory SSCP identified ALK F1174Cmutation. Treatment with second-generation ALK inhibitor alec-tinib resulted in radiographic stable disease for over 6 months,symptomatic improvement, and significant molecular responseas reflected bydeclining ctDNAallele fraction. Analysis of prostatecancer datasets showed that ALK amplification was associatedwith poor outcome. In prostate cancer cells and organoids, ALKF1174C expression enhanced growth and induced expression ofthe neuroendocrine marker neuron-specific enolase. Alectinibwas more effective than crizotinib in inhibiting ALK F1174C–expressing cell growth.

Conclusions: These findings implicate ALK-activating muta-tions in SCCP pathogenesis and suggest the therapeutic potentialof targeting ALK molecular alterations in some patients withSCCP. Clin Cancer Res; 24(12); 2732–9. �2018 AACR.

IntroductionSmall cell carcinoma of the prostate (SCCP) accounts for 0.5%

to2%of prostate cancers anddisplays an aggressive clinical course

(1). Although many patients present with de novo disease, 40% to50% of SCCP patients have a history of prostate adenocarcinomaand prior androgen-deprivation therapy (ADT; ref. 2). Despiteinitial response to platinum-based chemotherapy, metastaticSCCP has a poor prognosis with median overall survival of 9 to16 months related to molecular disruption of several pathwaysincluding amplification of Aurora Kinase A (AURKA) andMYCNgenes (3, 4). Advances in the understanding of molecular path-ogenesis of SCCP have not translated into meaningful clinicalbenefit to patients. Hence, the important need to identify noveltherapeutic targets.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosinekinase involved in the pathogenesis of neuroblastoma, non–small cell lung cancer (NSCLC) as well as other malignancies,but its importance in SCCP or adenocarcinoma of the prostatehas not been documented (5). Aberrant ALK activation due topoint mutations or gene fusions stimulates kinase activityand oncogenic transformation through downstream signalingpathways including RAS/ERK, JAK/STAT, and PI3K/AKT/MTOR. Although ALK mutations have been described as onco-genic drivers in neuroblastoma and anaplastic thyroid cancer,ALK fusion proteins have been associated with NSCLC andnon-Hodgkin lymphoma (NHL; ref. 6). Specifically, ALKF1174L mutation induces autophosphorylation and constitu-tive activation of ALK and is associated with resistance to ALK

1Division of Hematology/Oncology, Lifespan Cancer Institute, theWarren AlpertMedical School, Brown University, Providence, Rhode Island. 2The Robert H.Lurie Comprehensive Cancer Center, Northwestern University Feinberg Schoolof Medicine, Chicago, Illinois. 3Department of Urology, Northwestern UniversityFeinberg School of Medicine, Chicago, Illinois. 4Developmental TherapeuticsProgram, Division of Hematology/Oncology, Feinberg School of Medicine,Northwestern University, Chicago, Illinois. 5Department of Pathology, North-western University Feinberg School of Medicine, Chicago, Illinois. 6GuardantHealth, Inc., Redwood City, California. 7Rush University Medical Center, Divisionof Hematology/Oncology, Chicago, Illinois. 8Foundation Medicine Inc., Cam-bridge, Massachusetts. 9Upstate Medical University, Syracuse, New York.10Developmental Therapeutics Consortium, Chicago, Illinois.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

B.A. Carneiro and S. Pamarthy contributed equally to this article.

Corresponding Author: B.A. Carneiro, Lifespan Cancer Institute, Brown Uni-versity, 164 Summit St, Fain Building 2nd Floor, Providence, RI 02906. Phone:401-793-7151; Fax: 401-793-7132; E-mail: Benedito.carneiro@lifespan.org

doi: 10.1158/1078-0432.CCR-18-0332

�2018 American Association for Cancer Research.

ClinicalCancerResearch

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tyrosine kinase inhibitors (TKI). Activated full-length ALKregulates transcription of MYCN (gene encoding N-Myc), animportant mediator of neuroendocrine differentiation (4, 7).

