Anaplastic Lymphoma Kinase Mutation (ALK F1174C) in Small Cell Carcinoma of the Prostate and Molecular Response to Alectinib Benedito A

Published OnlineFirst March 20, 2018; DOI: 10.1158/1078-0432.CCR-18-0332

Clinical Trial Brief Report Clinical Cancer Research Anaplastic Lymphoma Kinase Mutation (ALK F1174C) in Small Cell Carcinoma of the Prostate and Molecular Response to Alectinib Benedito 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, and Sarki A. Abdulkadir2,3

Abstract

Purpose: Small cell carcinoma of the prostate (SCCP) is an Results: NGS analysis of the primary tumor and ctDNA of a 39- aggressive disease that can arise de novo or by transdiffer- year-old patient with refractory SSCP identified ALK F1174C entiation from prostate adenocarcinoma. Alterations in ana- mutation. Treatment with second-generation ALK inhibitor alec- plastic lymphoma kinase (ALK) gene are involved in neuro- tinib resulted in radiographic stable disease for over 6 months, blastoma, lung cancer, and other malignancies, but its role in symptomatic improvement, and significant molecular response SCCP has not been documented. We describe a patient with as reflected by declining ctDNA allele fraction. Analysis of prostate refractory de novo SCCP with ALK F1174C–activating muta- cancer datasets showed that ALK amplification was associated tion who obtained clinical benefit from treatment with ALK with poor outcome. In prostate cancer cells and organoids, ALK inhibitor. F1174C expression enhanced growth and induced expression of Experimental Design: Next-generation sequencing (NGS) was the neuroendocrine marker neuron-specific enolase. Alectinib used to analyze primary and circulating tumor DNA (ctDNA). was more effective than crizotinib in inhibiting ALK F1174C– Prostate cancer databases were queried for alterations in ALK gene, expressing cell growth. mRNA, and its impact in clinical outcomes. In vitro prostate cell Conclusions: These findings implicate ALK-activating muta- line/organoid models were generated by lentiviral-mediated tions in SCCP pathogenesis and suggest the therapeutic potential expression of ALK and ALK F1174C and assessed for response to of targeting ALK molecular alterations in some patients with ALK inhibitors crizotinib and alectinib. SCCP. Clin Cancer Res; 24(12); 2732–9. 2018 AACR.

Introduction (1). Although many patients present with de novo disease, 40% to 50% of SCCP patients have a history of prostate adenocarcinoma Small cell carcinoma of the prostate (SCCP) accounts for 0.5% and prior androgen-deprivation therapy (ADT; ref. 2). Despite to 2% of prostate cancers and displays an aggressive clinical course initial response to platinum-based chemotherapy, metastatic SCCP has a poor prognosis with median overall survival of 9 to 16 months related to molecular disruption of several pathways 1 Division of Hematology/Oncology, Lifespan Cancer Institute, the Warren Alpert including amplification of Aurora Kinase A (AURKA) and MYCN 2 Medical School, Brown University, Providence, Rhode Island. The Robert H. genes (3, 4). Advances in the understanding of molecular path- Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois. 3Department of Urology, Northwestern University ogenesis of SCCP have not translated into meaningful clinical fi Feinberg School of Medicine, Chicago, Illinois. 4Developmental Therapeutics bene t to patients. Hence, the important need to identify novel Program, Division of Hematology/Oncology, Feinberg School of Medicine, therapeutic targets. Northwestern University, Chicago, Illinois. 5Department of Pathology, North- Anaplastic lymphoma kinase (ALK) is a receptor tyrosine 6 western University Feinberg School of Medicine, Chicago, Illinois. Guardant kinase involved in the pathogenesis of neuroblastoma, non– 7 Health, Inc., Redwood City, California. Rush University Medical Center, Division small cell lung cancer (NSCLC) as well as other malignancies, of Hematology/Oncology, Chicago, Illinois. 8Foundation Medicine Inc., Cam- bridge, Massachusetts. 9Upstate Medical University, Syracuse, New York. but its importance in SCCP or adenocarcinoma of the prostate 10Developmental Therapeutics Consortium, Chicago, Illinois. has not been documented (5). Aberrant ALK activation due to point mutations or gene fusions stimulates kinase activity Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). and oncogenic transformation through downstream signaling pathways including RAS/ERK, JAK/STAT, and PI3K/AKT/ B.A. Carneiro and S. Pamarthy contributed equally to this article. MTOR. Although ALK mutations have been described as onco- Corresponding Author: B.A. Carneiro, Lifespan Cancer Institute, Brown Uni- genic drivers in neuroblastoma and anaplastic thyroid cancer, versity, 164 Summit St, Fain Building 2nd Floor, Providence, RI 02906. Phone: ALK fusion proteins have been associated with NSCLC and 401-793-7151; Fax: 401-793-7132; E-mail: [email protected] non-Hodgkin lymphoma (NHL; ref. 6). Specifically, ALK doi: 10.1158/1078-0432.CCR-18-0332 F1174L mutation induces autophosphorylation and constitu- 2018 American Association for Cancer Research. tive activation of ALK and is associated with resistance to ALK

