Identification of ALK Rearrangements in Malignant Peritoneal Mesothelioma

Research

JAMA Oncology | Brief Report Identification of ALK Rearrangements in Malignant Peritoneal Mesothelioma

Yin P. Hung, MD, PhD; Fei Dong, MD; Jaclyn C. Watkins, MD; Valentina Nardi, MD; Raphael Bueno, MD; Paola Dal Cin, PhD; John J. Godleski, MD; Christopher P. Crum, MD; Lucian R. Chirieac, MD

Supplemental content IMPORTANCE Malignant peritoneal mesothelioma is a rare, aggressive tumor arising from the peritoneal lining, induced by asbestos, therapeutic radiation, or germline mutations. Nevertheless, the molecular features remain largely unknown.

OBJECTIVE To investigate anaplastic lymphoma kinase (ALK) rearrangements in a large series of peritoneal mesothelioma and characterize the mutational landscape of these tumors.

DESIGN, SETTING, AND PARTICIPANTS We studied 88 consecutive patients (39 men, 49 women; median age 61, range 17-84 years) with peritoneal mesotheliomas diagnosed at a single institution between 2005 and 2015. We identified ALK-positive mesotheliomas by immunohistochemistry and confirmed ALK rearrangement by fluorescence in situ hybridization (FISH). In ALK-rearranged cases, we characterized the fusion partners using targeted next-generation sequencing of both tumor DNA and RNA. In select cases, we quantified asbestos fibers by combined scanning electron microscopy and x-ray spectroscopy. We also explored ALK rearrangement in a separate series of 205 patients with pleural mesothelioma.

MAIN OUTCOMES AND MEASURES Identification and characterization of novel ALK rearrangements and correlations with clinicopathologic characteristics.

RESULTS Anaplastic lymphoma kinase was positive by immunohistochemistry in 11 (13%) peritoneal mesotheliomas (focal weak in 8, diffuse strong in 3). In focal weak ALK-positive cases, no ALK rearrangement was detected by FISH or next-generation sequencing. In strong diffuse ALK-positive cases, FISH confirmed ALK rearrangements, and next-generation sequencing identified novel fusion partners ATG16L1, STRN, and TPM1. Patients with ALK-rearranged peritoneal mesotheliomas were women and younger than patients without ALK rearrangement (median age 36 vs 62; Mann-Whitney test, P = .02), but all other clinicopathologic characteristics (size of tumor nodules, histology, treatment, and survival) were not different. No asbestos fibers were detected in ALK-rearranged cases. Furthermore, loss of chromosomal region 9p or 22q or genetic alterations in BAP1, SETD2,orNF2 typically present in peritoneal mesothelioma were absent in the ALK-rearranged cases. All pleural mesotheliomas were ALK-negative by immunohistochemistry.

CONCLUSIONS AND RELEVANCE We identified unique ALK rearrangements in a subset of patients with peritoneal mesothelioma, each lacking asbestos fibers, therapeutic radiation, Author Affiliations: Department of and cytogenetic and molecular alterations typically found in these tumors. Identification of Pathology, Brigham and Women's clinically actionable ALK rearrangements may represent a novel pathogenetic mechanism of Hospital and Harvard Medical School, malignant peritoneal mesothelioma with promise for targeted therapy. Boston (Hung, Dong, Watkins, Dal Cin, Godleski, Crum, Chirieac); Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston (Nardi); Department of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston (Bueno). Corresponding Author: Lucian R. Chirieac, MD, Department of Pathology, Brigham and Women's JAMA Oncol. 2018;4(2):235-238. doi:10.1001/jamaoncol.2017.2918 Hospital, 75 Francis St, Boston, MA Published online September 14, 2017. 02115 ([email protected]).

