Pan-Cancer Landscape Analysis Reveals Recurrent KMT2A-MAML2 Gene Fusion in Aggressive Histologic Subtypes of Thymoma

Abstract

PURPOSE

Thymomas are epithelial neoplasms that represent the most common thymic tumors in adults. These tumors have been shown to harbor a relatively low mutational burden. As a result, there is a lack of genetic alterations that may be used prognostically or targeted therapeutically for this disease. Here, we describe a recurrent gene rearrangement in type B2 + B3 thymomas.

PATIENTS AND METHODS

A single index case of thymoma was evaluated by an RNA-based solid fusion assay. Separately, tissues from 255,008 unique advanced cancers, including 242 thymomas, were sequenced by hybrid capture–based next-generation DNA sequencing/comprehensive genomic profiling of 186 to 406 genes, including lysine methyltransferase 2A (KMT2A) rearrangements, and a portion were evaluated for RNA of 265 genes. We characterized molecular and clinicopathologic features of the pertinent fusion-positive patient cases.

RESULTS

We identified 11 patients with thymomas harboring a gene fusion of KMT2A and mastermind-like transcriptional coactivator 2 (MAML2). Fusion breakpoints were identified between exon 8, 9, 10, or 11 of KMT2A and exon 2 of MAML2. Fifty-five percent were men, with a median age of 48 years at surgery (range, 29-69 years). Concurrent genomic alterations were infrequent. The 11 thymomas were of B2 or B3 type histology, with 1 case showing foci of thymic carcinoma. The frequency of KMT2A-MAML2 fusion was 4% of all thymomas (10 of 242) and 6% of thymomas of B2 or B3 histology (10 of 169).

CONCLUSION

KMT2A-MAML2 represents the first recurrent fusion described in type B thymoma. The fusion seems to be specific to type B2 and B3 thymomas, the most aggressive histologic subtypes. The identification of this fusion offers insights into the biology of thymoma and may have clinical relevance for patients with disease refractory to conventional therapeutic modalities.

INTRODUCTION

Thymoma, a neoplasm that arises from or exhibits thymic epithelial differentiation, is the most common tumor of the adult thymus.¹ Thymoma has a strong association with autoimmune diseases, particularly myasthenia gravis, which typically resolve upon successful tumor resection. The most significant prognostic factors in thymoma are WHO histologic type, tumor stage, and completeness of surgical resection. WHO histologic types A and AB thymomas are associated with a favorable clinical course, with a nearly 100% 5-year overall survival rate.¹ Types B1, B2, and B3 show progressively worse survival, with type B3 demonstrating a 5-year survival rate ranging from 43% to 70%.¹ Surgery is the standard of care for localized tumors, with radiotherapy and chemotherapy reserved for advanced stages.¹

Recent genomic studies of thymic epithelial tumors have demonstrated a high frequency of a thymoma-specific codon mutation (L424H) in the GTF2I gene in type A and AB thymomas.^2,3 In contrast, whole- and arm-level copy-number alterations, but no specific recurrently mutated genes, have been described in type B2 and B3 thymomas.^2,3 Thymoma has demonstrated a relatively low overall mutational burden, and reports of effective targeted therapies are sparse.^2,4,5 Only rare reports of gene fusions in thymoma are present in the literature, and no recurrent fusion has been reported in any major thymoma subtype to date.^3,6 Herein, we describe a novel recurrent gene fusion in 11 patient cases of type B2 or B3 thymoma, involving the lysine methyltransferase 2A (KMT2A) and mastermind-like transcriptional coactivator 2 (MAML2) genes.