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dant360, Guardant Health, Inc.). Guardant360 is a 73-gene Translational Relevance ctDNA NGS panel from a Clinical Laboratory Improvement Small cell carcinoma of the prostate (SCCP) is an androgen Amendments (CLIA)-licensed, CAP-accredited laboratory. It signaling–independent lethal subtype of prostate cancer with provides complete exon sequencing of 19 cancer genes, critical limited treatment options. The molecular drivers responsible exons of 54 genes and amplifications (18 genes), fusions (6 for de novo small cell or treatment-emergent neuroendocrine genes), and indels (23 genes) with high clinical sensitivity rates phenotype of prostate cancer are not well understood. We (85% in stage III/IV solid tumors) and ultrahigh specificity as report the first case of an activating ALK F1174C mutation- described previously (11). driven de novo small cell carcinoma of prostate. We observed a remarkable molecular response to second-generation ALK Tissue biopsy NGS inhibitor alectinib and clinical benefitasreflected by declining NGS of DNA from primary tumor site was performed by circulating tumor DNA allele fraction and stable disease. FoundationOne panel as described (12). Database and functional analyses in vitro support the role of ALK in de novo and treatment-emergent neuroendocrine pros- Cell culture and reagents tate cancer and the therapeutic potential of ALK tyrosine kinase LNCaP, DU145, 22Rv1, and PC-3 cells were obtained from the inhibitors. 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 37C and 5% CO2 atmosphere. For ALK inhibition, crizotinib and alectinib tyrosine kinase inhibitors (TKI). Activated full-length ALK (Selleck Chemicals) were dissolved in DMSO and used at the regulates transcription of MYCN (gene encoding N-Myc), an indicated concentrations. important mediator of neuroendocrine differentiation (4, 7). Although ALK TKIs are used to treat NSCLC positive for ALK Lentiviral vectors and preparation rearrangements such as EML4-ALK, their efficacy in tumors har- R777-E007 Hs.ALK was a gift from Dominic Esposito (Addgene boring ALK point mutations has been described only in neuro- plasmid # 70291). FM1 lentiviral vector was kindly provided by blastoma and anaplastic large-cell lymphoma (8). The F1174C Dr. Jeffrey Milbrandt, which we modified to express CFP reporter. mutation has been associated with resistance to crizotinib and Human ALK cDNA was amplified from R777-E007, mutagenesis ceritinib, but maintained sensitivity to alectinib in patients with was performed at c.3521T, and both WT and ALK mutants were NSCLC (9). Comprehensive genomic profiling of 114,200 clinical subcloned into FM1 CFP at BstbI restriction site (Genscript). cases identified ALK rearrangements in a variety of tumor types Lentiviruses were prepared as previously described. Briefly, lenti- beyond NSCLC and highlighted ALK as a therapeutic target in viral titers were calculated infecting HEK293T cell line with tumors with small cell and neuronal differentiation (10). We lentivirus-expressing CFP (FM1-CFP, FM1-ALK WT-CFP, and report the first case of a potentially oncogenic ALK point mutation FM1-ALK F1174C-CFP). Titer is expressed as transducing units in SCCP associated with molecular response to alectinib as (TU)/mL calculated from CFP-positive cells (%) measured by flow reflected by declining cell-free circulating tumor DNA (ctDNA) cytometry, and cells were infected with 50 and 10 and 1 multi- allele fraction and stable radiologic disease. Database and func- plicity of infection for all lentiviruses. Lentiviral infection was tional analyses in vitro support the role of ALK in the pathogenesis performed with 6 mg/mL polybrene by centrifugation at 1,200 of SCCP. rpm for 3 hours at room temperature.