Although ALK TKIs are used to treat NSCLC positive for ALKrearrangements such as EML4-ALK, their efficacy in tumors har-boring ALK point mutations has been described only in neuro-blastoma and anaplastic large-cell lymphoma (8). The F1174Cmutation has been associated with resistance to crizotinib andceritinib, but maintained sensitivity to alectinib in patients withNSCLC (9). Comprehensive genomic profiling of 114,200 clinicalcases identified ALK rearrangements in a variety of tumor typesbeyond NSCLC and highlighted ALK as a therapeutic target intumors with small cell and neuronal differentiation (10). Wereport the first case of a potentially oncogenicALK pointmutationin SCCP associated with molecular response to alectinib asreflected by declining cell-free circulating tumor DNA (ctDNA)allele fraction and stable radiologic disease. Database and func-tional analyses in vitro support the role of ALK in the pathogenesisof SCCP.

Materials and MethodscBioportal and CCLE data analysis

To determine the spectrum of genomic alterations andexpression pattern of ALK in prostate cancer patients, datasetsavailable in cbioportal were used (www.cbioportal.org).Patients were stratified based on the presence ALK alterations,and survival data were extracted for available datasets. Muta-tional frequencies of ALK were compared in COSMIC (www.cancer.sanger.ac.uk/cosmic). The gene expression of ALK wascompared among prostate cancer cell lines in Comprehensivecell line encyclopedia cell line (CCLE) database (www.broadinstitute.org/ccle/home) by extracting gene-centric RMA-nor-malized mRNA expression data. ALK gene expression in pros-tate cancer PDX data was analyzed by extracting mRNA expres-sion data deposited in NCBI Geoprofile database with acces-sion number GSE41193.

Liquid biopsy NGSctDNA analysis was performed using a commercially avail-

able digital next-generation sequencing (NGS) assay (Guar-

dant360, Guardant Health, Inc.). Guardant360 is a 73-genectDNA NGS panel from a Clinical Laboratory ImprovementAmendments (CLIA)-licensed, CAP-accredited laboratory. Itprovides complete exon sequencing of 19 cancer genes, criticalexons of 54 genes and amplifications (18 genes), fusions (6genes), and indels (23 genes) with high clinical sensitivity rates(85% in stage III/IV solid tumors) and ultrahigh specificity asdescribed previously (11).

Tissue biopsy NGSNGS of DNA from primary tumor site was performed by

FoundationOne panel as described (12).

Cell culture and reagentsLNCaP, DU145, 22Rv1, and PC-3 cells were obtained from the

American Type Culture Collection and grown in RPMI medium(Gibco, Thermofisher Scientific) supplemented with 10% FBS,100 units/mL penicillin G, and 100 mg/mL streptomycin sulfate.Cells were maintained in a humidified incubator at 37�C and 5%CO2 atmosphere. For ALK inhibition, crizotinib and alectinib(Selleck Chemicals) were dissolved in DMSO and used at theindicated concentrations.

Lentiviral vectors and preparationR777-E007Hs.ALKwas a gift fromDominic Esposito (Addgene

plasmid # 70291). FM1 lentiviral vector was kindly provided byDr. Jeffrey Milbrandt, which wemodified to express CFP reporter.Human ALK cDNA was amplified from R777-E007, mutagenesiswas performed at c.3521T, and both WT and ALK mutants weresubcloned into FM1 CFP at BstbI restriction site (Genscript).Lentiviruses were prepared as previously described. Briefly, lenti-viral titers were calculated infecting HEK293T cell line withlentivirus-expressing CFP (FM1-CFP, FM1-ALK WT-CFP, andFM1-ALK F1174C-CFP). Titer is expressed as transducing units(TU)/mL calculated fromCFP-positive cells (%)measuredbyflowcytometry, and cells were infected with 50 and 10 and 1 multi-plicity of infection for all lentiviruses. Lentiviral infection wasperformed with 6 mg/mL polybrene by centrifugation at 1,200rpm for 3 hours at room temperature.

Organoid cultureLNCaP cells were plated at 2� 105/well density in 6-well plates

in RPMI complete media and allowed to adhere overnight. Cellswere then transduced with Control (FM1-CFP), ALK WT, or ALKF1174C lentivirus particles. For matrigel basement growth, trans-duced cells were seeded at 5,000 cells/well density in ultralowattachment 96-well plates (Coring) and cultured in Hepatocytegrowth media (Corning) containing primocin (Invitrogen) andmatrigel (BD Biosciences) as described previously (13). At day 8,organoids were treated with drugs, and at day 15, viability wasassessed as per the manufacturer's protocol (Cell Titer Glo-3Dviability assay, Promega). Organoids were imaged, and area wasmeasured using ImageJ.