PATIENTS AND METHODS

Index Case

One patient case of thymoma (index case) from Massachusetts General Hospital (MGH) underwent solid fusion assay analysis (MGH Solid Fusion Assay; MGH, Boston, MA; Data Supplement provides list of gene targets) and single-nucleotide variant and insertion/deletion analysis (SNaPshot DNA-based assay; Thermo Fisher Scientific, Waltham, MA; Data Supplement provides list of gene targets).⁷ The solid fusion assay used RNA-based fusion-targeted anchored multiplex polymerase chain reaction (ArcherDx primers, Boulder, CO) and Illumina (San Diego, CA) sequencing. Review of pathology report, tumor histopathology (L.R.M., R.P.H., and A.L.), and patient clinical data (age at diagnosis, sex, site of tumor biopsy, stage at diagnosis, and clinical outcome) was performed. Approval for this study, including a waiver of additional informed consent and a Health Insurance Portability and Accountability Act (HIPAA) waiver of authorization, was obtained from the Partners Institutional Review Board (protocol No. 2011P001749).

CONTEXT

• Key Objective

• Type B thymoma, an aggressive subset of thymic tumors, has been noted to show a paucity of genomic alterations. We aim to identify recurrent genetic alterations present in this tumor.

• Knowledge Generated

• A subset of type B2 and B3 thymomas harbor recurrent gene fusions between lysine methyltransferase 2A (KMT2A) and mastermind-like transcriptional coactivator 2 (MAML2). Aside from rare case reports of hematologic neoplasms in the literature, the KMT2A-MAML2 rearrangement seems specific to type B2 and B3 thymomas among human tumors.

• Relevance

• The finding of recurrent KMT2A-MAML2 fusion provides insights into tumor biology and potential therapeutic targets in type B2 and B3 thymomas, which are clinically aggressive with limited curative options.

Cohort

Comprehensive genomic profiling (CGP) was performed in a Clinical Laboratory Improvement Amendments–certified, College of American Pathologists–accredited laboratory (Foundation Medicine, Cambridge, MA). Approval for this study, including a waiver of informed consent and a HIPAA waiver of authorization, was obtained from the Western Institutional Review Board (protocol No. 20152817). The pathologic diagnosis of each patient case was confirmed on routine hematoxylin and eosin (HE) –stained slides. In brief, ≥ 60 ng of DNA was extracted from 255,008 cancer specimens, including 242 thymoma specimens, in 40 μm of formalin-fixed, paraffin-embedded tissue blocks. The samples were assayed by CGP using adaptor ligation, and hybrid capture was performed for all coding exons from 287 (version 1) to 315 (version 2) cancer-related genes plus select introns from 19 (version 1) to 28 (version 2) genes frequently rearranged in cancer (Data Supplement). GTF2I was not included on any list of cancer-related genes. Various samples were similarly assayed but performed in DNA on 406 genes and selected introns of 31 genes involved in rearrangements (Data Supplement) and in RNA on 265 genes commonly rearranged in cancer. Sequences were analyzed for all classes of genomic alterations, including short variant alterations (base substitutions, insertions, and deletions), copy-number alterations (focal amplifications and homozygous deletions), and select gene fusions or rearrangements by methods previously described.^8-10

The cohort of thymomas harboring a KMT2A-MAML2 fusion comprised 10 patient cases assayed with CGP (Foundation Medicine) during clinical care at other institutions. Clinicopathologic data, including patient age, sex, tumor site, and modified Masaoka stage, were extracted from the accompanying pathology reports. Histopathology was assessed on routine HE-stained slides of tissue sections submitted for genomic profiling by 2 board-certified hematopathologists (R.P.H. and A.L.).