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alignant mesothelioma is a rare aggressive neo- plasm arising from the pleural, peritoneal, or peri- Key Points cardial lining. Approximately 3000 new cases pre- M Question Is malignant peritoneal mesothelioma associated with 1 sent annually in the United States. Most mesotheliomas arise anaplastic lymphoma kinase (ALK) rearrangements? from the pleura, but 10% develop in the peritoneum.2 Findings In a large series of 88 consecutive patients with Despite various treatment modalities (cytoreductive surgery, peritoneal mesothelioma, we identified ALK rearrangements in 3% radiation, and hyperthermic intraoperative/adjuvant of cases that (1) present in young women (25% of women younger 3 chemotherapy), patients with mesothelioma have a poor than 40 years), (2) lack asbestos fibers, (3) have no history of prognosis.4 Mesothelioma is associated with exposure to therapeutic radiation, and (4) lack the typical cytogenetic and asbestos, less commonly with therapeutic radiation for molecular abnormalities usually present in peritoneal prior malignant neoplasms and BAP1 tumor predisposition mesothelioma. 5-7 syndrome. However, in cases without an identifiable cause, Meaning Identification of clinically actionable ALK the pathogenesis remains unknown. Cytogenetically, 40% to rearrangements reveals a novel pathogenetic mechanism of 70% of both pleural and peritoneal mesotheliomas harbor loss malignant peritoneal mesothelioma with promise for targeted of 9p including CDKN2A or 22q including NF2.8,9 Whereas therapy. somatic mutations in CDKN2A, BAP1, NF2, and SETD2 have been commonly identified in pleural mesothelioma,8 molecular features of peritoneal mesothelioma remain largely In all 3 cases with diffuse strong ALK expression, FISH con- unknown. firmed the presence of ALK rearrangements (eFigure 1C in the After encountering 1 index case of peritoneal mesothe- Supplement). In contrast, in the cases with focal weak ALK ex- lioma harboring ALK rearrangement, we systematically inves- pression (7 cases with available material), no bona fide ALK re- tigated the presence of ALK alterations in a large series of 88 arrangements were found, by FISH (4 cases) or targeted next patients with peritoneal mesothelioma and characterized the generation sequencing (OncoPanel, 3 cases). Instead, perito- mutational landscape of these tumors. neal mesotheliomas with focal weak ALK expression dis- played an abnormal FISH pattern indicative of polysomy for the corresponding region of 2p23 (eFigure 1D in the Supple- Methods ment). The ALK-negative cases had no ALK rearrangements. Using targeted next generation sequencing of both tu- This study was approved by the institutional review board of mor DNA and RNA, we identified novel ALK fusion partners: the Brigham and Women’s Hospital. We reviewed clinicopatho- STRN, ATG16L1, and TPM1 in 1 case each of the ALK- logic characteristics and performed immunohistochemistry for rearranged peritoneal mesotheliomas (Figure 1). The pres- ALK in all peritoneal mesotheliomas retrieved from the ar- ence of STRN-ALK translocation has been rarely reported in chives of Brigham and Women’s Hospital (eTable 1 in the carcinomas.10 To our knowledge, ATG16L1-ALK fusion has not Supplement). In ALK-positive cases, ALK rearrangement was been described to date; whereas TPM1-ALK fusion has been confirmed by FISH (eMethods in the Supplement). Anaplas- uncommonly described in bladder carcinomas.11 The ALK tic lymphoma kinase fusion partners were identified using 2 breakpoint was mapped to intron 19 in all 3 cases, the break- next generation sequencing platforms targeting tumor DNA point of STRN to intron 3 (Figure 1A), the breakpoint of ATG16L1 (OncoPanel) and RNA (AMP translocation) (eMethods in the to intron 2 (Figure 1B), and the breakpoint of TPM1 to the be- Supplement). In select cases, we quantified asbestos fibers by ginning of exon 9 (Figure 1C). Both STRN-ALK and TPM1-ALK combined scanning electron microscopy and x-ray spectros- fusions were in-frame alterations, predicted by DNA sequenc- copy (eMethods in the Supplement). Immunohistochemistry ing and confirmed by RNA sequencing (Figures 1A and C). For for ALK was also performed in 205 pleural mesotheliomas the ATG16L1-ALK fusion, RNA sequencing demonstrated a 122- (eMethods in the Supplement). base-pair insertion leading to an in-frame fusion between ATG16L1 and ALK (Figure 1B). In this case, karyotyping demonstrated t(2;2)(p23;q35), confirming rearrangement of ALK Results at 2p23 (eFigure 2 in the Supplement). Identification and Characterization of ALK Rearrangements Clinicopathologic Characteristics of Peritoneal The characteristics of the study group are detailed in Mesotheliomas With ALK Rearrangements eResults in the Supplement. Screening the cohort using im- All patients with ALK-rearranged peritoneal mesotheliomas munohistochemistry for ALK, we identified 3 cases of perito- were women and significantly younger than patients without neal mesothelioma with strong and diffuse cytoplasmic ex- ALK rearrangement (median age, 36 vs 62 years; Mann- pression (eFigure 1A in the Supplement), 8 cases with focal and Whitney test, P = .02) (eTable 1 in the Supplement). All other weak expression (eFigure 1B in the Supplement), and the re- clinicopathologic characteristics including tumor size, histol- maining 77 cases were lacking ALK expression. In contrast, all ogy, treatment, and survival were not different between pleural mesotheliomas were ALK-negative, consistent with cases with and without ALK rearrangement (eTable 1 in the published sequencing results.8 We explored the molecular Supplement), although the small number of cases with features of the ALK-positive peritoneal mesotheliomas. ALK rearrangement precluded definitive comparison. Each

