Oncogenic ALK Fusion in Rare and Aggressive Subtype of Colorectal Adenocarcinoma As a Potential Therapeutic Target Evgeny Yakirevich1, Murray B

Published OnlineFirst March 1, 2016; DOI: 10.1158/1078-0432.CCR-15-3000

Personalized Medicine and Imaging Clinical Cancer Research Oncogenic ALK Fusion in Rare and Aggressive Subtype of Colorectal Adenocarcinoma as a Potential Therapeutic Target Evgeny Yakirevich1, Murray B. Resnick1, Shamlal Mangray1, Michael Wheeler1, Cynthia L. Jackson1, Kara A. Lombardo1, Jeeyun Lee2, Kyoung-Mee Kim3, Anthony J. Gill4, Kai Wang5, Kyle Gowen5, James Sun5, Vincent A. Miller5, Philip J. Stephens5, Siraj M. Ali5, Jeffrey S. Ross5,6, and Howard Safran7

Abstract

Purpose: Chromosomal translocations in the anaplastic lym- EML4, C2orf44, CAD,andthenovelSTRN, PPP1R21, SENPF, phoma kinase (ALK) gene have been identified as oncogenic MAPRE3,andPRKAP1B partners. These advanced-stage colo- drivers in lung adenocarcinomas and other tumors, recently rectal carcinomas lacked mutations in other oncogenic drivers, including rare cases of colorectal carcinoma. We identified a predominantly involved the proximal colon, and often exhib- patient with refractory metastatic colorectal carcinoma harboring ited MSI and mucinous phenotype. The index patient was a STRN–ALK gene fusion who achieved an exceptional clinical treated with the ALK inhibitor ceritinib, resulting in a marked benefit to the ALK inhibitor ceritinib. Our goal was to further decrease in size of a skin metastasis, and resolution by com- define the clinicopathologic features of ALK-rearranged colorectal puterized tomography of all contrast enhancing tumor. After 9 carcinoma in a large cohort. months of treatment, biopsy of progressive disease demon- Experimental Design: Clinical cases of colorectal carcinoma strated a KRAS mutation, consistent with acquired resistance to evaluated by comprehensive genomic profiling (CGP) or by ALK ceritinib. immunohistochemistry (IHC) were reviewed retrospectively. Conclusions: Colorectal carcinoma harboring ALK fusions FISHandmicrosatelliteinstability(MSI) analyseswere performed. represent a rare aggressive subtype of colorectal carcinoma with Results: Nine colorectal carcinoma cases harbored ALK gene distinct clinicopathologic features. This report provides the first fusions. Six cases were identified by CGP of 3,157 colorectal clinical evidence that such patients may benefit from targeted carcinoma (0.2%) and three by IHC of 2,980 colorectal carci- monotherapy with ALK inhibitors. ClinCancerRes; 22(15); 3831–40. noma (0.1%). The ALK fusions involved known ALK partners 2016 AACR.

Introduction 132,700 new cases in 2015 (1). Approximately 20% to 25% of patients with colorectal carcinoma present with hepatic metasta- Colorectal cancer is the third most common cancer diagnosed ses on initial diagnosis, and a further 40% to 50% of patients will in both men and women in the United States with an estimated develop hepatic metastases within 3 years of their primary surgery (2). With recent advances in chemotherapy, the median survival rate for patients with metastatic colorectal carcinoma is approx- 1 Department of Pathology, Rhode Island Hospital, and Alpert Medical imately 25 to 30 months (3, 4). Recent genome-scale molecular School at Brown University, Providence, Rhode Island. 2Department of Medicine, Division of Hematology-Oncology, Samsung Medical Cen- analyses have uncovered several potential molecular targets in this ter, Sungkyunkwan University School of Medicine, Seoul, Republic of disease, including ERBB2 ampliﬁcations and sequence mutations, 3 Korea. Department of Pathology and Translational Genomics, Sam- as well as recurrent gene fusions that lead to oncogenic tyrosine sung Medical Center, Sungkyunkwan University School of Medicine, ALK, RET, ROS1 Seoul, Republic of Korea. 4Cancer Diagnosis and Pathology Group, kinase activation, including those involving , and Kolling Institute of Medical Research, Royal North Shore Hospital, St NTRK (5–7). Given the poor prognosis of advanced colorectal Leonards and University of Sydney, Sydney NSW, Australia. 5Founda- carcinoma, investigation of the druggability of these altered tion Medicine Inc., Cambridge, Massachusetts. 6Department of Pathol- ogy and Laboratory Medicine, Albany Medical College, Albany, New kinases is important to address this unmet clinical need. York. 7Oncology Division, Department of Internal Medicine, Rhode Gene rearrangements involving ALK receptor tyrosine kinase Island Hospital, and Alpert Medical School at Brown University, Prov- were ﬁrst discovered in anaplastic lymphoma, and later found in idence, Rhode Island. several tumor types from different lineages, including hemato- Note: Supplementary data for this article are available at Clinical Cancer lymphoid, mesenchymal, neural, and epithelial (8, 9). The fre- Research Online (http://clincancerres.aacrjournals.org/). quency of ALK fusions varies according to the tumor type. Among Corresponding Author: Evgeny Yakirevich, Rhode Island Hospital and Alpert the epithelial cancers they are most common in non–small cell Medical School at Brown University, Department of Pathology, APC12, 593 Eddy lung cancer (NSCLC), comprising approximately 3% to 7% of Street, Providence, RI 02903. Phone: 401-444-2780; Fax: 401-444-8514; E-mail: lung adenocarcinoma (10), while only nine cases of colorectal [email protected] carcinoma harboring ALK fusions have been described in seven doi: 10.1158/1078-0432.CCR-15-3000 previous studies (refs. 6, 7, 11–15; Supplementary Table 1). 2016 American Association for Cancer Research. Tumors with ALK rearrangements express activated fusion kinases

