Advertisement

Somatic Mutations in LRRK2 Identify a Subset of Invasive Mammary Carcinomas Associated with High Mutation Burden

Open ArchivePublished:September 12, 2020DOI:https://doi.org/10.1016/j.ajpath.2020.08.010
      Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson disease. Although LRRK2-related Parkinson disease patients have a heightened risk of certain nonskin cancers, including breast cancer, it is unknown whether LRRK2 somatic mutations occur and are associated with breast cancer. The objective of this study was to evaluate the occurrence of LRRK2 somatic mutations in breast cancer and the clinicopathologic features associated with LRRK2-mutated tumors. Using The Cancer Genome Atlas Breast Cancer Project, somatic LRRK2 DNA sequence information was obtained for 93 cases, of which 17 cases (18%) with 18 mutations were identified. LRRK2-mutated mammary carcinomas are enriched with stop-gain, truncating mutations predicted to result in loss of function; missense mutations frequently targeted the GTPase and kinase domains. Tumors displayed predominantly high-grade morphology with abundant granular eosinophilic cytoplasm, resembling mitochondria-rich apocrine-like carcinomas. Exploration of the genomic landscape of LRRK2-mutated carcinomas yielded frequent TP53 deactivation and a remarkably high tumor mutation burden. More important, breast cancers with LRRK2 mutations are associated with reduced patient survival compared with The Cancer Genome Atlas Breast Cancer Project cohort. These findings, for the first time, show that somatic LRRK2 mutations occur frequently in breast cancer, and the high mutation burden seen in this subset of tumors suggests that LRRK2 mutations may herald benefit from immune checkpoint inhibition.
      Cancer and Parkinson disease (PD), the most common movement neurodegenerative disorder, at first glance seem pathophysiologically dissimilar. Although cancer results from an uncontrolled regenerative process, PD is characterized by neuronal cell death. Although these disease processes appear to be mechanistically contradictory, an association between PD and cancer has been established, suggesting overlap in the underlying biochemical dysfunction.
      • West A.B.
      • Dawson V.L.
      • Dawson T.M.
      To die or grow: Parkinson's disease and cancer.
      Studies have shown both increases and decreases in cancer incidence among PD subjects, depending on the cancer type and whether PD was familial or idiopathic.
      • Rugbjerg K.
      • Friis S.
      • Lassen C.F.
      • Ritz B.
      • Olsen J.H.
      Malignant melanoma, breast cancer and other cancers in patients with Parkinson's disease.
      • Bajaj A.
      • Driver J.A.
      • Schernhammer E.S.
      Parkinson's disease and cancer risk: a systematic review and meta-analysis.
      • Agalliu I.
      • Ortega R.A.
      • Luciano M.S.
      • Mirelman A.
      • Pont-Sunyer C.
      • Brockmann K.
      • Vilas D.
      • Tolosa E.
      • Berg D.
      • Waro B.
      • Glickman A.
      • Raymond D.
      • Inzelberg R.
      • Ruiz-Martinez J.
      • Mondragon E.
      • Friedman E.
      • Hassin-Baer S.
      • Alcalay R.N.
      • Mejia-Santana H.
      • Aasly J.
      • Foroud T.
      • Marder K.
      • Giladi N.
      • Bressman S.
      • Saunders-Pullman R.
      Cancer outcomes among Parkinson's disease patients with leucine rich repeat kinase 2 mutations, idiopathic Parkinson's disease patients, and nonaffected controls.
      • Zhang Q.
      • Guo S.
      • Zhang X.
      • Tang S.
      • Shao W.
      • Han X.
      • Wang L.
      • Du Y.
      Inverse relationship between cancer and Alzheimer's disease: a systemic review meta-analysis.
      Interestingly, breast cancer is consistently observed to be increased among PD subjects compared with controls.
      Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial PD, and like idiopathic PD, carriers of LRRK2 mutations are at increased risk for breast cancer.
      • Rugbjerg K.
      • Friis S.
      • Lassen C.F.
      • Ritz B.
      • Olsen J.H.
      Malignant melanoma, breast cancer and other cancers in patients with Parkinson's disease.
      ,
      • Agalliu I.
      • San Luciano M.
      • Mirelman A.
      • Giladi N.
      • Waro B.
      • Aasly J.
      • Inzelberg R.
      • Hassin-Baer S.
      • Friedman E.
      • Ruiz-Martinez J.
      • Marti-Masso J.F.
      • Orr-Urtreger A.
      • Bressman S.
      • Saunders-Pullman R.
      Higher frequency of certain cancers in LRRK2 G2019S mutation carriers with Parkinson disease: a pooled analysis.
      • Waro B.J.
      • Aasly J.O.
      Exploring cancer in LRRK2 mutation carriers and idiopathic Parkinson's disease.
      • Saunders-Pullman R.
      • Barrett M.J.
      • Stanley K.M.
      • Luciano M.S.
      • Shanker V.
      • Severt L.
      • Hunt A.
      • Raymond D.
      • Ozelius L.J.
      • Bressman S.B.
      LRRK2 G2019S mutations are associated with an increased cancer risk in Parkinson disease.
      • Inzelberg R.
      • Cohen O.S.
      • Aharon-Peretz J.
      • Schlesinger I.
      • Gershoni-Baruch R.
      • Djaldetti R.
      • Nitsan Z.
      • Ephraty L.
      • Tunkel O.
      • Kozlova E.
      • Inzelberg L.
      • Kaplan N.
      • Fixler Mehr T.
      • Mory A.
      • Dagan E.
      • Schechtman E.
      • Friedman E.
      • Hassin-Baer S.
      The LRRK2 G2019S mutation is associated with Parkinson disease and concomitant non-skin cancers.
      Among the subset of PD patients with an LRRK2 mutation, cancer may arise before a PD diagnosis, suggesting that this may be the sentinel event in at least a subset of LRRK2 mutation carriers.
      • Waro B.J.
      • Aasly J.O.
      Exploring cancer in LRRK2 mutation carriers and idiopathic Parkinson's disease.
      Despite this observation, the link between LRRK2 mutations and breast cancer has been largely unexplored. We report, for the first time, somatic LRRK2 mutations in breast cancer, and that this subset of tumors displays high-risk features with a high mutation burden, making them promising candidates for immune checkpoint therapy.