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Figure 1. DNA and Amino Acid Sequences of the 3 ALK Fusion Proteins in Peritoneal Mesothelioma

A STRN-ALK fusion

STRN intron 3 … T G C T G T A T C A C A C T T T T T T G T A A T G T C G G T A G A A G G G A G A T G G C A G C A C A C A C T … ALK intron 19

Striatin Tyrosine kinase domain

…S Y D S VYRR … STRN exon 3 … A G C T A T G A T T C T G T G T A C C G C C G G … ALK exon 20

B ATG16L1-ALK fusion

ATG16L1 intron 2 … C A A G T A T T T C T G A G C GGG T T T G T G T A T G G A G A T C C A G G G A G G C T T C C T G T A … ALK intron 19

ATG16L1 Tyrosine kinase domain ATG16L1 exon 2 …H E I RQVFLSGF V Y……AV Y R R… … C A C G A G A T A A G A C A A G T A T T T C T G A G C G G G T T T G T G TAT ……G C A G T G T A C C G C C G G … ALK exon 20

C TPM1-ALK fusion

TPM1 intron 8 … A T A C T A A G G T T T T C T T C T T T G G C A T G A A T T G A A A T G T G T A A A T T G C C G A G C A C G … ALK intron 19

Tropomyosin Tyrosine kinase domain

…D D L E VYRR … TPM1 exon 8 … G A T G A C T T A G A A G T G T A C C G C C G G … ALK exon 20

A, Peritoneal mesothelioma with STRN-ALK fusion, with (top) DNA sequencing ATG16L1 exon 2 and ALK exon 20 secondary to alternative splicing, leading to mapping breakpoints to intron 3 of STRN and intron 19 of ALK, and (bottom) in-frame fusion of exon 2 of ATG16L1 followed by additional 40 amino acids to RNA sequencing confirming in-frame fusion of exon 3 of STRN to exon 20 of exon 20 of ALK. C, Peritoneal mesothelioma with TPM1-ALK fusion, with (top) ALK. B, Peritoneal mesothelioma with ATG16L1-ALK fusion, with (top) DNA DNA sequencing mapping breakpoints to intron 8/exon 9 of TPM1 and intron 19 sequencing mapping breakpoints to intron 2 of ATG16L1 and intron 19 of ALK, of ALK, and (bottom) RNA sequencing confirming in-frame fusion of exon 8 of and (bottom) RNA sequencing showing insertion of 122 base pairs between the TPM1 to exon 20 of ALK.