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biologic behavior of this rare aggressive subtype of colorectal Translational Relevance carcinoma. ALK rearrangement defines a rare molecular subtype of colorectal carcinoma with distinct clinical and pathological Patients and Methods features, including advanced stage, proximal colon location, Clinical trial often microsatellite instability, and mucinous phenotype. The index patient was enrolled in the Novartis Signature Trial Although ALK fusions occur in 0.1% to 0.2% of patients with within the arm "Ceritinib (LDK378) for Patients Who Have colorectal carcinoma, this equates to approximately 265 new Tumors with ALK or ROS1 Gene Alterations (Signature)" (Clin- cases of colorectal carcinoma per year in the United States and icalTrials.gov identifier NCT02186821). Comprehensive geno- approximately 2,800 new cases of colorectal carcinoma per mic profiling was performed after written informed consent for year worldwide. We provided the first clinical evidence that participation in the Rhode Island Hospital Institutional Review patients with ALK-rearranged colorectal carcinoma may Board (IRB) protocol "Molecular Testing of Cancer by Integrated achieve an exceptional clinical benefit from targeted mono- Genomic, Transcriptomic, and Proteomic Analysis" (Clinical- therapy with the ALK inhibitor ceritinib. These data support Trials.gov identifier NCT02213822). Response to ceritinib was further evaluation of patients with colorectal carcinoma har- classified by Response Evaluation Criteria in Solid Tumors boring ALK fusions in early-phase clinical trials and also (RECIST), version 1.1 (19). suggest that such patients today may benefit from already- approved ALK inhibitors as well as investigational agents. Study population A database of 3,117 clinical cases of colorectal carcinoma evaluated by CGP in the course of clinical care at Foundation Medicine (Cambridge, MA) was reviewed retrospectively to composed of the intact ALK tyrosine kinase domain fused with identify cases harboring ALK gene fusions, somatic mutations, different unrelated gene partners. The resulting ALK gene fusion and copy number alterations (amplifications and deletions). creates a constitutively active kinase that drives tumor cell growth All cases were clinically advanced-stage III or IV tumors at the through activation of MAP kinase, STAT3, and phosphoinositide time of sequencing. Local site permissions to use clinical 3-kinase (PI3K) pathways, among others (9). samples were obtained for this study. Four previously reported The discovery of ALK rearrangements in NSCLC has initiated a ALK-rearranged colorectal carcinoma cases (12, 14, 15) were new era of targeted therapy in lung cancer (16). The ALK inhibitors also included for the analysis. One of these cases was identified crizotinib and ceritinib have become standard of care for ALK- by the comprehensive genomic profiling of 40 colorectal driven lung cancer, with superior efficacy and improved tolera- carcinoma cases (12), another case by immunohistochemical bility, in comparison with cytotoxic drugs (17, 18). In two (IHC) screening of 1,889 colorectal carcinoma cases at the preclinical studies, the growth of colorectal carcinoma cell lines Department of Anatomical Pathology, Royal North Shore harboring EML4–ALK fusions was inhibited by the ALK inhibitors Hospital, Sydney, Australia (14), and two cases by IHC screen- crizotinib and entrectinib (7, 15). ing of 222 colorectal carcinoma cases at the Department of This article describes the first patient with advanced meta- Pathology and Translational Genomics, Samsung Medical static colorectal carcinoma harboring a STRN–ALK gene fusion Center, Sungkyunkwan University School of Medicine, Seoul, identified by comprehensive genomic profiling (CGP) after Republic of Korea (15). For this study, the database of Aus- failing standard therapies, who achieved an exceptional clinical tralian cases was updated by IHC screening of 719 additional response to ceritinib, a second-generation ALK inhibitor. In a colorectal carcinoma cases, and 150 cases were screened at retrospective analysis, we identified eight additional cases of Rhode Island and Miriam Hospitals, Providence, RI. The colorectal carcinoma with ALK fusions and present a detailed presence of ALK fusions in all three cases identified by IHC analysis of the molecular, clinical, histopathologic features, and was further confirmed by CGP.