      Materials and Methods

      Clinical information and simple somatic mutations, including single-nucleotide variants and small insertions/deletions, from whole exome sequencing and copy number information available through The Cancer Genome Atlas (TCGA) Breast Cancer Project were downloaded from the Genomic Data Commons Data Portal. For an extensive description of the tumor variant annotation workflow performed by the Genomic Data Commons DNA-sequencing analysis pipeline, please see National Cancer Institute Genomic Data Commons (https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/DNA_Seq_Variant_Calling_Pipeline/#tumor-only-variant-annotation-workflow, last accessed July 10, 2020). The simple somatic mutations, single-nucleotide variants, and small insertions/deletions were subjected to the current guidelines by the American College of Medical Genetics. Briefly, LRRK2 nonsense, frameshift, and canonical splice-site sequence variants were considered pathologically significant because all of these types of mutations lead to either a stop codon or truncated, incomplete, or typically nonfunctional protein product. The remaining variants were compared with published data or were evaluated using in silico analyses. SIFT, MutationTaster, and PolyPhen were used to evaluate missense variants, which were only considered pathogenic if consistently predicted deleterious by all three programs (Table 1). Those variants that did not meet these strict criteria for pathogenicity and are of unknown significance are included in Supplemental Table S1. No germ-line variants were included in the analysis.
      Table 1LRRK2 Mutations, Predicted Protein Change, Variant Type, and Domain Affected from TCGA
      Case no.BarcodeCodingProteinVariant typeDomain
      1TCGA-E9-A243c.1274T>Cp.L425PMissenseArmadillo
      2TCGA-AN-A0AKc.1605delp.K535Nfs∗13FrameshiftArmadillo
      3TCGA-AC-A23Hc.2573C>Gp.S858
      Denotes a stop codon.
      NonsenseInterdomain
      4TCGA-C8-A132c.2765T>Ap.L922
      Denotes a stop codon.
      NonsenseInterdomain
      5TCGA-AN-A046c.3266A>G