Materials and Methods Organoid culture LNCaP cells were plated at 2 105/well density in 6-well plates cBioportal and CCLE data analysis in RPMI complete media and allowed to adhere overnight. Cells To determine the spectrum of genomic alterations and were then transduced with Control (FM1-CFP), ALK WT, or ALK expression pattern of ALK in prostate cancer patients, datasets F1174C lentivirus particles. For matrigel basement growth, trans- available in cbioportal were used (www.cbioportal.org). duced cells were seeded at 5,000 cells/well density in ultralow ﬁ Patients were strati ed based on the presence ALK alterations, attachment 96-well plates (Coring) and cultured in Hepatocyte and survival data were extracted for available datasets. Muta- growth media (Corning) containing primocin (Invitrogen) and tional frequencies of ALK were compared in COSMIC (www. matrigel (BD Biosciences) as described previously (13). At day 8, cancer.sanger.ac.uk/cosmic). The gene expression of ALK was organoids were treated with drugs, and at day 15, viability was compared among prostate cancer cell lines in Comprehensive assessed as per the manufacturer's protocol (Cell Titer Glo-3D cell line encyclopedia cell line (CCLE) database (www.broad viability assay, Promega). Organoids were imaged, and area was institute.org/ccle/home) by extracting gene-centric RMA-nor- measured using ImageJ. malized mRNA expression data. ALK gene expression in prostate cancer PDX data was analyzed by extracting mRNA expres- Cell proliferation ﬁ sion data deposited in NCBI Geopro le database with acces- Cell proliferation was measured using the CellTiter 96 AQue- sion number GSE41193. ous One Solution Cell Proliferation Assay (Promega). A total of 5,000 cells/well were seeded in 96-well and incubated with DMSO Liquid biopsy NGS control, crizotinib, and alectinib and indicated concentrations. ctDNA analysis was performed using a commercially avail- Absorbance was measured at 72 hours at 490 nm in a micro- able digital next-generation sequencing (NGS) assay (Guar- plate reader, and cell viability was calculated as follows: cell