Table 1. Molecular alterations in clinical cases of colorectal carcinoma harboring ALK fusions Median exon Number of genomic ALK fusion Ampliﬁcations Patient coverage alterations detected partner Mutations (copy number) 1 555 11 STRN KRAS R164Q, STK11, TP53, AEID1A, BCOR, CDH2, MLL2, MLL3, PAX5, RAD50 2 1509 7 SENPF TP53, BRCA2 AURKA (6), CCND2 (7), CDK6 (6), AKT3 (6) 3 824 7 MAPRE3 CTNNB1 GNAS (7), AURKA (7), ZNF217 (7), BCL2L1 (7), TOP1 (7) 4543 5EML4 TOP2A, TP53, APC MYST3 5525 4PRKAR1B TP53, SMAD4 GATA6 6 C2orf44 7 609 23 PPP1R21 CDKN2A, FBXW7, MLL3, MSH6, TP532, ACVR1B, ARID1A, ASXL1, ATR, ATRX, BCORL1, CASP8, CDH4, EP300, FAM123B, KDM5C, MLL22, PAX5, PTPRD, QKI 8 453 7 CAD FBXW7, ARID1A x2, CTNNB1, IKZF2, MLL2 9507 3EML4 TP53, FANCA

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Comprehensive genomic profiling The histologic grade of the tumors was determined according to Hybrid capture–based NGS was performed using DNA the WHO classification as well differentiated (grade 1) for those extracted from formalin-fixed paraffin-embedded sections cut with 5% or less of solid growth, moderately differentiated (grade at 10-mm thickness in a CLIA-certified laboratory (Foundation 2) for those with 6% to 50% of solid growth, and poorly Medicine). The sample submitted for DNA extraction con- differentiated (grade 3) for those with more than 50% solid tained a minimum of 20% DNA derived from tumor cells. growth (22). The proportion of the mucinous component was DNA sequencing was performed for the entire coding region of determined by quantitation of the percentage of extracellular 315 cancer-related genes plus introns from 28 genes frequently mucin in H&E-stained slides and calculation of the mean. rearranged in cancer on an indexed, adaptor-ligated, hybridization-captured library and fully sequenced using 49-bp reads IHC (Illumina HiSeq 2500) to a median exon coverage of 555X for IHC was performed on 4-mm formalin-fixed paraffin-embed- fi this speci c case. These 315 genes included key genes involved ded tissue sections using a BenchMark Ultra autostainer, version KRAS, BRAF, PIK3CA, in colorectal carcinogenesis, such as 12.3 (Ventana Medical Systems). Tissue sections were deparaffi- PTEN, CTNNB1, APC, MLH1, PMS2, MSH2, MSH6, HER2, nized and pretreated in CC1 solution (EDTA, pH9), extended ALK, ROS1, and NTRK (a full list is available at foundatio- regime. The primary rabbit monoclonal anti-ALK antibody clone none.com). Sequence reads were mapped to the reference D5F3 (Cell Signaling Technology) was applied at a 1:50 dilution, human genome (hg19) and analyzed for genomic alterations, and the slides were incubated at 37C for 60 minutes. The Ultra- including base substitutions, small insertions and deletions View DAB detection kit was used with the amplification kit. IHC fi (indels), copy number alterations (ampli cations and homo- staining for CDX2 and TTF1 was performed with the Dako zygous deletions), and select gene fusions/rearrangements as Autostainer Link 48 (Dako) and EnVision Flex detection system previously described (20). (Dako) with DAB (Dako). The anti-CDX2 mouse monoclonal antibody clone CDX2-88 (Biogenex) was used at 1:50 dilution. Assessment of MSI by NGS-based approach The anti-TTF1mouse monoclonal antibody clone SPT24 (Vector A novel computational method was developed to assess a Laboratories) was used at a 1:1,000 dilution. All cases were sample's MSI status by examining indel characteristics at 114 evaluated and scored as either positive or negative by two pathol- homopolymer repeat loci in or near the targeted gene regions of ogists (S. Mangray and E. Yakirevich). the FoundationOne gene panel. This method utilizes the same CGP sequencing data and eliminates the need for standard in situ pentaplex PCR or IHC, thus conserving the tissue. This novel Fluorescence hybridization (FISH) m fi methodology has high sensitivity (95%) and specificity (98%) FISH was performed on 5- mparafnsectionsusingthe when compared with standard PCR or IHC and is 97% concor- Vysis LSI ALK Dual Color Break Apart rearrangement probe dant with these techniques (21). (Abbott Molecular). FISH analysis was considered positive for ALK > The 114 loci were selected from a total set of 1,897 micro- rearrangement if 15% of tumor cells ( 50 tumor cells satellites that have adequate sequence coverage and maximized counted) showed isolated red signals and/or split red and green variability between samples. Each chosen locus was intronic signals. and had an hg19 reference repeat length of 10 to 20 bp. This range of repeat lengths was chosensuchthatthemicrosatellites Results are long enough to produce a high rate of DNA polymerase Index patient presentation slippage, while short enough to be well within the 49-bp read The patient is an 87-year-old woman without significant past length of NGS to facilitate alignment to the human reference medical history who presented 3 years ago with fatigue, hemo- genome. Using the 114 loci, the repeat length in each read that globin of 8.6 g/dL, and iron deficiency. Colonoscopy revealed spans the locus was calculated for each sample. The means and a cecal mass, and the patient underwent a right hemicolectomy. variances of repeat lengths across the reads were recorded, Pathologic examination of the specimen revealed a 5.1-cm forming 228 data points per sample. In a large training set of moderately differentiated adenocarcinoma with a prominent data from clinical specimens, the principal components anal- mucinous component that penetrated to the visceral peritoneal ysis (PCA) was applied to project the 228-dimension data onto surface and involved 7 of 28 lymph nodes (pathologic stage asingledimension(thefirst principal component) that max- pT4aN2b; Fig. 1A, B). Analysis of mismatch repair proteins by imizes the data separation, producing an NGS-based "MSI IHC revealed loss of expression of MLH1 and PMS2 with score." Ranges of the MSI score were assigned as MSI-high retained expression of MSH2 and MSH6. Staging chest com- (MSI-H), MSI-ambiguous, or microsatellite stable (MSS) by puted tomography (CT) revealed small bilateral pulmonary manual unsupervised clustering of specimens for which MSI nodules suspicious for metastatic disease. The patient's disease status was previously assessed either by IHC if available or was staged clinically as T4N2M1 (stage IV). approximated by the number of homopolymer indel mutations The patient received 10 cycles of mFOLFOX6 with resolution of detected by standard pipeline. pulmonary nodules. She was without evidence of disease until she presented with peritoneal carcinomatosis 18 months later. The Histologic assessment patient subsequently received eight treatments of FOLFIRI fol- The tumor histology in all cases with ALK translocations was lowed by maintenance fluorouracil and leucovorin. She then reviewed by two pathologists (S.M. Ali and E. Yakirevich). In five experienced further peritoneal progression including a metastasis cases, the hematoxylin and eosin (H&E)-stained sections were to a periumbilical area that extended through the skin obliterating available for microscopic analysis. Digital images of the tumors the umbilicus. mFOLFOX was reinstituted, but after an initial were reviewed in four cases in which the tissue was not available. response to six cycles of treatment, there was further tumor