      c.4469C>T
      p.N1089S

      p.A1490V
      Missense

      Missense
      Leucine-rich repeat and

      ROC
      6TCGA-D8-A1JPc.3910C>Tp.L1304FMissenseLeucine-rich repeat
      7TCGA-D8-A1X5c.4334C>Tp.S1445FMissenseROC
      8TCGA-BH-A0E9c.4514_4520delp.I1505Rfs∗16FrameshiftROC
      9TCGA-D8-A1XKc.4915dupp.R1639Kfs∗13FrameshiftCOR
      10TCGA-AN-A0XOc.5072T>Cp.I1691TMissenseCOR
      11TCGA-AO-A0J4c.5117C>Gp.S1706
      Denotes a stop codon.
      NonsenseCOR
      12TCGA-AN-A0XUc.5312G>Cp.R1771TMissenseCOR
      13TCGA-D8-A1XZc.5455G>Cp.G1819RMissenseCOR
      14TCGA-S3-AA11c.5861C>Tp.S1954FMissenseKinase
      15TCGA-C8-A3M7c.6322G>Ap.E2108KMissenseKinase
      16TCGA-B6-A0I8c.7153G>Ap.G2385RMissenseWD40
      17TCGA-AC-A3QPc.958+1G>Ap.?Splice siteArmadillo
      COR, C-terminal of Roc; ROC, Ras of complex; TCGA, The Cancer Genome Atlas.
      Denotes a stop codon.
      Processed, normalized, and segmented data from Affymetrix SNP 6.0 (Applied Biosystems, Foster City, CA) arrays were used to generate masked (E.R.P.C.) copy number segment files, and numeric focal-level copy number variation values were obtained using GISTIC2 (Genomic Data Commons Copy Number Variation Analysis Pipeline). Copy number variation with values <−0.3 were categorized as loss and values >0.3 were categorized as gain. Nonsynonymous mutation rates from 988 of 1098 cases computed by the Broad Genome Data Analysis Center using MutSigCV version 0.9 (Broad Institute, Cambridge, MA) were obtained from FireBrowse.
      • Carrion M.D.P.
      • Marsicano S.
      • Daniele F.
      • Marte A.
      • Pischedda F.
      • Di Cairano E.
      • Piovesana E.
      • von Zweydorf F.
      • Kremmer E.
      • Gloeckner C.J.
      • Onofri F.
      • Perego C.
      • Piccoli G.
      The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.
      The rate of nonsynonymous gene mutations was determined by the Broad Genome Data Analysis Center using MutSigCV version 0.9.
      Whole slide images of diagnostic sections were downloaded from the Genomic Data Commons Data Portal and assessed by at least one breast pathologist on ImageScope. Select clinical data, follow-up times, and vital status were available for 1096 of 1098 patients. Overall survival was defined as date of diagnosis to the date of last clinical follow-up or death. Simple somatic mutations were also downloaded and analyzed from the cBio Cancer Genomics Portal.
      • Cerami E.
      • Gao J.
      • Dogrusoz U.
      • Gross B.E.
      • Sumer S.O.
      • Aksoy B.A.
      • Jacobsen A.
      • Byrne C.J.
      • Heuer M.L.
      • Larsson E.
      • Antipin Y.
      • Reva B.
      • Goldberg A.P.
      • Sander C.
      • Schultz N.
      The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.
      ,
      • Gao J.
      • Aksoy B.A.
      • Dogrusoz U.
      • Dresdner G.
      • Gross B.
      • Sumer S.O.
      • Sun Y.
      • Jacobsen A.
      • Sinha R.
      • Larsson E.
      • Cerami E.
      • Sander C.
      • Schultz N.
      Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.
      Statistical analysis included comparisons between the groups using the χ2 test, U-test (where tests for homoscedasticity failed to demonstrate equal variances), and Mantel-Cox log-rank test.