Cell proliferationCell proliferation was measured using the CellTiter 96 AQue-

ous One Solution Cell Proliferation Assay (Promega). A total of5,000 cells/wellwere seeded in96-well and incubatedwithDMSOcontrol, crizotinib, and alectinib and indicated concentrations.Absorbance was measured at 72 hours at 490 nm in a micro-plate reader, and cell viability was calculated as follows: cell

Translational Relevance

Small cell carcinoma of the prostate (SCCP) is an androgensignaling–independent lethal subtype of prostate cancer withlimited treatment options. The molecular drivers responsiblefor de novo small cell or treatment-emergent neuroendocrinephenotype of prostate cancer are not well understood. Wereport the first case of an activating ALK F1174C mutation-driven de novo small cell carcinoma of prostate. We observed aremarkable molecular response to second-generation ALKinhibitor alectinib and clinical benefit as reflected by decliningcirculating tumor DNA allele fraction and stable disease.Database and functional analyses in vitro support the role ofALK in de novo and treatment-emergent neuroendocrine pros-tate cancer and the therapeutic potential of ALK tyrosine kinaseinhibitors.

ALK in Small Cell Prostate Cancer

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viability ¼ absorbance of test group/absorbance of control cellgroup � 100%.

Western blottingCells transduced with ALK-expressing lentivirus were harvested

after lentivirus transduction for 7 days. Total proteinwas extractedwith RIPA buffer (Invitrogen), and protein concentration wasdetermined by BCA assay. Protein extracts were separated by 4%to 20% SDS-PAGE and transferred to PVDF membranes. Themembranes were blocked with 3% BSA in Tris-buffered salinewith 0.1% Tween 20 (TBST) for 1 hour at 37�C, and thenincubated overnight at 4�C in blocking buffer with primaryantibody including ALK (#3333; Cell Signaling Technology),pALK (#3341, Cell Signaling Technology), pMAPK (#9101; CellSignaling Technology), NSE (MAB324; Millipore), and GAPDH(V18, sc20357, Santa Cruz Biotechnology). Following incubationwith horseradish peroxidase–conjugated goat anti-rabbit/mousesecondary antibody (Biorad) for 1 hour, the membranes weredetected using a Supersignal West Femto kit (Thermofisher).

ImmunohistochemistryFormalin-fixed paraffin-embedded (FFPE) sections were depar-

affinized and stained using hematoxylin and eosin (H&E) orspecific antibodies. Sections were incubated in citrate buffer (pH6) for antigen retrieval, followed by3%H2O2 toblock peroxidase.Biotinylated secondary antibodies (Vector Labs) were used fol-lowed by incubation with ABC reagent (Vector Labs, PK-7100)and 3,30-diaminobenzidine (Sigma; Cat#D4168-50SET). Sec-tions were counterstained with hematoxylin, dehydrated, andmounted.

Statistical analysisAll experiments were repeated at least 3 times and conducted in

triplicates each time. The results are expressed as mean values ofSEM. Groups were compared by one-way or two-way ANOVAfollowed by appropriate posttests formultiple comparisons usingGraphPad Prism 7.0 software. Differences between groups wereconsidered statistically significant at P < 0.05.

Patient studiesInformed consent was obtained from the patient described in

this study. This research was approved by Quorum IRB for thegeneration of deidentified datasets for research purposes (Guar-dant protocol) and Northwestern University IRB (protocolSTU00205723).

ResultsA 39-year-old man with no family history of malignancies

presented with urinary urgency, and physical examinationrevealed nodularity of the prostate. Prostate-specific antigen(PSA) was 1.6 ng/mL. Transrectal ultrasound showed a rightprostate mass (4.1 � 1.8 � 1.9 cm), and biopsy revealed smallcell carcinoma with immunohistochemistry (IHC) panel positivefor AE1/AE3 and synaptophysin, focally positive for neuron-specific enolase (NSE), negative for chromogranin, with a Ki-67indexof 30%(Fig. 1A). PET-CT showedhypermetabolic activity ofthe right prostate tumor and extensive bone metastases withoutvisceral disease. The patient was treated with palliative radiother-apy to the thoracic spine, denosumab, and four cycles of cisplatinand etoposide. Repeat PET-CT showedprogression of diseasewith