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Figure 1. Histologic, molecular, and IHC analyses of the tumor. A, gross image of a 5.1-cm cecal tumor. B, a representative histologic section of the tumor shows moderately differentiated adenocarcinoma containing glands exhibiting prominent infoldings, pseudopapillary structures, and a prominent mucinous component (H&E). C, screenshot of Integrative Genomics Viewer demonstrating uniquely mapped paired-end reads in the intronic regions of STRN (intron 3) and ALK (intron 19) on chromosome 2. D, schematic representation of the STRN–ALK fusion of the N-terminal portion of STRN and C-terminal portion of ALK. Exons 1 to 3 of STRN containing the caveolin-binding domain (CB) and coiled-coil (CC) domain are shown in blue. Exons 20 to 29 of ALK containing the tyrosine kinase domain (TK) are shown in orange. Both fusion partners are located on the short arm of chromosome 2 (2p22.2 and 2p23, separated by 7.69 Mb), indicating that the fusion is a result of intrachromosomal paracentric rearrangement. The coiled-coil STRN domain may act as a dimerization motif that could constitutively activate ALK tyrosine kinase. E, IHC with the D5F3 anti-ALK antibody showing strong diffuse intracytoplasmic immunoreactivity conﬁrming ALK overexpression as a result of STRN–ALK fusion. The loss of ALK extracellular and intramembrane domains detected by sequencing is associated with intracellular localization of the fusion protein. F, dual-color break-apart FISH analysis of the tumor cells performed with a 50 ALK probe (green) and 30 ALK probe (red). Single isolated red probe signals (arrow) indicate the ALK chromosomal rearrangement. An unsplit red and green probe signals indicate the non-rearranged wild-type ALK locus (arrowhead).

growth. To identify avenues for potential benefit from targeted Comprehensive genomic profiling, IHC, and FISH therapy, archival formalin-fixed paraffin-embedded tumor tissue CGP of the primary tumor sample identified 14 genomic altera- obtained from the colectomy specimen was submitted for hybrid- tions: a STRN–ALK fusion (Fig. 1C) and mutations in KRAS capture–based comprehensive genomic profiling utilizing the R164Q, STK11, TP53, AEID1A, BCOR, CDH2, MLL2, MLL3, PAX5, FoundationOne (Foundation Medicine) assay platform. and RAD50 genes. Although no BRAF mutations were identified,

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Figure 2. Umbilical skin metastasis and imaging studies before and after ceritinib treatment. A, a pretreatment photograph shows the tumor metastasis involving periumbilical skin. B, skin metastasis signiﬁcantly decreased within 7 weeks of treatment with ceritinib. C, after an additional 2 weeks of treatment, there is marked decrease in periumbilical thickening. D, at 6-month follow-up, the mass protruding through the skin resolved, leaving a scar. E, pretreatment CT scan shows large pelvic tumor mass. F, 6 months after ceritinib therapy, a CT scan demonstrates resolution of all contrast-enhancing tumor. G, 9 months after ceritinib therapy, the periumbilical metastasis increased in size. H, CT scan at 9 months revealing peritoneal carcinomatosis and enhancement of the tumor.