      Results

      Curated clinical, mutation, and copy number information was available for 1098 of TCGA project-level breast cancer cases. Because of the large size of LRRK2 (51 exons spanning over 148 Kb) and the stringency of gene-level curation of TCGA bioinformatics pipeline, only 93 cases with complete LRRK2 sequence were available. A total of 28 LRRK2 somatic variants (synonymous, nonsynonymous, and deep intronic) were identified, of which 18 were considered potentially pathogenic in 17 cases, including 11 missense, 3 nonsense, 3 frameshift, and 1 splice-site variant (Table 1). LRRK2 encodes a multidomain protein with an enzymatic core composed of a Ras of complex GTPase domain in tandem with a C-terminal of Roc domain and a kinase domain, which localizes to the cytoplasm and mitochondrial membrane.
      • Biskup S.
      • Moore D.J.
      • Celsi F.
      • Higashi S.
      • West A.B.
      • Andrabi S.A.
      • Kurkinen K.
      • Yu S.W.
      • Savitt J.M.
      • Waldvogel H.J.
      • Faull R.L.
      • Emson P.C.
      • Torp R.
      • Ottersen O.P.
      • Dawson T.M.
      • Dawson V.L.
      Localization of LRRK2 to membranous and vesicular structures in mammalian brain.
      A large fraction of the alterations were stop-gain, inactivating mutations (41% of cases). Interestingly, missense variants commonly targeted the Ras of complex, C-terminal of Roc, and kinase domains (Table 1). A single case was characterized by the PD-associated LRRK2 p.G2385R mutation. Cases with wild-type/polymorphic variant LRRK2 sequences and cases with variants with insufficient evidence for pathogenicity are listed in Supplemental Tables S1 and S2.
      All patients with tumors harboring LRRK2 mutations were female, had a median age at diagnosis of 61 (range, 41 to 90) years, and most presented with stage II or III disease (82%); these characteristics were similar to the study cohort (Supplemental Table S3).
      • Amin M.B.
      • Edge S.B.
      AJCC Cancer Staging Manual.
      High-grade, Nottingham grade 3, ductal and pleomorphic lobular morphologies prevailed (82% and 18%, respectively) (Table 2)
      • Amin M.B.
      • Edge S.B.
      AJCC Cancer Staging Manual.
      ; interestingly, a granular eosinophilic cytoplasm, consistent with mitochondrial-rich apocrine-like features, was present in 71% of LRRK2-mutated cases (Figure 1A and Table 2). Clinical predictive marker information was available for 15 patients with LRRK2 mutations. Most cases were estrogen receptor–positive disease (67%), including three cases that were additionally positive for human epidermal growth factor receptor 2 (Supplemental Table S3), four cases that were triple negative, and no case that was positive for human epidermal growth factor receptor 2 in the absence of estrogen receptor expression (Supplemental Table S3). Androgen receptor expression information was not available.
      Table 2Clinicopathologic Summary of Breast Cancer Patients Harboring an LRRK2 Mutation
      Case no.Age, yearsHistologyGradeLateralityQuadrantpTpNpMStage
      • Amin M.B.
      • Edge S.B.
      AJCC Cancer Staging Manual.
      152Ductal with apocrine features and zonal necrosis3RightUIQT2N0M0IIA
      277Ductal with apocrine and micropapillary features and zonal necrosis3LeftUOQT2N0M0IIA
      390Ductal with apocrine features3RightUIQ, LIQT2NXM0IIA
      456Ductal with apocrine features3LeftNOST2N1M0IIB
      569Ductal with apocrine features3RightLIQT2N0M0IIA
      674Ductal with clear cell features3LeftUIQT1N0M0IA
      781Ductal with apocrine features and zonal necrosis3RightUOQ, LIQT2N3MXIIIC
      853Pleomorphic lobular with apocrine features3RightNOST2N1M0IIB
      955Ductal with prominent lymphocytic infiltrate and zonal necrosis3LeftLIQT2N1MXIIB
      1059Ductal with apocrine and cribriform features2LeftUOQT2N1M0IIIA
      1141Ductal with prominent lymphocytic infiltrate and zonal necrosis3LeftUOQT1N0M0IA
      1255Ductal with clear cell features and zonal necrosis3LeftUOQT2N0M0IIA
      1382Ductal with apocrine features, prominent lymphocytic infiltrate, and zonal necrosis3RightLIQT1N2M0IIIA
      1468Ductal, NOS3RightNOST2N0M0IIA
      1561Pleomorphic lobular with apocrine features2LeftUIQT4N0M0IIIB
      1647Ductal with apocrine features3RightUIQT1NXM0IA
      1780Pleomorphic lobular with apocrine features2RightNOST2N1MXIIB
      Staging information per American Joint Committee on Cancer, eighth edition.
      • Amin M.B.
      • Edge S.B.
      AJCC Cancer Staging Manual.
      LIQ, lower inner quadrant; NOS, not otherwise specified; UIQ, upper inner quadrant; UOQ, upper outer quadrant.
      Figure thumbnail gr1
      Figure 1Clinicopathologic features of LRRK2-mutated invasive mammary carcinomas. A: Abundant granular eosinophilic cytoplasm, with mitochondrial-rich apocrine-like features, prevailed in LRRK2-mutated tumors. B: Box plot of nonsilent mutations per megabase of DNA in tumors with LRRK2 mutations compared with The Cancer Genome Atlas Breast Cancer Project (TCGA-BRCA) cohort, including cases with no LRRK2 mutations or indeterminate LRRK2 status. C: Kaplan-Meier overall survival (OS) stratified by LRRK2 mutation status. Patients with tumors harboring an LRRK2 mutation display an OS inferior to patients relative to TCGA-BRCA cohort, including cases with no LRRK2 mutations or indeterminate LRRK2 status, with 3-year OS of 31% (95% CI, 0.9%–74%) versus 84% (95% CI, 79%–87%), respectively. Asterisks represent extreme outliers, as defined as three times the interquartile range. Of note, breast cancer–specific survival data are not available in TCGA-BRCA cohort. n = 16 tumors with LRRK2 mutations (B); n = 972 for TCGA-BRCA cohort (B); n = 17 patients with tumors harboring an LRRK2 mutation (C); n = 1079 cases with no LRRK2 mutations or indeterminate LRRK2 status (C). Original magnification, ×200 (A). HR, hazard ratio.
      To explore the genomic landscape of LRRK2-mutated tumors, single-nucleotide variants and copy number changes in commonly altered genes in breast cancer were assessed.
      Cancer Genome Atlas Network
      Comprehensive molecular portraits of human breast tumours.
      TP53 alterations, including stop-gain mutations and copy number losses, were the most common event (65%) (Supplemental Figure S1). Furthermore, LRRK2-mutated carcinomas displayed a high mutation burden compared with the remainder of the breast cancer cohort (mean, 16.6 versus 1.4 nonsilent mutations per megabase, respectively; U-test P = 0.003) (Figure 1B). In fact, the two cases with the highest tumor mutation burdens in TCGA Breast Cancer Project harbored LRRK2 mutations. LRRK2-mutated carcinomas showed similarly high mutation burdens when only cases with complete LRRK2 sequence were considered, despite the small sample size (Supplemental Figure S2). Consistent with a high-risk phenotype, subjects with LRRK2 mutations had a statistically significant inferior overall survival, including 3-year survival rates of 31% and 84% among patients with LRRK2 mutations and the remainder of the TGCA Breast Cancer Project, respectively, with a hazard ratio of 3.42 (95% CI, 1.08–10.80; P = 0.036) (Figure 1C). Similar overall survival trends were observed when LRRK2-sequenced cases alone were examined, albeit this study was underpowered (Supplemental Figure S3).