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viability ¼ absorbance of test group/absorbance of control cell new bone metastases. He received three cycles of topotecan, but group 100%. the prostate tumor and bone metastases progressed further. Next, he received nivolumab/ipilimumab with imaging at 12 weeks Western blotting showing stable disease. After 18 weeks of immunotherapy, the Cells transduced with ALK-expressing lentivirus were harvested patient developed symptoms of radiculopathy caused by pro- after lentivirus transduction for 7 days. Total protein was extracted gression of an epidural cervical lesion and right brachial plexus with RIPA buffer (Invitrogen), and protein concentration was involvement. At the same time, a brain MRI showed 3 new determined by BCA assay. Protein extracts were separated by 4% subcentimeter metastases. He was treated with gamma knife to 20% SDS-PAGE and transferred to PVDF membranes. The stereotactic radiosurgery and intensity-modulated radiotherapy membranes were blocked with 3% BSA in Tris-buffered saline (IMRT) to the cervical lesion. Shortly thereafter, he had pro- with 0.1% Tween 20 (TBST) for 1 hour at 37C, and then gression of bone metastases in the pelvis and lumbar-sacral incubated overnight at 4C in blocking buffer with primary spine with compression of his cauda equina requiring radio- antibody including ALK (#3333; Cell Signaling Technology), therapy (Fig. 1B). pALK (#3341, Cell Signaling Technology), pMAPK (#9101; Cell Genomic profiling of prostate biopsy specimen by NGS Signaling Technology), NSE (MAB324; Millipore), and GAPDH revealed the following mutations: ALK F1174C, ATM R2060H, (V18, sc20357, Santa Cruz Biotechnology). Following incubation ARID1A Q268 , FAS L12FS 12, and TP53 R248Q. Variants of with horseradish peroxidase–conjugated goat anti-rabbit/mouse unknown significance were identified on the genes AXIN1, FAT1, secondary antibody (Biorad) for 1 hour, the membranes were PIK3R2, SMARCA4, KDM5A, and JAK2. Germline testing revealed detected using a Supersignal West Femto kit (Thermofisher). an ATM p.R2060H germline variant of unknown significance (germline panel included the following genes ATM, BRCA1, Immunohistochemistry BRCA2, EPCAM, MLH1, MSH2, MSH6, PMS2, CHEK2, HOXB13, Formalin-fixed paraffin-embedded (FFPE) sections were depar- NBN, TP53, and SMARCA4). affinized and stained using hematoxylin and eosin (H&E) or ctDNA was evaluated by NGS after initial progression on specific antibodies. Sections were incubated in citrate buffer (pH cisplatin and etoposide, revealing the same ALK F1174C muta- 6) for antigen retrieval, followed by 3% H2O2 to block peroxidase. tion detected on primary tumor. This mutation had not been Biotinylated secondary antibodies (Vector Labs) were used fol- described in prostate cancer or small cell carcinoma despite lowed by incubation with ABC reagent (Vector Labs, PK-7100) being reported as the most frequent ALK mutation in neuro- and 3,30-diaminobenzidine (Sigma; Cat#D4168-50SET). Sec- blastoma (44.5%; ref. 5). The ALK F1174 variant allele fre- tions were counterstained with hematoxylin, dehydrated, and quency (VAF) in peripheral blood fell from 10.0% prior to mounted. immunotherapy to 6.4% after 12 weeks of immunotherapy. It rose to 12.2% at progression of epidural disease and dipped to Statistical analysis 7.8% after completing radiotherapy to several areas of disease All experiments were repeated at least 3 times and conducted in (Fig. 1C). After progression on combination immunotherapy triplicates each time. The results are expressed as mean values of and radiotherapy, the patient was not eligible for available SEM. Groups were compared by one-way or two-way ANOVA clinical trials. Based on the ALK F1147C mutation and its followed by appropriate posttests for multiple comparisons using association with sensitivity to alectinib in NSCLC setting, the GraphPad Prism 7.0 software. Differences between groups were patient started treatment with alectinib 150 mg daily. After considered statistically significant at P < 0.05. 1 month of therapy, the patient's ALK F1174C VAF decreased to 0.5% and the patient described significant improvement Patient studies of fatigue and appetite with 10 lb weight gain. PET-CT (after Informed consent was obtained from the patient described in 2 months of treatment) and CT scans of chest/abdomen/pelvis this study. This research was approved by Quorum IRB for the after 4 and 6 months of therapy demonstrated overall stable generation of deidentified datasets for research purposes (Guar- disease (Fig. 1B). The patient remained on alectinib at the time dant protocol) and Northwestern University IRB (protocol of submission of article (7 months of treatment) with contin- STU00205723). ued clinical benefit. The somatic alteration frequencies of ALK F1174C and other mutations detected through serial liquid biopsy are listed in Supplementary Table S1. Results To explore the role of ALK in prostate cancer, we first analyzed A 39-year-old man with no family history of malignancies several pan-cancer datasets in cBioPortal. The MSK-IMPACT Clin- presented with urinary urgency, and physical examination ical Sequencing Cohort consists of sequencing and copy-number revealed nodularity of the prostate. Prostate-specific antigen alterations (CNA) data for 10,336 patients representing 62 tumor (PSA) was 1.6 ng/mL. Transrectal ultrasound showed a right types (14). Analyses of MSK-IMPACT dataset identified ALK prostate mass (4.1 1.8 1.9 cm), and biopsy revealed small alteration in 4% of cases, occurring most frequently in neuro- cell carcinoma with immunohistochemistry (IHC) panel positive blastoma (also known as embryonal tumor; 12.8% mutations for AE1/AE3 and synaptophysin, focally positive for neuron- and 3.5% amplifications) followed by small cell lung cancer specific enolase (NSE), negative for chromogranin, with a Ki-67 (12.2% mutations; Supplementary Fig. S1A). Among 717 cases index of 30% (Fig. 1A). PET-CT showed hypermetabolic activity of of prostate cancer, ALK was altered in 20% of SCCP cases (1 deep the right prostate tumor and extensive bone metastases without deletion in 5 cases) and 1.1% in prostate adenocarcinoma (1 deep visceral disease. The patient was treated with palliative radiother- deletion, 1 truncating, and 6 missense mutations among 705 apy to the thoracic spine, denosumab, and four cycles of cisplatin cases). Further analysis of missense mutations revealed only one and etoposide. Repeat PET-CT showed progression of disease with putative driver mutation (R1275Q) mapping to the tyrosine