genomic alterations were not detected in genes encoding the (Fig. 1E). This staining pattern conﬁrmed the loss of ALK mismatch repair proteins MLH1, PMS2, MSH2, and MSH6, con- extracellular and intramembrane domains as detected by sistent with a sporadic microsatellite instable (MSI) colorectal sequencing, and thus the positioning of the fusion protein in cancer. The KRAS R164Q mutation present in this case was the intracellular compartment. FISH analysis also showed the reported as non-activating and essentially equivalent to wild-type presence of ALK gene rearrangement in 74% of the tumor cells KRAS status (23). (Fig. 1F). A STRN–ALK fusion involved the intrachromosomal translocation of exons 1–3ofSTRN to exons 20–29 of ALK (Fig. 1D) Clinical response of the patient with STRN–ALK fusion and within the short arm of chromosome 2 (2p22.2 and 2p23, development of resistance to ceritinib separated by 7.69 Mb), consistent with an intrachromosomal Based on the STRN–ALK fusion, the patient was treated with paracentric rearrangement. The predicted fusion protein does not ceritinib, an orally available ALK inhibitor, 750 mg/day, on the have a calmodulin-binding domain and the tryptophan-aspartic Novartis Signature Trial. The tumor metastasis involving perium- acid (WD) repeats, but retains the N-terminal caveolin-binding bilical skin before ceritinib therapy is shown in Fig. 2A. This and the coiled-coil binding domains of STRN, fused to the periumbilical cutaneous metastasis signiﬁcantly decreased in size intracellular juxtamembrane region of ALK, containing the kinase within 7 weeks of treatment (Fig. 2B). After an additional 2 weeks domain (Fig. 1D). of treatment there was marked decrease in periumbilical thick- IHC staining of the primary tumor demonstrated diffuse ening (Fig. 2C). At 6 months of follow-up, the mass protruding intracytoplasmic immunoreactivity with the anti-ALK antibody through the skin could no longer be seen (Fig. 2D). Although the

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size of the pelvic tumor by CT scan did not change (deemed stable for further assays were ALK positive by IHC, while all five cases disease by RECIST), there has been resolution of all contrast- (100%) were ALK rearranged by FISH. enhancing tumor by CT scan (Fig. 2E and F). The treatment was accompanied by a clinical benefit with the disappearance Non-fusion ALK genomic alterations in colorectal carcinoma of tumor-related abdominal and periumbilical pain. However, In addition to ALK fusions, we retrospectively analyzed a after 9 months of treatment, the disease progressed with an database of 3,117 clinical cases of colorectal carcinoma evaluated increase in size of existing masses and the development of new by CGP at Foundation Medicine for other ALK genomic altera- contrast-enhancing peritoneal implants and ascites consistent tions, including point mutations and copy number alterations. with progressive peritoneal carcinomatosis (Fig. 2G and H). At Somatic point mutations in ALK were identified in 10 cases that time, the patient underwent a biopsy of a periumbilical (0.3%; Supplementary Table S2). All mutations were nonsynon- metastasis, and posttargeted therapy CGP was performed, which ymous base substitutions. Three mutations (T1151M, A1200V, now identified a new KRAS G13D mutation in addition to the and R1209Q) involved the intracellular tyrosine kinase domain. STRN–ALK fusion. One recurrent mutation R401 involving the ligand-binding domain was identified in three cases. The remaining two muta- Molecular and clinicopathologic features of a subset of tions (recurrent A585T and V757M) did not involve critical colorectal carcinoma with ALK fusions domains. To further define the frequency and molecular and clinicopath- Gene amplifications at the ALK locus were observed in two ologic features of colorectal carcinoma harboring ALK fusions, we cases (0.06%; Supplementary Table S2). Twenty-five of the 29 ALK identified five additional ALK-rearranged colorectal carcinoma, exons gained eight gene copies. No ALK deletions were present. including four previously unreported cases identified by CGP in a retrospective analysis of 3,157 relapsed and refractory clinical cases of colorectal carcinoma originally evaluated at Foundation Discussion Medicine (estimated incidence 0.2%). Three previously published This is the first report of treatment and response to an ALK cases (14, 15) that were identified by IHC (estimated incidence inhibitor in a patient with ALK-rearranged colorectal carcinoma 0.1%) and further confirmed by CGP were also included in the and a detailed analysis of molecular and clinicopathologic char- analysis. The colorectal origin of the tumors was confirmed by an acteristics of the largest series of colorectal carcinoma harboring IHC staining pattern with positivity for CDX2, a marker of ALK fusions to date. intestinal differentiation, and negative staining for TTF-1, a mark- The ALK gene encodes a receptor tyrosine kinase protein with a er of pulmonary differentiation. crucial role in the pathogenesis of various hematologic malig- The ALK gene rearrangements in the series of nine cases, nancies and solid tumors. Since the discovery of ALK as a gene including the current case involved known ALK fusion partners, target of the t(2;5) chromosomal translocation in anaplastic large such as EML4 (in two cases), C2orf44, CAD, as well as the novel cell lymphoma in 1994, ALK gene alterations, including gene STRN, PPP1R21, SENPF, MAPRE3, and PRKAP1B fusion partners fusions involving the ALK locus at 2p23, point mutations, and reported here for the first time (Table 1 and Fig. 3). All ALK fusions gene amplifications have been described in 18 different tumor were in-frame starting at the canonical exon 20 recombination types (8, 9). The term "ALKoma" was suggested in order to unify site, as has been previously reported in other cancers (10). these various tumor types arising in different organs but sharing Intrachromosomal paracentric translocations involving fusion ALK oncogenic activation as an essential tumor growth driver partners EML4, STRN, C2orf44, CAD, PPP1R21, and MAPRE3 (24). Among epithelial tumors, ALK gene rearrangements are were the most common type of chromosomal rearrangements. most common in lung carcinomas, varying from 4% to 7%, and In two cases, interchromosomal reciprocal translocations rare in kidney, breast, colorectal, esophageal, thyroid, ovarian, involved the CENPF gene on chromosome 1 and the PRKAR1B and bladder carcinomas (8, 9). gene on chromosome 7. None of the ALK-rearranged tumors Our findings confirm that ALK fusions are rare in patients with harbored mutations in other oncogenic drivers, including EGFR, colorectal carcinoma with a frequency of only 0.2% in a large KRAS, BRAF, and ERBB2 as well as in other kinase fusions such as retrospective series of colorectal carcinoma assayed by CGP. ROS1, RET, and NTRK (Table 1). Lower detection rates were observed by IHC screening of colo- This cohort of ALK-driven colorectal carcinoma included rectal carcinoma (0.1%), indicative of a higher sensitivity of CGP six females and three males with a mean age of 61 (range, 43– in identification of ALK fusions in colorectal carcinoma. Although 87; Table 2). Eight patients had stage IV disease with metastases to the frequency of ALK fusions in colorectal carcinoma is low, the liver and lung, and one patient had locally advanced tumor at the incidence of 0.2% translates into approximately 265 patients in time of CGP. Eight (89%) of the ALK fusion–positive colorectal the United States each year and approximately 2,800 patients carcinoma had a site of origin in the proximal colon, while one worldwide each year. The relatively broad range of ALK fusion (11%) originated in the rectum. Three (33%) of the nine cases frequencies in colorectal cancer varying from 0.13% to 2.5% tested positively for MSI with loss of MLH1 and PMS2 mismatch described in previous studies (Supplementary Table S1) may be repair proteins by IHC. The MSI status for these three cases was related to different patient cohorts analyzed, in some studies further confirmed by an NGS-based test at 114 microsatellite loci. limited to 40 cases, and different testing methodologies, includ- Four tumors, including two with MSI, exhibited mucinous ing IHC, FISH, real-time PCR, exon array, comprehensive genomic differentiation, one MSI case had medullary carcinoma features profiling, and transcriptome analysis (6, 7, 11–15). In contrast, in (patient 8), one had signet ring cell features (patient 9), and the this study, ALK rearrangements were identified in a cohort of remainder were moderately differentiated adenocarcinomas, with 3,157 patients with colorectal carcinoma using CGP and cohort of morphology typical of primary colorectal carcinoma. Four of five 2,980 patients with colorectal carcinoma by IHC. It has yet to be (80%) ALK-rearranged colorectal carcinomas with tissue available determined what screening test should be used clinically in order