      Discussion

      We uncovered a previously unrecognized role of LRRK2 in breast cancer, whereby tumors harboring a somatic LRRK2 mutation may constitute a distinct clinicopathologic entity with increased tumor mutation burden and high-risk clinical behavior. Immune checkpoint inhibitors have emerged as effective oncologic therapy, leveraging adaptive immune responses, and detecting error-prone DNA repair deficiencies predicts responses to these drugs, presumably due to the production of mutation-associated neoantigens.
      • Le D.T.
      • Durham J.N.
      • Smith K.N.
      • Wang H.
      • Bartlett B.R.
      • Aulakh L.K.
      • et al.
      Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.
      As a corollary, tumor mutation burden has been used as a predictive biomarker in this therapeutic context, albeit with imperfect correlation with responses.
      • Chan T.A.
      • Yarchoan M.
      • Jaffee E.
      • Swanton C.
      • Quezada S.A.
      • Stenzinger A.
      • Peters S.
      Development of tumor mutation burden as an immunotherapy biomarker: utility for the oncology clinic.
      Detailed biochemical studies will be necessary to dissect whether LRRK2 alterations are causative of increased mutation burdens or a sensitive marker of LRRK2-independent mutagenesis. Nonetheless, the high fraction of inactivating stop-gain mutations in LRRK2 would suggest a mechanistic relationship, not a bystander effect, and evidence exists supporting a DNA maintenance role for LRRK2.
      • Chen Z.
      • Cao Z.
      • Zhang W.
      • Gu M.
      • Zhou Z.D.
      • Li B.
      • Li J.
      • Tan E.K.
      • Zeng L.
      LRRK2 interacts with ATM and regulates Mdm2-p53 cell proliferation axis in response to genotoxic stress.
      ,
      • Sanders L.H.
      • Laganiere J.
      • Cooper O.
      • Mak S.K.
      • Vu B.J.
      • Huang Y.A.
      • Paschon D.E.
      • Vangipuram M.
      • Sundararajan R.
      • Urnov F.D.
      • Langston J.W.
      • Gregory P.D.
      • Zhang H.S.
      • Greenamyre J.T.
      • Isacson O.
      • Schule B.
      LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.
      Assessing for LRRK2 integrity may thus identify a subset of genomically unstable breast tumors with vulnerability to immune checkpoint inhibition.
      A recent study reported that PD patients with germ-line LRRK2 p.G2019S mutations were at an increased risk for leukemia and colon cancer, suggesting that the overall baseline cancer risk may be elevated among LRRK2 mutation carriers.
      • Agalliu I.
      • Ortega R.A.
      • Luciano M.S.
      • Mirelman A.
      • Pont-Sunyer C.
      • Brockmann K.
      • Vilas D.
      • Tolosa E.
      • Berg D.
      • Waro B.
      • Glickman A.
      • Raymond D.
      • Inzelberg R.
      • Ruiz-Martinez J.
      • Mondragon E.
      • Friedman E.
      • Hassin-Baer S.
      • Alcalay R.N.
      • Mejia-Santana H.
      • Aasly J.
      • Foroud T.
      • Marder K.
      • Giladi N.
      • Bressman S.
      • Saunders-Pullman R.
      Cancer outcomes among Parkinson's disease patients with leucine rich repeat kinase 2 mutations, idiopathic Parkinson's disease patients, and nonaffected controls.
      Of note, a PD-associated variant, p.G2385R, was somatically detected in one breast cancer case, although the functional impact of this particular variant is still poorly understood.
      • Carrion M.D.P.
      • Marsicano S.
      • Daniele F.
      • Marte A.
      • Pischedda F.
      • Di Cairano E.
      • Piovesana E.
      • von Zweydorf F.
      • Kremmer E.
      • Gloeckner C.J.
      • Onofri F.
      • Perego C.
      • Piccoli G.
      The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.
      ,
      • Ho D.H.
      • Jang J.
      • Joe E.H.
      • Son I.
      • Seo H.
      • Seol W.
      G2385R and I2020T mutations increase LRRK2 GTPase activity.
      ,