new bone metastases. He received three cycles of topotecan, butthe prostate tumor and bone metastases progressed further. Next,he received nivolumab/ipilimumab with imaging at 12 weeksshowing stable disease. After 18 weeks of immunotherapy, thepatient developed symptoms of radiculopathy caused by pro-gression of an epidural cervical lesion and right brachial plexusinvolvement. At the same time, a brain MRI showed 3 newsubcentimeter metastases. He was treated with gamma knifestereotactic radiosurgery and intensity-modulated radiotherapy(IMRT) to the cervical lesion. Shortly thereafter, he had pro-gression of bone metastases in the pelvis and lumbar-sacralspine with compression of his cauda equina requiring radio-therapy (Fig. 1B).

Genomic profiling of prostate biopsy specimen by NGSrevealed the following mutations: ALK F1174C, ATM R2060H,ARID1A Q268�, FAS L12FS�12, and TP53 R248Q. Variants ofunknown significance were identified on the genes AXIN1, FAT1,PIK3R2, SMARCA4, KDM5A, and JAK2. Germline testing revealedan ATM p.R2060H germline variant of unknown significance(germline panel included the following genes ATM, BRCA1,BRCA2, EPCAM, MLH1, MSH2, MSH6, PMS2, CHEK2, HOXB13,NBN, TP53, and SMARCA4).

ctDNA was evaluated by NGS after initial progression oncisplatin and etoposide, revealing the same ALK F1174C muta-tion detected on primary tumor. This mutation had not beendescribed in prostate cancer or small cell carcinoma despitebeing reported as the most frequent ALK mutation in neuro-blastoma (44.5%; ref. 5). The ALK F1174 variant allele fre-quency (VAF) in peripheral blood fell from 10.0% prior toimmunotherapy to 6.4% after 12 weeks of immunotherapy. Itrose to 12.2% at progression of epidural disease and dipped to7.8% after completing radiotherapy to several areas of disease(Fig. 1C). After progression on combination immunotherapyand radiotherapy, the patient was not eligible for availableclinical trials. Based on the ALK F1147C mutation and itsassociation with sensitivity to alectinib in NSCLC setting, thepatient started treatment with alectinib 150 mg daily. After1 month of therapy, the patient's ALK F1174C VAF decreased to0.5% and the patient described significant improvementof fatigue and appetite with 10 lb weight gain. PET-CT (after2 months of treatment) and CT scans of chest/abdomen/pelvisafter 4 and 6 months of therapy demonstrated overall stabledisease (Fig. 1B). The patient remained on alectinib at the timeof submission of article (7 months of treatment) with contin-ued clinical benefit. The somatic alteration frequencies of ALKF1174C and other mutations detected through serial liquidbiopsy are listed in Supplementary Table S1.

To explore the role of ALK in prostate cancer, we first analyzedseveral pan-cancer datasets in cBioPortal. TheMSK-IMPACT Clin-ical Sequencing Cohort consists of sequencing and copy-numberalterations (CNA) data for 10,336 patients representing 62 tumortypes (14). Analyses of MSK-IMPACT dataset identified ALKalteration in 4% of cases, occurring most frequently in neuro-blastoma (also known as embryonal tumor; 12.8% mutationsand 3.5% amplifications) followed by small cell lung cancer(12.2% mutations; Supplementary Fig. S1A). Among 717 casesof prostate cancer, ALKwas altered in 20% of SCCP cases (1 deepdeletion in 5 cases) and 1.1% in prostate adenocarcinoma (1 deepdeletion, 1 truncating, and 6 missense mutations among 705cases). Further analysis of missense mutations revealed only oneputative driver mutation (R1275Q) mapping to the tyrosine

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kinase domain of ALK (Fig. 2B). No alterations were detected inALK in samples of castration-resistant prostate cancer with neu-roendocrine differentiation (t-NEPC; 7 cases) andmixed prostatecarcinoma patients (5 cases) in this dataset. However, analysis of

ALK aberrations among prostate cancer cases in cBioportal data-base that included cases of pure castration-resistant adenocarci-noma prostate cancer (CRPC) or NEPC revealed significantlyhigher ALK amplification: 27% in NEPC (9 of 30 cases) as

Figure 1.