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Figure 3. Putative ALK fusion diagrams for each patient based on DNA sequencing data; all fusions include the ALK tyrosine kinase domain. Genomic partner is on the left and ALK is on the right. Interchromosomal partners are shown in orange, intrachromosomal partners in yellow, ALK in green, and protein domains in purple. Arrows indicate the direction of transcription for each gene. Previously reported cases are indicated by asterisks. 1, STRN–ALK fusion containing a portion of the STRN Striatin domain. 2, CENPF–ALK fusion. 3, MAPRE3–ALK fusion containing the MAPRE3 Calponin homology domain and EB1- like C-terminal motif. 4, EML4–ALK fusion. 5, PRKAR1B–ALK fusion containing the PRKAR1B regulatory subunit of type II PKA R-subunit (RIIa). 6, C2orf44–ALK fusion. 7, PPP1R21– ALK fusion. 8, CAD–ALK fusion. 9, EML4–ALK fusion containing the EML4 Hydrophobic EMAP-like protein motif and a portion of the WD40 domain.

to identify potential candidates for treatment with ALK inhibitors. in 0.06% of colorectal carcinoma cases. Three of these mutations Currently, detection of ALK rearrangements is established as a were located within the tyrosine kinase domain and may poten- routine clinical test in cases of NSCLC. Screening for ALK fusions tially alter tyrosine kinase activity of ALK. Of note, one of these or ALK protein will help to identify potential candidates for ALK mutations, T1151M, was previously reported in neuroblastoma inhibitor therapy in colorectal carcinoma. and was associated with modest constitutive activation of the In addition to ALK rearrangements, we found somatic ALK tyrosine kinase domain (25, 26). Based on the data available from point mutations in 0.3% of cases and very rare ALK ampliﬁcations large-scale cancer genomics datasets, including The Cancer