      • Rudenko I.N.
      • Kaganovich A.
      • Hauser D.N.
      • Beylina A.
      • Chia R.
      • Ding J.
      • Maric D.
      • Jaffe H.
      • Cookson M.R.
      The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation.
      It will be informative to investigate whether LRRK2 mutations occur in other primary sites, supporting a broader tumor suppressor function for LRRK2, and future mechanistic studies may examine a direct role in cancer pathogenesis.
      • Greenman C.
      • Stephens P.
      • Smith R.
      • Dalgliesh G.L.
      • Hunter C.
      • Bignell G.
      • et al.
      Patterns of somatic mutation in human cancer genomes.
      Several PD-associated genes, including LRRK2, have been implicated in cell cycle control and tied to DNA integrity, which are classic tumor suppressor functions frequently targeted for inactivation in cancer.
      • West A.B.
      • Dawson V.L.
      • Dawson T.M.
      To die or grow: Parkinson's disease and cancer.
      ,
      • Chen Z.
      • Cao Z.
      • Zhang W.
      • Gu M.
      • Zhou Z.D.
      • Li B.
      • Li J.
      • Tan E.K.
      • Zeng L.
      LRRK2 interacts with ATM and regulates Mdm2-p53 cell proliferation axis in response to genotoxic stress.
      ,
      • Sanders L.H.
      • Laganiere J.
      • Cooper O.
      • Mak S.K.
      • Vu B.J.
      • Huang Y.A.
      • Paschon D.E.
      • Vangipuram M.
      • Sundararajan R.
      • Urnov F.D.
      • Langston J.W.
      • Gregory P.D.
      • Zhang H.S.
      • Greenamyre J.T.
      • Isacson O.
      • Schule B.
      LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.
      The somatic LRRK2 variants identified herein were predicted to be loss of function, including a high proportion of stop-gain mutations, albeit biochemical functional characterizations are still necessary to substantiate the pathogenic nature of these variants. Yet, biochemical studies examining the common PD-linked p.G2019S mutation have described a gain of function,
      • Kett L.R.
      • Dauer W.T.
      Leucine-rich repeat kinase 2 for beginners: six key questions.
      and clinical trials for PD using LRRK2 kinase inhibitors and antisense oligonucleotides are currently underway (clinicaltrials.gov, last accessed July 10, 2020). In light of our results, it may be prudent to assess the impact of LRRK2 dysregulation on carcinogenesis as an adverse effect of LRRK2-directed PD therapy.
      One of the limitations of our study is that of the 1098 breast cancer cases in TCGA, only a fraction had available LRRK2 sequence information, limiting the statistical power of these results. To address this, somatic LRRK2 pathogenic variants were identified in the cBio Cancer Genomics Portal in primary and metastatic breast cancer (Supplemental Table S4), showing some overlap with TCGA Breast Cancer Project, including another missense variant targeting leucine 425 and nonsense mutation p.S858∗. The fact that stop-gain mutations and homozygous deletions are reported in this external data set additionally supports our view that LRRK2 inactivation is a breast cancer–associated phenomenon (Supplemental Table S4). Furthermore, as a retrospective study, the findings were limited by the paucity of clinical information available for the evaluation of therapeutic outcomes. Notwithstanding these limitations, the relative frequency of LRRK2 mutations detected is strong evidence for its biomedical and oncologic relevance. On the basis of our collective findings, the inclusion of LRRK2 in commercial somatic sequencing panels may be warranted.
      In conclusion, somatic LRRK2 mutations are associated with breast cancer, and these findings shed light onto the mechanistic underpinnings of LRRK2-related neurodegeneration versus neoplastic disease. The identification of LRRK2 mutations in breast cancer may provide an opportunity for risk stratification and new treatment paradigms.