Clinical, histopathologic and molecular features of the patient. A, IHC shows characteristics of small cell by H&E and staining of ki67, PSA, synaptophysin,chromogranin, and NSE. Brown staining, DAB; counterstain, hematoxylin. B, PET and CT scans during course of treatment. C, Patient treatment timeline andcorresponding tumor response map of ctDNA NGS findings is shown. Alterations detected in primary tumor tissue NGS are shown in the box.

ALK in Small Cell Prostate Cancer

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compared with 13% in CRPC (7 of 51 cases). ALK mRNAexpressionwas also higher inNEPC.Only onemissense passengermutation was identified in NEPC (Fig. 2A; ref. 15). The Kaplan–Meier survival curves of TCGA prostate adenocarcinoma (PRAD)dataset (NCI genomic commons) showed that patients withamplification in ALK gene exhibited significantly shorter dis-ease-free survival (DFS) as compared with those with no altera-tions in ALK (P < 0.0001). DFS and overall survival rates followedthe same trend in MSKCC PRAD (16) and MSK-IMPACT prostatecancer (14) datasets (Fig. 2B). The most frequent ALK mutationsoccur at residues F1174 and R1275 with a reported 95 and 83occurrences in the Catalog of Somatic Mutations in Cancer(COSMIC) database, respectively. Both mutations map to thetyrosine kinase domain of ALK (Fig. 2C) and induce autopho-sphorylation leading to constitutive activation of ALK (6). Anal-ysis of ALK F1174 mutation in databases from Guardant Healthand Foundation Medicine identified the current report to be theonly case of small cell carcinoma of the prostate with the ALKF1174 mutation. Among 2,182 prostate cancer cases in GuardantHealth database, 54 had nonsynonymous ALK mutations; 13 ofthese, including the current F1174 and a single case of R1275Q,described below were in the TKI domain and 39 were outside ofthe TKI domain. The other 11 were variants of uncertain signif-

icance. The patient with the ALK R1275Q mutation had a diag-nosis of prostate adenocarcinoma 8 years prior to his ctDNAanalysis, but never had a rebiopsy to confirm small cell transfor-mation. His ctDNA results did show two RB1 loss–of-functionmutations which are suggestive of a small cell phenotype (17).Together, these results suggest the potential role of ALK pointmutations as prognostic marker in SCCP.

We extracted mRNA expression data from the Geoprofile data-base related to a recent study on patient-derived xenografts (PDX)of prostate cancer (18) and identified higher expression of ALK inNEPC (Supplementary Fig. S1B). NEPCPDX in the reported studywere derived from prolonged exposure to androgen withdrawalleading to CRPC which eventually transdifferentiated into NEPC.A similar association between NEPC and high ALK levels wasconfirmed by analyzing gene expression data for all the prostatecancer cell lines within the CCLE database where NEPC cell lineNCI H660 showed highest expression followed by PC-3 cellswhich also exhibit features of small cell carcinoma (Supplemen-tary Fig. S1D). To assess protein expression of ALK in prostatecancer, we performed immunoblotting of LNCaP (hormone-sensitive prostate cancer), NE1.3 (LNCaP cell derivative isolatedby chronic growth in androgen-deprived conditions to modelNEPC), and PC3 cells (prostate cancer with small cell–like

Figure 2.

ALK alteration in prostate cancer is associated with poor survival. A, ALK gene amplification frequency and comparative mRNA expression in CRPC andNEPC cohorts of Trenton/Broad/Cornell dataset from cBioportal database. B, Kaplan–Meier survival curves relative to ALK alteration from prostate cancerdatasets in cBioportal were generated. Total number of patients in the two categories is shown. C, ALK protein domains and the location of the two identifiedactivating mutations are shown. � , P � 0.10 for Fisher exact test at 90% CI for ALK amplification. � , P � 0.05 for t test of ALK mRNA expression.

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features). As anticipated, PC-3 cells showed elevated expression oftotal and phosphorylated ALK (Supplementary Fig. S1C).

Next, to explore the effects of ALK in prostate tumorigenesisand response to therapy, we transduced LNCaP cells withlentivirus overexpressing ALK Wild Type (ALK WT) or ALK

mutant (ALK F1174C). Consistent with previous reports, weobserved increased activity of ALK shown by higher levels ofphosphorylated ALK and phosphorylated MAP kinase, a keydownstream mediator of ALK signaling pathway (6). Wealso observed an increase in NSE, a neuroendocrine marker

Figure 3.