ALK-rearranged peritoneal mesothelioma lacked a history of Figure 2. Genetic Alterations in Peritoneal Mesothelioma therapeutic radiation, had multiple confluent peritoneal nodules (eFigure 3A in the Supplement), showed a tubulopapil- Case No. 1 2 3 4 5 6 7 8 9 Molecular alterations Rearrangement lary or solid histology (eFigure 3 in the Supplement), ex- Histology E E B E E E E E B Nonsense mutation pressed mesothelial markers (keratin AE1/AE3, calretinin, and ALK Missense mutation WT1), and retained BAP1 expression (eFigure 4 and eTable 2 BAP1 Splice site mutation in the Supplement). Ultrastructural studies in 2 available cases One-copy loss SETD2 demonstrated apical microvilli and numerous desmosomes, Two-copy loss confirming a mesothelial phenotype (eFigure 5 in the Supple- NF2 ment). No asbestos fibers were detected in any of the ALK- rearranged cases by combined scanning electron microscopy Heat map comparing the distribution of recurrent single nucleotide and copy and x-ray spectroscopy.12 Furthermore, the typical loss of chro- number variations in ALK-rearranged peritoneal mesotheliomas (cases 1-3) and peritoneal mesotheliomas with no ALK rearrangement (cases 4-9) using mosomal region 9p or 22q was not detected in any of ALK- targeted next generation DNA sequencing. Peritoneal mesotheliomas with ALK rearranged cases using FISH or karyotyping. All 3 ALK- rearrangements comprise a distinct genetic cluster lacking the most common rearranged peritoneal mesotheliomas lacked molecular molecular alterations typically found in peritoneal mesotheliomas (BAP1, SETD2, and NF2). E indicates epithelioid mesothelioma; B, biphasic mesothelioma. alterations in BAP1, SETD2,orNF2 that are typically present in other peritoneal mesotheliomas (Figure 2). No significant difference in overall survival was noted with survival, 4.8, 38.4, and 79.2 months, respectively; 3-way analy- age, history of therapeutic radiation, tumor histology, or BAP1 sis, P = .03) (eResults and eFigure 7 in the Supplement). expression (eResults, eTable 3, and eFigure 6 in the Supple- ment). Although overall survival was not affected by the presence of ALK rearrangement (present vs absent; HR, 0.5; 95% Discussion CI, 0.1-1.8; P = .29) or ALK protein expression (present vs absent; HR, 1.5; 95% CI, 0.6-4.2; P = .40), patients with weak fo- Oncogenic fusions of ALK to diverse partners have been cal ALK expression have a shorter overall survival than pa- reported in carcinomas, mesenchymal tumors, and tients with absent or diffuse strong ALK expression (median lymphomas.10,11,13 In some ALK-rearranged tumors, ALK is a

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pivotal therapeutic target.14 However, the ALK status in me- suggest that the ALK alterations uncovered in this study rep- sotheliomas remains unexplored. Recently, ALK rearrange- resent functional and clinically-actionable alterations (Figure 1) ment has been described in 1 pediatric peritoneal mesothe- (eFigure 1A and eFigure 8 in the Supplement). Furthermore, lioma, although the fusion partner was not investigated.15 ALK-rearranged cases lacked asbestos fibers, a history of therapeutic radiation, and loss of chromosomal region 9p or 22q or Limitations the genetic alterations in BAP1, SETD2,orNF2 that are typi- Although most studies have not found ALK rearrangement in cally present in peritoneal mesothelioma (Figure 2). Our find- mesotheliomas, this may be owing to the tumor location (pleu- ings suggest that oncogenic ALK fusion may represent a novel ral vs peritoneal) and the magnitude of the study cohort. Our causation mechanism for a subset of patients with peritoneal study was limited by the low incidence of peritoneal meso- mesothelioma. These patients can be readily identified using thelioma and the low prevalence of patients with ALK re- ALK immunohistochemistry and triaged toward a personal- arrangement. Larger multiinstitutional prospective studies are ized targeted treatment. needed to confirm our correlative findings and explore the ef- ficacy of ALK-targeted therapy. Genome-wide analysis of 216 8 pleural mesotheliomas has found no ALK-rearranged cases. Conclusions Indeed, our study did not identify any ALK-positive pleural mesotheliomas. In contrast, in peritoneal mesothelioma, we iden- We identified ALK rearrangements in 3% of patients with peri- tified ALK rearrangements in 3 of 88 (3%) patients, including toneal mesotheliomas that (1) present in younger women, (2) 2 of 8 (25%) women under 40, suggesting that ALK rearrange- lack asbestos fibers, (3) have no history of radiation expo- ment may be more prevalent in young women with perito- sure, and (4) lack the typical genetic abnormalities present in neal mesothelioma. We identified (1) the precise ALK break- peritoneal mesothelioma. We describe clinically actionable ALK points by DNA sequencing, (2) the in-frame mutant ALK fusion rearrangements as a novel pathogenetic mechanism in a sub- transcripts by RNA sequencing, and (3) ALK protein overex- set of peritoneal mesotheliomas with promise for targeted pression by immunohistochemistry, which together strongly therapy.