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Table 2. Clinicopathologic characteristics of patients with colorectal carcinoma harboring ALK fusions ALK fusion Primary Tumor ALK ALK RFS OS Patient partner Age Sex site size, cm Grade Stageb Metastases MSI Mucinous IHC FISH (mo) (mo) Reference 1 STRN 84 F Cecum 5.1 2 IV Lung, MSI 30% Pos Pos (74%) 18 AWD39 Umbilicus 2 SENPF 62 M Proximal 7.0 2 IV Liver MSS 0 NA NA NA transverse 3 MAPRE3 43 F Ascending 3.5 2 IV Liver MSS 0 NA NA NA NA 4 EML4 53 F Right colon NA 2 IV Lung MSS 0 NA NA NA NA 5 PRKAR1B 55 M Right colon NA 2 IV Omentum, MSS 0 NA NA NA NA peritoneum 6a C2orf44 58 F Ascending 6.1 2 IV Liver MSS 60% Neg Pos (69.5%) 0 DOD 3 12 7a PPP1R21 87 F Distal 4.8 2 II No MSI 70% Pos Pos 17.6 LTF 17.6 14 transverse 8a CAD 46 F Rectum 6.5 3 IV Lung, MSI 0 Pos Pos (64%) 8 DOD18 15 pericardium 9a EML4 65 M Ascending 4.0 3 IV Liver MSS 40% Pos Pos (66%) 1 DOD5 15 Abbreviations: MSI, microsatellite instable; MSS, microsatellite stable; Pos, positive; Neg, negative; NA, not available; RFS, relapse-free survival; OS, overall survival; mo, months; AWD, alive with disease; DOD, death of disease, LTF, lost to follow-up. aPreviously reported cases with material available for pathologic review and additional testing. bStage is provided at the time of comprehensive genomic proﬁling.

Genome Atlas (TCGA), Genentech, and MSKCC, the rate of ALK agents appear to have, at best, static in colon cancer cell lines as mutations in colorectal carcinoma cases is estimated as 5.5%, reported by Garcia-Carbonero and colleagues (31). Furthermore, 11.1%, and 2.2%, respectively (5, 27, 28). Although these fre- as reported by Cutsem and colleagues, the addition of an IGF-1R quencies are higher than in this report, ALK mutations involving inhibitor, ganitumab, to the EGFR inhibitor panitumumab did not the tyrosine kinase domain were present in only one colorectal increase response rate in patients with advanced colon cancer as carcinoma case from the TCGA dataset (0.4%), and none of the compared with panitumumab alone (32). Therefore, it is unlikely Genentech or MSKCC cases. Therefore, the rate of clinically the marked clinical effect observed in the case was due to ceritinib significant ALK mutations in colorectal carcinoma is unclear and inhibition of IGF-1R. Further confirmation of the driving onco- needs to be determined in additional studies. genic properties of the ALK fusion observed in this case is the Our data provide additional detailed molecular characteriza- development of resistance to ceritinib accompanied by acquisition tion of ALK-rearranged colorectal carcinoma. In contrast to pre- of a KRAS oncogenic mutation. The early clinical and radiographic vious reports addressing ALK alterations only, we sequenced all response to ceritinib in this case is similar to rapid responses exons of a broad panel of 315 cancer-related genes and 28 genes observed to ceritinib therapy in lung cancer, as is the chronology frequently rearranged in cancer (20). The lack of mutations in of acquired resistance (18, 29). This effect represents a major known oncogenic drivers, including KRAS, BRAF, EGFR, and limitation of its long-term clinical benefit and requires develop- ERBB2, indicates that ALK fusions are indeed driving oncogenic ment of new therapeutic strategies to overcome the resistance. events in colorectal carcinoma and support the study of mono- Importantly, this finding is one of the few, initial descriptions therapy with ALK inhibitors targeting ALK fusion products in of benefit from targeted monotherapy for advanced colorectal colorectal carcinoma. carcinoma. The failure of vemurafenib treatment for BRAF V600E Ceritinib (LDK378, Zykadia, Novartis Pharmaceuticals) is an colorectal carcinoma has been held up as a counterexample to the orally administered, small-molecule, ATP-competitive, second- success of targeted therapy (33). Recent data indicating benefit generation ALK inhibitor (18). In the preclinical setting, cer- from dabrafenib and trametinib for these patients highlight the itinib is 20 times as potent as crizotinib against ALK (18). In potential benefit of combination targeted therapy for colorectal addition to ALK inhibitor activity, both crizotinib and ceritinib carcinoma (34). However, the index case presented here suggests inhibit ROS1. In contrast to crizotinib, ceritinib does not that a sufficiently potent targeted therapy may be effective as inhibit kinase activity of MET, a tyrosine kinase that can be monotherapy in advanced colorectal carcinoma with a targetable activated in NSCLC but does inhibit insulin-like growth factor 1 oncogenic driver, such as a kinase fusion. This is borne out by the receptor (IGF-1R), and insulin receptor (InsR; ref.18). In a recent report of an NTRK fusion expressing colorectal carcinoma phase I study, ceritinib was highly effective in patients with responding to entrectinib, a specific NTRK inhibitor (35), and advanced ALK-rearranged NSCLC, with an overall response rate hinted at in a report of a RET fusion colorectal carcinoma (36). of 58% (29). Moreover, responses were observed in patients In this case, an ALK fusion event involving the STRN gene was with various resistance mutations in ALK acquired during detected for the first time in colorectal carcinoma. This fusion crizotinib treatment (29). leads to constitutive activation of ALK kinase via dimerization The patient's clinical protocol did not include a tumor biopsy mediated by the coiled-coil domain of STRN, transforms cells in that would have been required to demonstrate loss of ALK-related vitro, and induces tumor formation in nude mice (37). In addi- activity within the tumor following the treatment with ceritinib. tion, STRN–ALK-driven cells are sensitive to ALK inhibitors (37). Because ceritinib may also target IGF-1R, it is possible that some of STRN–ALK fusions have been reported most frequently in thyroid the clinical benefit could have been related to IGF-1R inhibition. carcinoma patients, but were also identified in lung carcinoma Patel and colleagues have observed that colon cancer cells surviving and papillary renal cell carcinoma (6, 37). Interestingly, similar to chemotherapy are enriched in colon cancer stem cells that express this case, thyroid tumors harboring STRN–ALK present with increased levels of IGF-1R (30). However, in vitro effects of IGF-1R aggressive features, such as extrathyroidal extension and lymph