Alectinib suppresses ALK F1174C–induced proliferation in prostate cancer cells. A,Western blot analysis of ALK, pALK, pMAPK, and NSE in LNCaP cell infected withALK WT– and ALK F1174C–expressing lentivirus. GAPDH is internal loading control. Data represent three independent experiments. B, Representative imagesshowing growth of LNCaP organoids. Cells were infected with ALK WT or ALK F1174C and grown in matrigel supplemented media to form organoidsfor 7 days. At day 8, organoids were treated with 5 mm crizotinib or alectinib for 7 days. Scale bars, 100 mm. C, Quantification of percent 3D cell viability assay.D, Quantification of organoid area (mm2). E, Percent cell viability after 72-hour treatment with 10 mm crizotinib or alectinib in PC-3, LNCaP and 22Rv1cells. Data represent mean � SE, n ¼ 6. Ns, nonsignificant; � , P � 0.05; �� , P � 0.01; ��� , P � 0.001; and ��� , P � 0.001.

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in both ALK WT– and ALK F1174C–infected cells (Fig. 3A).These results support the hypothesis that upregulation of ALKcontributes to development of de novo and treatment-emergentSCCP.

In in vitro organoid cultures, ALK F1174C–overexpressingLNCaP cells displayed enhanced proliferation with significantincrease in organoid area and percent cell viability. A similar trendwas also observedwithALKWT–overexpressing cells (Fig. 3B).Wethen tested the capability of ALK inhibitors on ALK-driven pro-liferation. Both ALK WT and ALK F1174C were significantlysensitive to treatment with the second-generation inhibitor alec-tinib in organoid culture. ALK F1174C was less sensitive tocrizotinib when compared with ALK WT (Fig. 3C and D) consis-tent with clinical experience in neuroblastoma (9). Westernblotting of hormone-sensitive LNCaP cells and CRPC 22Rv1 cellstreated with crizotinib or alectinib further demonstrated theefficacy of alectinib in inhibiting ALK pathway as seen by decreasein pALK and pMAPK (Supplementary Fig. S2A and S2B). We thentested various prostate cancer cell lines for their sensitivity to ALKinhibitors. Although both drugs induced dose-dependent cyto-toxicity in LNCaP cells, alectinib had lower IC50 in both ALKWTand ALK F1174C cells (Supplementary Fig. S2C). PC-3 cells havehigher endogenous ALK and respond to both drugs. Hormone-sensitive LNCaP cells were the most sensitive to ALK inhibitors.CRPC cell line 22Rv1 was sensitive to alectinib but not crizotinib(Fig. 3E). AR-negative DU145 cells responded equally to bothcrizotinib and alectinib, and attributed to the expression of c-Metin these cells, an additional target of crizotinib (SupplementaryFig. S2D; ref. 19). In summary, these data suggest that ALKexpression induces cell growth, and alectinib treatment resultsin favorable response in both ALKWT– and ALK F1174C–expres-sing prostate cancer cells.

DiscussionThe results describe for the first time the presence of ALK

F1174C mutation in a patient with de novo small cell carcinomaof the prostate. They also demonstrate the higher frequency ofalterations and elevated expression of ALK in NEPC. These find-ings led to analysis of a large plasma-based NGS database toidentify a likely second case, highlighting the importance ofpublically searchable large NGS databases to identify novel tar-getable genomic alterations (20).

In NSCLC, ALK translocation leads to dimerization with fusionproteins and stimulates kinase activity and oncogenic transfor-mation involving downstream signaling pathways including theRAS/ERK, JAK/STAT, and PI3K/AKT/MTOR pathways (5). Recentcase reports discussed the unusual transformation of ALK-positiveNSCLC or lung adenocarcinoma to small cell lung cancer follow-ing treatment with alectinib illustrating the complex resistancemechanisms to ALK inhibitors while also highlighting the role ofALK in small cell transformation (21, 22).Our data in LNCaP cellssuggest that similar mechanisms involving ALK might regulatetransdifferentiation to neuroendocrine phenotype in prostatecancer.