RESULTS

Index Case: Identification of Novel KMT2A-MAML2 Fusion in Aggressive Type B3 Thymoma

A 29-year-old woman presented with a thymic mass soon after being diagnosed with myasthenia gravis. Upon surgical resection of the mass, histologic review revealed combined type B2 and B3 thymoma with negative surgical margins. The patient’s myasthenia gravis symptoms resolved postoperatively. Seven years later, the patient developed recurrent thymoma, with direct invasion of the lung, pericardium, and distal left main pulmonary artery, as well as metastasis to paratracheal lymph nodes (Fig 1A). The patient received induction chemotherapy and underwent surgical resection (radical thymectomy, radical pericardiectomy, and left pneumonectomy), followed by paratracheal lymph node excision and radiotherapy. Histopathology was again diagnostic of type B2 + B3 thymoma (Fig 1B-1D). Within 1 year, the patient’s thymoma recurred and progressed, despite additional radiotherapy, chemotherapy, and multiple investigational drug therapies. Her clinical course was complicated by Good syndrome (a rare combined B- and T-cell immunodeficiency), pure red cell aplasia, paraneoplastic intestinal pseudo-obstruction, and recurrence of myasthenia gravis. The patient died as a result of complications of her thymoma 15 years after initial diagnosis.

FIG 1.

Radiology and pathology from index patient. (A) Computed tomography of the chest from the patient’s first recurrence demonstrates a 7.8-cm anterior mediastinal mass (arrow) in contact with the pericardium, aorta, and portions of the main and left pulmonary arteries. (B) Histopathologic examination of the tumor shows sheets of large epithelioid cells lacking significant lymphocytic infiltration, consistent with type B3 thymoma (hematoxylin and eosin stain; magnification 400×). (C) Immunohistochemistry (IHC) for a wide-spectrum keratin (MNF116) is diffusely positive, highlighting neoplastic cell membranes (magnification 400×). (D) IHC for terminal deoxynucleotidyl transferase highlights scattered nuclei of T-cell lineage thymocytes associated with the tumor (magnification 400×).

Given the aggressive nature of this thymoma, molecular genetic assays were performed on the initial recurrence to lung, in an attempt to identify potentially targetable genetic alterations. This patient case was found to have an in-frame KMT2A-MAML2 fusion using the MGH Solid Fusion Assay (Fig 2). This fusion assay result was reproduced on the patient’s subsequent paratracheal lymph node excision.

FIG 2.

Schematic of KMT2A-MAML2 fusion from index patient, featuring KMT2A exons 1 to 10 and MAML2 exons 2 to 5. Exon numbers are shown above their respective boxes for reference sequence KMT2A transcript variant 2 (NM_005933) and MAML2 (NM_032427). The fusion breakpoint is shown as a dashed line. The fusion protein contains the CXXC-type DNA binding domain of KMT2A, as well as the central and C-terminal acidic domains of MAML2. These domains were preserved across all 11 patient cases.

KMT2A-MAML2 Fusion Occurs in Aggressive Histologic Subtypes of Thymoma

To determine the frequency of KMT2A-MAML2 fusion in a cohort specifically enriched in clinically more aggressive cases of thymoma, we reviewed a set of 242 patient cases of thymoma from the Foundation Medicine archives. The cohort included the following thymoma histologic subtypes: 8 type A (3%), 13 type AB (5%), 43 type B1 (18%), 10 type B1 + B2 (4%), 66 type B2 (27%), 21 type B2 + B3 (9%), and 72 type B3 subtypes (30%; including 2 patient cases with components of thymic carcinoma). An additional 9 patient cases (4%) could not be histologically subtyped for technical reasons or unusual case features. The median age of the cohort was 53 years, and 118 patients (51%) were women. An in-frame KMT2A-MAML2 fusion was identified in 10 patient cases (4.1%). A reverse query for KMT2A-MAML2 fusion was performed on 255,008 Foundation Medicine cases of all tumor categories, including 366 thymic carcinomas. Across all 255,008 samples, only 1 additional patient case, diagnosed as plasmacytoma, was identified as harboring a KMT2A-MAML2 fusion. The patient case was reviewed, and the original diagnosis was confirmed.

