Extreme Outlier Analysis Identifies Occult Mitogen-Activated Protein Kinase Pathway Mutations in Patients With Low-Grade Serous Ovarian Cancer

Abstract

Purpose: No effective systemic therapy exists for patients with metastatic low-grade serous (LGS) ovarian cancers. BRAF and KRAS mutations are common in serous borderline (SB) and LGS ovarian cancers, and MEK inhibition has been shown to induce tumor regression in a minority of patients; however, no correlation has been observed between mutation status and clinical response. With the goal of identifying biomarkers of sensitivity to MEK inhibitor treatment, we performed an outlier analysis of a patient who experienced a complete, durable, and ongoing (> 5 years) response to selumetinib, a non-ATP competitive MEK inhibitor.

Patients and methods: Next-generation sequencing was used to analyze this patient's tumor as well as an additional 28 SB/LGS tumors. Functional characterization of an identified novel alteration of interest was performed.

Results: Analysis of the extraordinary responder's tumor identified a 15-nucleotide deletion in the negative regulatory helix of the MAP2K1 gene encoding for MEK1. Functional characterization demonstrated that this mutant induced extracellular signal-regulated kinase pathway activation, promoted anchorage-independent growth and tumor formation in mice, and retained sensitivity to selumetinib. Analysis of additional LGS/SB tumors identified mutations predicted to induce extracellular signal-regulated kinase pathway activation in 82% (23 of 28), including two patients with BRAF fusions, one of whom achieved an ongoing complete response to MEK inhibitor-based combination therapy.

Conclusion: Alterations affecting the mitogen-activated protein kinase pathway are present in the majority of patients with LGS ovarian cancer. Next-generation sequencing analysis revealed deletions and fusions that are not detected by older sequencing approaches. These findings, coupled with the observation that a subset of patients with recurrent LGS ovarian cancer experienced dramatic and durable responses to MEK inhibitor therapy, support additional clinical studies of MEK inhibitors in this disease.

Fig 1.

Analysis of extraordinary responder to selumetinib identifies 15–base pair deletion in MAP2K1 gene. (A) Radiographic response of extraordinary responder to selumetinib therapy. Comparative computed tomography scan images at baseline and after treatment with selumetinib confirming complete radiographic response after 17 months of therapy, which was durable at 4 and 5 years. (B) MAP2K1 deletion in outlier patient's tumor, as displayed using Integrated Genomics Viewer software (Broad Institute, Cambridge, MA). Sixty (8.3%) of 725 reads harbored deletion in tumor tissue, whereas no evidence of deletion was found in 434 reads from germline DNA derived from blood. Schematic below shows 15 nucleotides spanning six codons that were deleted, resulting in loss of five amino acids (QKQKV, underlined) within negative regulatory region of MEK1. (C) Two ribbon diagrams representing crystal structure of wild-type MEK1 on left and predicted structure of MEK1 Q56_V60 deletion on right. Deletion of residues 56 to 60 in MEK1 significantly alters interactions of inhibitory N-terminal helix A with the core kinase (right). In wild-type MEK1 structure, selected helix A residues that interact with core kinase are labeled as follows: MEK1 core kinase (pink), helix A (orange, with residues 56 to 60 in gray), allosteric inhibitor (gold), and guanosine diphosphate (GDP; white sticks). Consequent to deletion of five residues at C-terminus of helix A, registration of helix has shifted, resulting in nonconservative changes to helix A residues that interact with core kinase domain. In this predicted structure, the following are displayed: MEK1 core kinase (teal), truncated helix A (purple), allosteric inhibitor (yellow), and GDP (white sticks). (D) Schematic of MEK1 with alterations identified across cancer types is displayed. Major domains of protein are also shown. Horizontal bars below schematic represent deletions. Triangles represent point mutations and are distributed based on involved residue. Alterations displayed were chosen based on frequency of > 5% observed in COSMIC (Catalogue of Somatic Mutations in Cancer). CRC, colorectal cancer; ERK, extracellular signal-regulated kinase; LCH, Langerhans cell histiocytosis; NSCLC, non–small-cell lung cancer.

Fig 2.

Functional characterization of MEK1 Q56_V60 deletion. (A) Immunoblots showing levels of phosphorylated MEK (pMEK), phosphorylated extracellular signal-regulated kinase (pERK), and phosphorylated ribosomal protein S6 kinase (pRSK) in 293H cells expressing MEK1 Q56_V60 deletion, MEK1 F53L, and wild-type MEK1. (B) Exposure to selumetinib of 293H cells expressing either wild-type MEK1 or MEK1 Q56_V60 deletion results in potent downregulation of pERK levels. (C) Soft agar colony formation assay after stable transfection of NIH-3T3 cells with vector alone, wild-type MEK1, two MEK1 oncogenic mutants (F53L and K57N), and MEK1 Q56_V60 deletion. Number of colonies resulting from MEK1 deletion was significantly increased as compared with vector (P = .032), wild-type MEK1, or F53L and K57N MEK1 mutants (P = .045). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein.

Fig 3.

Low-grade serous (LGS)/serous borderline (SB) ovarian cancers are characterized by frequent mitogen-activated protein kinase pathway alterations. (A) Oncoprint displaying selected genetic alterations identified in cohort of 29 SB and LGS ovarian tumors. (B) Fisher's exact test was used to compare frequency of selected genetic alterations between 11 SB samples, 18 LGS ovarian tumors, and 316 high-grade serous (HGS) ovarian tumors analyzed by The Cancer Genome Atlas. (*) P < .05.

Fig A1.

MEK1 Q56_V60 deletion induces anchorage-independent growth in vitro and accelerates tumor growth in xenograft models. (A) Colony growth in soft agar of NIH-3T3 cells transfected with MEK1 Q56_V60 deletion in presence or absence of AZD6244. (B) Tumor volume of xenografts generated by transfection of NIH-3T3 cells with empty vector, wild-type MEK1, MEK1 Q56_V60 deletion, or MEK1 F53L. Measurements were taken at day 11 postimplantation.

Fig A2.

Schematic of two novel paracentric fusions involving BRAF in low-grade serous tumors. (A) To form MKRN1:BRAF fusion, internal tandem duplication of region of chromosome 7 containing exons 1 to 4 of MKRN1 (blue) and exons 11 to 18 of BRAF (gold) occurs first (shaded box), followed by insertion of this region into intron 4 of MKRN1, juxtaposing exons 1 to 4 of MKRN1 and exons 11 to 18 of BRAF (kinase domain). (B) Schematic of paracentric inversion in chromosome 7, which results in CUL1:BRAF fusion. CUL1 and BRAF are located on sense and antisense strands, respectively. After breakpoint occurs within exon 7 of CUL1 and exon 8 of BRAF, this region undergoes inversion, leading to juxtaposition of exons 1 to 7 of CUL1 and exons 9 to 18 (containing intact kinase domain) of BRAF. Representative sequences from RNA sequencing of tumor harboring fusion are shown below.