ALK mutation F1174L has been shown to induce constitutiveactivation, regulate MYCN, and enhance its oncogenic activity inneuroblastoma (23). Although little is known about the molec-ular drivers of SCCP, it has been suggested that MYCN drivesneuroendocrine differentiation of prostate (24) and that AURKAand MYCN amplifications co-occur and cooperate to induce a

small cell phenotype in prostate cells (25). In anaplastic large-celllymphoma, AURKA ismore highly expressed inALK-positive thanin ALK-negative tumors (26). These reports support the notionthat ALK, AURKA, andMYCN form a key signaling axis in SCCP,and our findings highlight the need to further explore the role ofALK to delineate the molecular mechanisms underlying SCCPprogression.

Several ALK inhibitors have been developed, among whichcrizotinib, ceritinib, brigatinib, alectinib, and lorlatinib are themost extensively studied. Alectinib has emerged as a highly potentand selective ALK inhibitor capable of overcoming resistance tocrizotinib treatment in neuroblastoma and NSCLC (27). In arecent clinical trial, alectinib showed superior efficacy and lowertoxicity in primary treatment of ALK-fusion positive NSCLC (28).ForALK F1174C in particular, studies have shown that this variantis resistant to crizotinib and ceritinib but sensitive to alectinib(29). The remarkable molecular response we observed in thepatient and our results from prostate cancer cell lines supportthe efficacy of alectinib over crizotinib in ALK WT as well asALK-activating mutant cancers. This report also highlights theclinical utility of ctDNA to identify novel therapeutic targetsand monitor response to treatment by providing an indirectmeasure of tumor burden.

In conclusion, we identified for the first time ALK F1174Cmutation in a de novo SCCP patient who responded to alectinibwith stable radiographic findings after 6 months of therapy,molecular response as reflected by declining ctDNA allele fractionand symptomatic improvement. Further studies are needed torefine the role of ALK in the pathogenesis of SCCP and confirm itsrole as a therapeutic target in this disease.

Disclosure of Potential Conflicts of InterestR.B. Lanman holds ownership interest (including patents) in Guardant

Health, Inc. J.S. Ross and L. Gay hold ownership interest (including patents)in Foundation Medicine. S.M. Ali holds ownership interest (includingpatents) in Foundation Medicine, and is a consultant/advisory board mem-ber for Incysus. Y.K. Chae reports receiving speakers bureau honoraria fromGenentech. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: B.A. Carneiro, S. Pamarthy, X.J. Yang, R.B. Lanman,J.S. Ross, F.J. Giles, S.A. AbdulkadirDevelopment of methodology: B.A. Carneiro, S. Pamarthy, H. Han,R.B. Lanman, J.S. Ross, F.J. Giles, S.A. AbdulkadirAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.A. Carneiro, S. Pamarthy, A.N. Shah, V. Sagar,R.J. Nagy, R.B. Lanman, T.M. Kuzel, J.S. Ross, L. Gay, J.A. Elvin, S.A.AbdulkadirAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B.A. Carneiro, S. Pamarthy, V. Sagar, X.J. Yang,T.M. Kuzel, J.S. Ross, L. Gay, S.M. Ali, M. Cristofanilli, F.J. Giles,S.A. AbdulkadirWriting, review, and/or revisionof themanuscript:B.A.Carneiro, S. Pamarthy,A.N. Shah, V. Sagar, K. Unno, R.B. Costa, R.J. Nagy, R.B. Lanman, T.M. Kuzel,J.S. Ross, S.M. Ali, M. Cristofanilli, Y.K. Chae, F.J. Giles, S.A. AbdulkadirAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B.A. Carneiro, S. Pamarthy, V. Sagar, F.J. Giles,S.A. AbdulkadirStudy supervision: B.A. Carneiro, S. Pamarthy, F.J. Giles, S.A. AbdulkadirOther (made plasmid construct names as FM1-CFP in this article): K. Unno

AcknowledgmentsWe extend our sincerest gratitude to the patient and his family for their

contribution to this study. We thank Dr. Devalingam Mahalingam for helpful

Clin Cancer Res; 24(12) June 15, 2018 Clinical Cancer Research2738

Carneiro et al.

on October 5, 2020. © 2018 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

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comments. We thank the Flow Cytometry Core Facility and the Pathology Coreat Northwestern University for their technical help.

Thisworkwas supported byNCI grants 1P50CA180995, R01CA123484, andR01 CA167966 (to S.A. Abdulkadir).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 26, 2018; revised February 27, 2018; accepted March 15,2018; published first March 20, 2018.

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