Among thymomas, the KMT2A-MAML2 fusion was restricted to the most aggressive histologic thymoma subtypes, being present in 10 (5.9%) of 169 thymomas that included B2 or B3 components, but 0 (0%) of 64 of the remaining thymomas (types A, AB, and B1). No other known or likely pathogenic alterations were identified in 7 of 10 patient cases with a KMT2A-MAML2 fusion, whereas a concurrent mutation in TP53, ARID1A, or SF3B1 was identified in 1 patient case each. The clinical, histologic, and molecular features of all 11 patient cases of KMT2A-MAML2–rearranged thymoma are listed in Tables 1 and 2. A single patient case with a KMT2A-MAML2 fusion showed foci of thymic carcinoma.

TABLE 1.

Clinical Characteristics of Patients With KMT2A-MAML2–Rearranged Thymomas

TABLE 2.

Molecular Characteristics of KMT2A-MAML2–Rearranged Thymomas

Among the entire cohort, no additional recurrent fusions were identified. In-frame KMT2A fusions with the partner gene MLLT3 or MTMR2 were identified in 2 additional patient cases of thymoma (both type B2-B3). No additional fusions involving MAML2 and another partner were found. TP53 and HRAS pathogenic genomic alterations were identified in 7% and 3% of all patient cases of thymoma, respectively.

DISCUSSION

In the 243 patient cases of thymoma evaluated, we discovered a recurrent fusion of KMT2A and MAML2 in 11. This fusion seems to be highly specific to thymomas with aggressive histologic features, because all fusion-positive patient cases contained type B2 and/or B3 histology. The striking restriction of KMT2A-MAML2 fusion to thymomas was underscored by the absence of the fusion in approximately 255,000 patient cases of diverse tumor types, including 366 thymic carcinomas, with the exception of a single patient case of plasmacytoma.

KMT2A, first described in 1991, was initially termed mixed-lineage leukemia-1 because of its frequent appearance as a translocation partner in acute myeloid and lymphoid leukemias.¹² The 36-exon gene is located on chromosome 11q23. The encoded protein binds DNA and methylates histone H3 at lysine-4 to regulate other genes, including several homeobox (HOX) genes.¹³ KMT2A has been found to be a promiscuous translocation partner, with > 80 unique fusion partners identified.¹⁴ Currently, there is no unifying theory on how KMT2A rearrangements lead to neoplasia.¹⁴ MAML2 is a 5-exon gene residing on chromosome 11q21. MAML2 and other MAML family proteins are involved in NOTCH pathway–mediated transcriptional activation.¹⁵ Recurrent gene rearrangements involving MAML2 have been described in mucoepidermoid carcinoma, in which fusion of the first exon of the cAMP response element-binding protein–regulated transcription coactivator-1 (CRTC1) with MAML2 exons 2 to 5 leads to NOTCH pathway disruption.¹⁶

The KMT2A-MAML2 gene fusion results from inv(11)(q21q23), a cytogenetic abnormality first reported in 1998 by Obama et al¹⁷ in a patient with therapy-related acute myeloid leukemia. Subsequent reports of the fusion are exceptionally rare and restricted to hematologic malignancies. To our knowledge, only 8 patient cases have been reported, including 2 of acute myeloid leukemia, 2 of myelodysplastic syndrome, and 4 of acute lymphoblastic leukemia.^17-22 Reported fusion proteins in these patient cases involved regions similar to those identified in our cohort, with the exception of 2 patient cases reported by Metlzer et al²¹ with MAML2 breakpoints in introns 2 and 3.

Prior functional studies of the KMT2A-MAML2 construct have shown evidence of disrupted NOTCH pathway signaling. In addition to describing the fusion in patient cases of therapy-related myeloid neoplasms, Nemoto et al¹⁹ demonstrated via luciferase assay that the KMT2A-MAML2 fusion suppresses promoter activation of the NOTCH1 target gene HES1.¹⁹ Gene expression profiles from 2 patient cases of KMT2A-MAML2–positive T-cell acute lymphoblastic leukemia showed differential expression patterns relative to controls, suggesting activation of genes downstream of NOTCH1.²¹ Another study demonstrated oncogenic activity by KMT2A-MAML2 fusion inserted into cell lines with sleeping-beauty vectors.²³ The sum of these studies has demonstrated oncogenic function of the KMT2A-MAML2 fusion, likely via disruption of NOTCH signaling. Further study is warranted to examine the impact of this fusion on NOTCH signaling.