3838 Clin Cancer Res; 22(15) August 1, 2016 Clinical Cancer Research

ALK Fusion in Colorectal Carcinoma

node metastasis (37, 38). In this study, seven additional ALK Disclosure of Potential Conflicts of Interest fusion partners were identified, including four novel ALK fusion K. Gowen, V.A. Miller, P.J. Stephens, and S.M. Ali have ownership interest partners, PPP1R21, SENPF, MAPRE3, and PRKAP1B, which add to (including patents) in Foundation Medicine. J.S. Ross reports receiving a the growing list of ALK fusion partners in colorectal carcinoma. commercial research grant from and has ownership interest (including patents) in Foundation Medicine. No potential conflicts of interest were disclosed by the This study provides important information on the distinct other authors. clinical features of ALK-rearranged subtype of colorectal carcinoma. All of these tumors are high-stage adenocarcinomas, most Authors' Contributions with distant metastases to liver and lung. This subtype has Conception and design: E. Yakirevich, M.B. Resnick, S.M. Ali, J.S. Ross predilection to right colon and often shows mucinous differen- Development of methodology: E. Yakirevich, M.B. Resnick, A.J. Gill, S.M. Ali, tiation and MSI phenotype, as seen also in the index case. MSI J.S. Ross colorectal carcinoma are found in 10% to 15% and usually exhibit Acquisition of data (provided animals, acquired and managed patients, an indolent clinical behavior and are less prevalent in more provided facilities, etc.): E. Yakirevich, M. Wheeler, C.L. Jackson, J. Lee, advanced stages of the disease, accounting for <5% in the met- K.-M. Kim, A.J. Gill, S.M. Ali, J.S. Ross astatic setting (5, 39). Identification of ALK-rearranged subtype of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, fi computational analysis): E. Yakirevich, M.B. Resnick, C.L. Jackson, A.J. Gill, MSI colorectal carcinoma may de ne a distinct aggressive subtype K. Wang, K. Gowen, J. Sun, S.M. Ali, J.S. Ross fi of MSI colorectal carcinoma, which may bene t from targeted Writing, review, and/or revision of the manuscript: E. Yakirevich, therapy with ALK inhibitors. Of note, analysis of 151 cell lines M.B. Resnick, S. Mangray, C.L. Jackson, K.-M. Kim, A.J. Gill, K. Wang, K. Gowen, identified one MSI cell line harboring EML4–ALK fusion gene (7). V.A. Miller, P.J. Stephens, S.M. Ali, J.S. Ross, H. Safran In a preclinical study, proliferation of this cell line was inhibited Administrative, technical, or material support (i.e., reporting or organizing by RNAi knockdown and the ALK inhibitor crizotinib. Moreover, data, constructing databases): E. Yakirevich, M. Wheeler, K.A. Lombardo, A.J. Gill, K. Wang, J.S. Ross crizotinib treatment downregulated the MAPK and PI3K path- Study supervision: E. Yakirevich, M.B. Resnick ways in this cell line (7). This observation also raises the possi- Other (interpretation of immunohistochemical stains): S. Mangray bility that combination treatment with an ALK inhibitor and a Other (formatting of images): S. Mangray checkpoint inhibitor in this subset of colorectal carcinoma might Other (treatment of patients): H. Safran result in more durable benefit (40). In summary, this is the first case of a patient with advanced The costs of publication of this article were defrayed in part by the paymentof metastatic colorectal cancer harboring an oncogenic ALK gene page charges. This article must therefore be hereby marked advertisement in fusion who achieved durable benefit following treatment with a accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tyrosine kinase inhibitor, ceritinib. Colorectal carcinoma harboring ALK fusions represent a rare aggressive subtype of colorectal Received December 14, 2015; revised February 1, 2016; accepted February 18, carcinoma that may be responsive to ALK inhibitors. 2016; published OnlineFirst March 1, 2016.

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3840 Clin Cancer Res; 22(15) August 1, 2016 Clinical Cancer Research

Oncogenic ALK Fusion in Rare and Aggressive Subtype of Colorectal Adenocarcinoma as a Potential Therapeutic Target

Evgeny Yakirevich, Murray B. Resnick, Shamlal Mangray, et al.

Clin Cancer Res 2016;22:3831-3840. Published OnlineFirst March 1, 2016.

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