      Acknowledgments

      We thank Drs. Michael Lutz, Daniel Laskowitz, and Rex Bentley for thoughtful manuscript review and discussion.

      Supplemental Data

      • Supplemental Figure S1

        Genomic landscape of LRRK2 carcinomas. ∗Nonsynonymous missense changes are represented by gray boxes, and stop-gain mutations (ie, nonsense, frameshift, and splice site) are black boxes. Copy number gains are represented by red boxes, and losses are blue boxes. ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; Mut., mutations; N, not available; PR, progesterone receptor.

      Figure thumbnail figs1
      Supplemental Figure S2Tumor mutation burden in LRRK2-mutated carcinomas. Box plot of nonsilent mutations per megabase of DNA in tumors with LRRK2 mutations compared with cases confirmed with no LRRK2 mutations and the remainder of The Cancer Genome Atlas Breast Cancer Project (TCGA-BRCA) cohort. The mean mutation burden among cases with a confirmed LRRK2 pathogenic mutation, compared with those with LRRK2 sequence information but without a pathogenic mutation or TCGA-BRCA cohort, was 16.6 versus 2.2 nonsilent mutations per megabase, respectively (U-test P = 0.377). Comparing the mean mutation burden among cases with a confirmed LRRK2 pathogenic mutation with TCGA-BRCA cohort was 16.6 versus 1.3 nonsilent mutations per megabase, respectively (U-test P = 0.002). Circles represent outliers (1.5 times interquartile range), and asterisks represent extreme outliers, as defined as three times the interquartile range. n = 16 tumors with LRRK2 mutations; n = 69 cases with no LRRK2 mutations; n = 903 for the remainder of TCGA-BRCA cohort.
      Figure thumbnail figs2
      Supplemental Figure S3Kaplan-Meier overall survival (OS) stratified by LRRK2 mutation status. Patients with tumors harboring an LRRK2 mutation (gray line) display an OS inferior to patients relative to both those with LRRK2 sequence and no pathogenic mutation (dashed line) and those with indeterminate LRRK2 status (black line). The 3-year OS rates were 47%, 93% and 90%, respectively, with the overall log-rank P = 0.082. n = 17 patients with tumors harboring an LRRK2 mutation; n = 76 patients with LRRK2 sequence and no pathogenic mutation; n = 1003 patients with indeterminate LRRK2 status.