In patients with malignancies not amenable to traditional surgical or chemoradiotherapy protocols, targeted therapy offers an additional potential opportunity for disease control. The finding of recurrent KMT2A-MAML2 fusion in a subset of thymomas predisposed to aggressive behavior may offer a potential target in patients with high-stage disease refractory to initial therapy. Early data suggest that MAML2 fusion–positive mucoepidermoid carcinoma may respond to targeted therapeutics, including epidermal growth factor receptor inhibitors such as gefitinib.^24-26

Given the low tumor mutational burden seen in thymoma, identification of this small but genomically distinctive subset of KMT2A-MAML2–rearranged tumors may provide a therapeutic target in patients not responsive to traditional therapy. This finding illustrates the importance of performing comprehensive genomic profiling to define treatment strategies, including molecular inclusion criteria for clinical trials and more fully informed personalized therapeutic options that could lead to improved patient outcomes.

AUTHOR CONTRIBUTIONS

Conception and design: Lucas R. Massoth, Daniel Duncan, Brendan J. Gillespie, Jeffrey S. Ross, Lawrence R. Zukerberg, Abner Louissaint Jr, Erik A. Williams

Financial support: Abner Louissaint Jr

Provision of study material or patients: Dora Dias-Santagata, Krzysztof Glomski, Abner Louissaint Jr

Collection and assembly of data: Lucas R. Massoth, Dora Dias-Santagata, Maristela Onozato, Nikunj Shah, Jeffrey S. Ross, Shannon K. Harkins, Krzysztof Glomski, Lawrence R. Zukerberg, Abner Louissaint Jr, Erik A. Williams

Data analysis and interpretation: Lucas R. Massoth, Yin P. Hung, Dora Dias-Santagata, Eric Severson, Daniel Duncan, Nathan F. Williams, Jeffrey S. Ross, Jo-Anne Vergilio, Shannon K. Harkins, Valentina Nardi, Lawrence R. Zukerberg, Robert P. Hasserjian, Abner Louissaint Jr, Erik A. Williams

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.

Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).

Dora Dias-Santagata

Consulting or Advisory Role: Rarecyte (I)

Maristela Onozato

Patents, Royalties, Other Intellectual Property: Patent pending for multiplex FISH assay, filed on October 25, 2019 (no direct compensation related to this patent)

Nikunj Shah

Employment: Foundation Medicine

Eric Severson

Employment: Foundation Medicine, Partners Healthcare

Stock and Other Ownership Interests: Foundation Medicine

Daniel Duncan

Employment: Foundation Medicine

Jeffrey S. Ross

Employment: Foundation Medicine

Leadership: Foundation Medicine

Stock and Other Ownership Interests: Foundation Medicine

Research Funding: Foundation Medicine

Jo-Anne Vergilio

Employment: Foundation Medicine

Stock and Other Ownership Interests: Foundation Medicine, Roche

Valentina Nardi

Consulting or Advisory Role: Thermo Fisher Scientific (I), Cell Signaling Technology (I)

Robert P. Hasserjian

Stock and Other Ownership Interests: Avanos Medical, Danaher, Henry Schein, Hologic, IDEXX Laboratories, Johnson & Johnson, Kimberly Clary, Medtronic, Stryker, Cooper Companies, Thermo Fisher Scientific

Consulting or Advisory Role: Jazz Pharmaceuticals, Promedior

Erik A. Williams

Employment: Foundation Medicine

Stock and Other Ownership Interests: F. Hoffmann-La Roche

No other potential conflicts of interest were reported.