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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 and corresponding tumor response map of ctDNA NGS ﬁndings is shown. Alterations detected in primary tumor tissue NGS are shown in the box.

kinase domain of ALK (Fig. 2B). No alterations were detected in ALK aberrations among prostate cancer cases in cBioportal data- ALK in samples of castration-resistant prostate cancer with neu- base that included cases of pure castration-resistant adenocarci- roendocrine differentiation (t-NEPC; 7 cases) and mixed prostate noma prostate cancer (CRPC) or NEPC revealed signiﬁcantly carcinoma patients (5 cases) in this dataset. However, analysis of higher ALK ampliﬁcation: 27% in NEPC (9 of 30 cases) as

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Figure 2. ALK alteration in prostate cancer is associated with poor survival. A, ALK gene amplification frequency and comparative mRNA expression in CRPC and NEPC cohorts of Trenton/Broad/Cornell dataset from cBioportal database. B, Kaplan–Meier survival curves relative to ALK alteration from prostate cancer datasets in cBioportal were generated. Total number of patients in the two categories is shown. C, ALK protein domains and the location of the two identified activating 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.

compared with 13% in CRPC (7 of 51 cases). ALK mRNA icance. The patient with the ALK R1275Q mutation had a diag- expression was also higher in NEPC. Only one missense passenger nosis of prostate adenocarcinoma 8 years prior to his ctDNA mutation was identified in NEPC (Fig. 2A; ref. 15). The Kaplan– analysis, but never had a rebiopsy to confirm small cell transfor- Meier survival curves of TCGA prostate adenocarcinoma (PRAD) mation. His ctDNA results did show two RB1 loss–of-function dataset (NCI genomic commons) showed that patients with mutations which are suggestive of a small cell phenotype (17). amplification in ALK gene exhibited significantly shorter dis- Together, these results suggest the potential role of ALK point ease-free survival (DFS) as compared with those with no altera- mutations as prognostic marker in SCCP. tions in ALK (P < 0.0001). DFS and overall survival rates followed We extracted mRNA expression data from the Geoprofile data- the same trend in MSKCC PRAD (16) and MSK-IMPACT prostate base related to a recent study on patient-derived xenografts (PDX) cancer (14) datasets (Fig. 2B). The most frequent ALK mutations of prostate cancer (18) and identified higher expression of ALK in occur at residues F1174 and R1275 with a reported 95 and 83 NEPC (Supplementary Fig. S1B). NEPC PDX in the reported study occurrences in the Catalog of Somatic Mutations in Cancer were derived from prolonged exposure to androgen withdrawal (COSMIC) database, respectively. Both mutations map to the leading to CRPC which eventually transdifferentiated into NEPC. tyrosine kinase domain of ALK (Fig. 2C) and induce autopho- A similar association between NEPC and high ALK levels was sphorylation leading to constitutive activation of ALK (6). Anal- confirmed by analyzing gene expression data for all the prostate ysis of ALK F1174 mutation in databases from Guardant Health cancer cell lines within the CCLE database where NEPC cell line and Foundation Medicine identified the current report to be the NCI H660 showed highest expression followed by PC-3 cells only case of small cell carcinoma of the prostate with the ALK which also exhibit features of small cell carcinoma (Supplemen- F1174 mutation. Among 2,182 prostate cancer cases in Guardant tary Fig. S1D). To assess protein expression of ALK in prostate Health database, 54 had nonsynonymous ALK mutations; 13 of cancer, we performed immunoblotting of LNCaP (hormone- these, including the current F1174 and a single case of R1275Q, sensitive prostate cancer), NE1.3 (LNCaP cell derivative isolated described below were in the TKI domain and 39 were outside of by chronic growth in androgen-deprived conditions to model the TKI domain. The other 11 were variants of uncertain signif- NEPC), and PC3 cells (prostate cancer with small cell–like