ARTICLE INFORMATION REFERENCES mutations, gene fusions and splicing alterations. Accepted for Publication: June 21, 2017. 1. Mazurek JM, Syamlal G, Wood JM, Hendricks SA, Nat Genet. 2016;48(4):407-416. Published Online: September 14, 2017. Weston A. Malignant mesothelioma 9. Singhi AD, Krasinskas AM, Choudry HA, et al. doi:10.1001/jamaoncol.2017.2918 mortality—United States, 1999-2015. MMWR Morb The prognostic significance of BAP1, NF2, and Mortal Wkly Rep. 2017;66(8):214-218. CDKN2A in malignant peritoneal mesothelioma. Author Contributions: Drs Hung and Chirieac had Mod Pathol. 2016;29(1):14-24. full access to all of the data in the study and take 2. Moolgavkar SH, Meza R, Turim J. Pleural and responsibility for the integrity of the data and the peritoneal mesotheliomas in SEER: age effects and 10. Kelly LM, Barila G, Liu P, et al. Identification of accuracy of the data analysis. temporal trends, 1973-2005. Cancer Causes Control. the transforming STRN-ALK fusion as a potential Concept and design: Hung, Chirieac. 2009;20(6):935-944. therapeutic target in the aggressive forms of Acquisition, analysis, or interpretation of data: All 3. Sugarbaker PH, Yan TD, Stuart OA, Yoo D. thyroid cancer. Proc Natl Acad SciUSA. 2014;111(11): authors. Comprehensive management of diffuse malignant 4233-4238. Drafting of the manuscript: Hung, Chirieac. peritoneal mesothelioma. Eur J Surg Oncol. 2006; 11. Stransky N, Cerami E, Schalm S, Kim JL, Critical revision of the manuscript for important 32(6):686-691. Lengauer C. The landscape of kinase fusions in intellectual content: All authors. 4. Magge D, Zenati MS, Austin F, et al. Malignant cancer. Nat Commun. 2014;5:4846. Statistical analysis: Hung, Chirieac. peritoneal mesothelioma: prognostic factors and 12. Cramer DW, Welch WR, Berkowitz RS, Godleski Administrative, technical, or material support: Dong, oncologic outcome analysis. Ann Surg Oncol. 2014; JJ. Presence of talc in pelvic lymph nodes of a Nardi, Bueno, Dal Cin, Godleski, Crum, Chirieac. 21(4):1159-1165. woman with ovarian cancer and long-term genital Study supervision: Hung, Godleski, Crum, Chirieac. 5. Carbone M, Emri S, Dogan AU, et al. exposure to cosmetic talc. Obstet Gynecol.2007; Conflict of Interest Disclosures: Dr Chirieac, A mesothelioma epidemic in Cappadocia: scientific 110(2 Pt 2):498-501. served on the Advisory board for Merck Sharp & developments and unexpected social outcomes. 13. Mariño-Enríquez A, Dal Cin P. ALK as a paradigm Dohme and undertakes medicolegal work related Nat Rev Cancer. 2007;7(2):147-154. of oncogenic promiscuity: different mechanisms of to mesothelioma and lung cancer. Dr Bueno activation and different fusion partners drive receives support from the National Cancer Institute, 6. Taylor S, Carpentieri D, Williams J, Acosta J, Southard R. Malignant Peritoneal Mesothelioma in tumors of different lineages. Cancer Genet. 2013; Verastem, Genentech-Roche, and Castle 206(11):357-373. Biosciences via research grants to Brigham and an Adolescent Male With BAP1 Deletion. J Pediatr Women's Hospital. The financial disclosures do not Hematol Oncol. 2015;37(5):e323-e327. 14. Cagle PT, Chirieac LR. Advances in treatment of apply to the current study, which is not associated 7. Chirieac LR, Barletta JA, Yeap BY, et al. lung cancer with targeted therapy. Arch Pathol Lab with a specific source of funding. Clinicopathologic characteristics of malignant Med. 2012;136(5):504-509. Additional Contributions: We thank Lauren mesotheliomas arising in patients with a history of 15. Loharamtaweethong K, Puripat N, Aoonjai N, Ritterhouse, MD, for technical discussion; Mei radiation for Hodgkin and non-Hodgkin lymphoma. Sutepvarnon A, Bandidwattanawong C. Anaplastic Zheng and the immunohistochemistry laboratory; J Clin Oncol. 2013;31(36):4544-4549. lymphoma kinase (ALK) translocation in paediatric Michele Baltay, at the Center for Advanced 8. Bueno R, Stawiski EW, Goldstein LD, et al. malignant peritoneal mesothelioma: a case report Molecular Diagnostics, Brigham and Women's Comprehensive genomic analysis of malignant of novel ALK-related tumour spectrum. Hospital, for technical support; and Linda Como for pleural mesothelioma identifies recurrent Histopathology. 2016;68(4):603-607. secretarial support. They were not compensated.

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