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features). As anticipated, PC-3 cells showed elevated expression of mutant (ALK F1174C). Consistent with previous reports, we total and phosphorylated ALK (Supplementary Fig. S1C). observed increased activity of ALK shown by higher levels of Next, to explore the effects of ALK in prostate tumorigenesis phosphorylated ALK and phosphorylated MAP kinase, a key andresponsetotherapy,wetransduced LNCaP cells with downstream mediator of ALK signaling pathway (6). We lentivirus overexpressing ALK Wild Type (ALK WT) or ALK also 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 with ALK WT– and ALK F1174C–expressing lentivirus. GAPDH is internal loading control. Data represent three independent experiments. B, Representative images showing growth of LNCaP organoids. Cells were infected with ALK WT or ALK F1174C and grown in matrigel supplemented media to form organoids for 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 22Rv1 cells. 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). small cell phenotype in prostate cells (25). In anaplastic large-cell These results support the hypothesis that upregulation of ALK lymphoma, AURKA is more highly expressed in ALK-positive than contributes to development of de novo and treatment-emergent in ALK-negative tumors (26). These reports support the notion SCCP. that ALK, AURKA, and MYCN form a key signaling axis in SCCP, In in vitro organoid cultures, ALK F1174C–overexpressing and our findings highlight the need to further explore the role of LNCaP cells displayed enhanced proliferation with significant ALK to delineate the molecular mechanisms underlying SCCP increase in organoid area and percent cell viability. A similar trend progression. was also observed with ALK WT–overexpressing cells (Fig. 3B). We Several ALK inhibitors have been developed, among which then tested the capability of ALK inhibitors on ALK-driven pro- crizotinib, ceritinib, brigatinib, alectinib, and lorlatinib are the liferation. Both ALK WT and ALK F1174C were significantly most extensively studied. Alectinib has emerged as a highly potent sensitive to treatment with the second-generation inhibitor alec- and selective ALK inhibitor capable of overcoming resistance to tinib in organoid culture. ALK F1174C was less sensitive to crizotinib treatment in neuroblastoma and NSCLC (27). In a crizotinib when compared with ALK WT (Fig. 3C and D) consis- recent clinical trial, alectinib showed superior efficacy and lower tent with clinical experience in neuroblastoma (9). Western toxicity in primary treatment of ALK-fusion positive NSCLC (28). blotting of hormone-sensitive LNCaP cells and CRPC 22Rv1 cells For ALK F1174C in particular, studies have shown that this variant treated with crizotinib or alectinib further demonstrated the is resistant to crizotinib and ceritinib but sensitive to alectinib efficacy of alectinib in inhibiting ALK pathway as seen by decrease (29). The remarkable molecular response we observed in the in pALK and pMAPK (Supplementary Fig. S2A and S2B). We then patient and our results from prostate cancer cell lines support tested various prostate cancer cell lines for their sensitivity to ALK the efficacy of alectinib over crizotinib in ALK WT as well as inhibitors. Although both drugs induced dose-dependent cyto- ALK-activating mutant cancers. This report also highlights the toxicity in LNCaP cells, alectinib had lower IC50 in both ALK WT clinical utility of ctDNA to identify novel therapeutic targets and ALK F1174C cells (Supplementary Fig. S2C). PC-3 cells have and monitor response to treatment by providing an indirect higher endogenous ALK and respond to both drugs. Hormone- measure of tumor burden. sensitive LNCaP cells were the most sensitive to ALK inhibitors. In conclusion, we identified for the first time ALK F1174C CRPC cell line 22Rv1 was sensitive to alectinib but not crizotinib mutation in a de novo SCCP patient who responded to alectinib (Fig. 3E). AR-negative DU145 cells responded equally to both with stable radiographic findings after 6 months of therapy, crizotinib and alectinib, and attributed to the expression of c-Met molecular response as reflected by declining ctDNA allele fraction in these cells, an additional target of crizotinib (Supplementary and symptomatic improvement. Further studies are needed to Fig. S2D; ref. 19). In summary, these data suggest that ALK refine the role of ALK in the pathogenesis of SCCP and confirm its expression induces cell growth, and alectinib treatment results role as a therapeutic target in this disease. in favorable response in both ALK WT– and ALK F1174C–expressing prostate cancer cells. Disclosure of Potential Conflicts of Interest R.B. Lanman holds ownership interest (including patents) in Guardant Health, Inc. J.S. Ross and L. Gay hold ownership interest (including patents) Discussion in Foundation Medicine. S.M. Ali holds ownership interest (including patents) in Foundation Medicine, and is a consultant/advisory board mem- The results describe for the first time the presence of ALK de novo ber for Incysus. Y.K. Chae reports receiving speakers bureau honoraria from F1174C mutation in a patient with small cell carcinoma Genentech. No potential conflicts of interest were disclosed by the other of the prostate. They also demonstrate the higher frequency of authors. alterations and elevated expression of ALK in NEPC. These findings led to analysis of a large plasma-based NGS database to Authors' Contributions identify a likely second case, highlighting the importance of Conception and design: B.A. Carneiro, S. Pamarthy, X.J. Yang, R.B. Lanman, publically searchable large NGS databases to identify novel tar- J.S. Ross, F.J. Giles, S.A. Abdulkadir getable genomic alterations (20). Development of methodology: B.A. Carneiro, S. Pamarthy, H. Han, In NSCLC, ALK translocation leads to dimerization with fusion R.B. Lanman, J.S. Ross, F.J. Giles, S.A. Abdulkadir Acquisition of data (provided animals, acquired and managed patients, proteins and stimulates kinase activity and oncogenic transfor- provided facilities, etc.): B.A. Carneiro, S. Pamarthy, A.N. Shah, V. Sagar, mation involving downstream signaling pathways including the R.J. Nagy, R.B. Lanman, T.M. Kuzel, J.S. Ross, L. Gay, J.A. Elvin, S.A. RAS/ERK, JAK/STAT, and PI3K/AKT/MTOR pathways (5). Recent Abdulkadir case reports discussed the unusual transformation of ALK-positive Analysis and interpretation of data (e.g., statistical analysis, biostatistics, NSCLC or lung adenocarcinoma to small cell lung cancer follow- computational analysis): B.A. Carneiro, S. Pamarthy, V. Sagar, X.J. Yang, ing treatment with alectinib illustrating the complex resistance T.M. Kuzel, J.S. Ross, L. Gay, S.M. Ali, M. Cristofanilli, F.J. Giles, S.A. Abdulkadir mechanisms to ALK inhibitors while also highlighting the role of Writing, review, and/or revision of the manuscript: B.A. Carneiro, S. Pamarthy, ALK in small cell transformation (21, 22). Our data in LNCaP cells A.N. Shah, V. Sagar, K. Unno, R.B. Costa, R.J. Nagy, R.B. Lanman, T.M. Kuzel, suggest that similar mechanisms involving ALK might regulate J.S. Ross, S.M. Ali, M. Cristofanilli, Y.K. Chae, F.J. Giles, S.A. Abdulkadir transdifferentiation to neuroendocrine phenotype in prostate Administrative, technical, or material support (i.e., reporting or organizing cancer. data, constructing databases): B.A. Carneiro, S. Pamarthy, V. Sagar, F.J. Giles, ALK mutation F1174L has been shown to induce constitutive S.A. Abdulkadir MYCN, Study supervision: B.A. Carneiro, S. Pamarthy, F.J. Giles, S.A. Abdulkadir activation, regulate and enhance its oncogenic activity in Other (made plasmid construct names as FM1-CFP in this article): K. Unno neuroblastoma (23). Although little is known about the molecular drivers of SCCP, it has been suggested that MYCN drives Acknowledgments neuroendocrine differentiation of prostate (24) and that AURKA We extend our sincerest gratitude to the patient and his family for their and MYCN amplifications co-occur and cooperate to induce a contribution to this study. We thank Dr. Devalingam Mahalingam for helpful

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comments. We thank the Flow Cytometry Core Facility and the Pathology Core advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate at Northwestern University for their technical help. this fact. This work was supported by NCI grants 1P50CA180995, R01CA123484, and R01 CA167966 (to S.A. Abdulkadir).

The costs of publication of this article were defrayed in part by the Received January 26, 2018; revised February 27, 2018; accepted March 15, payment of page charges. This article must therefore be hereby marked 2018; published ﬁrst March 20, 2018.

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www.aacrjournals.org Clin Cancer Res; 24(12) June 15, 2018 2739

Anaplastic Lymphoma Kinase Mutation (ALK F1174C) in Small Cell Carcinoma of the Prostate and Molecular Response to Alectinib

Benedito A. Carneiro, Sahithi Pamarthy, Ami N. Shah, et al.

Clin Cancer Res 2018;24:2732-2739. Published OnlineFirst March 20, 2018.

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