      References

        • West A.B.
        • Dawson V.L.
        • Dawson T.M.
        To die or grow: Parkinson's disease and cancer.
        Trends Neurosci. 2005; 28: 348-352
        • Rugbjerg K.
        • Friis S.
        • Lassen C.F.
        • Ritz B.
        • Olsen J.H.
        Malignant melanoma, breast cancer and other cancers in patients with Parkinson's disease.
        Int J Cancer. 2012; 131: 1904-1911
        • Bajaj A.
        • Driver J.A.
        • Schernhammer E.S.
        Parkinson's disease and cancer risk: a systematic review and meta-analysis.
        Cancer Causes Control. 2010; 21: 697-707
        • Agalliu I.
        • Ortega R.A.
        • Luciano M.S.
        • Mirelman A.
        • Pont-Sunyer C.
        • Brockmann K.
        • Vilas D.
        • Tolosa E.
        • Berg D.
        • Waro B.
        • Glickman A.
        • Raymond D.
        • Inzelberg R.
        • Ruiz-Martinez J.
        • Mondragon E.
        • Friedman E.
        • Hassin-Baer S.
        • Alcalay R.N.
        • Mejia-Santana H.
        • Aasly J.
        • Foroud T.
        • Marder K.
        • Giladi N.
        • Bressman S.
        • Saunders-Pullman R.
        Cancer outcomes among Parkinson's disease patients with leucine rich repeat kinase 2 mutations, idiopathic Parkinson's disease patients, and nonaffected controls.
        Mov Disord. 2019; 34: 1392-1398
        • Zhang Q.
        • Guo S.
        • Zhang X.
        • Tang S.
        • Shao W.
        • Han X.
        • Wang L.
        • Du Y.
        Inverse relationship between cancer and Alzheimer's disease: a systemic review meta-analysis.
        Neurol Sci. 2015; 36: 1987-1994
        • Agalliu I.
        • San Luciano M.
        • Mirelman A.
        • Giladi N.
        • Waro B.
        • Aasly J.
        • Inzelberg R.
        • Hassin-Baer S.
        • Friedman E.
        • Ruiz-Martinez J.
        • Marti-Masso J.F.
        • Orr-Urtreger A.
        • Bressman S.
        • Saunders-Pullman R.
        Higher frequency of certain cancers in LRRK2 G2019S mutation carriers with Parkinson disease: a pooled analysis.
        JAMA Neurol. 2015; 72: 58-65
        • Waro B.J.
        • Aasly J.O.
        Exploring cancer in LRRK2 mutation carriers and idiopathic Parkinson's disease.
        Brain Behav. 2018; 8: e00858
        • Saunders-Pullman R.
        • Barrett M.J.
        • Stanley K.M.
        • Luciano M.S.
        • Shanker V.
        • Severt L.
        • Hunt A.
        • Raymond D.
        • Ozelius L.J.
        • Bressman S.B.
        LRRK2 G2019S mutations are associated with an increased cancer risk in Parkinson disease.
        Mov Disord. 2010; 25: 2536-2541
        • Inzelberg R.
        • Cohen O.S.
        • Aharon-Peretz J.
        • Schlesinger I.
        • Gershoni-Baruch R.
        • Djaldetti R.
        • Nitsan Z.
        • Ephraty L.
        • Tunkel O.
        • Kozlova E.
        • Inzelberg L.
        • Kaplan N.
        • Fixler Mehr T.
        • Mory A.
        • Dagan E.
        • Schechtman E.
        • Friedman E.
        • Hassin-Baer S.
        The LRRK2 G2019S mutation is associated with Parkinson disease and concomitant non-skin cancers.
        Neurology. 2012; 78: 781-786
        • Carrion M.D.P.
        • Marsicano S.
        • Daniele F.
        • Marte A.
        • Pischedda F.
        • Di Cairano E.
        • Piovesana E.
        • von Zweydorf F.
        • Kremmer E.
        • Gloeckner C.J.
        • Onofri F.
        • Perego C.
        • Piccoli G.
        The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.
        Sci Rep. 2017; 7: 5377
        • Cerami E.
        • Gao J.
        • Dogrusoz U.
        • Gross B.E.
        • Sumer S.O.
        • Aksoy B.A.
        • Jacobsen A.
        • Byrne C.J.
        • Heuer M.L.
        • Larsson E.
        • Antipin Y.
        • Reva B.
        • Goldberg A.P.
        • Sander C.
        • Schultz N.
        The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.
        Cancer Discov. 2012; 2: 401-404
        • Gao J.
        • Aksoy B.A.
        • Dogrusoz U.
        • Dresdner G.
        • Gross B.
        • Sumer S.O.
        • Sun Y.
        • Jacobsen A.
        • Sinha R.
        • Larsson E.
        • Cerami E.
        • Sander C.
        • Schultz N.
        Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.
        Sci Signal. 2013; 6: pl1
        • Biskup S.
        • Moore D.J.
        • Celsi F.
        • Higashi S.
        • West A.B.
        • Andrabi S.A.
        • Kurkinen K.
        • Yu S.W.
        • Savitt J.M.
        • Waldvogel H.J.
        • Faull R.L.
        • Emson P.C.
        • Torp R.
        • Ottersen O.P.
        • Dawson T.M.
        • Dawson V.L.
        Localization of LRRK2 to membranous and vesicular structures in mammalian brain.
        Ann Neurol. 2006; 60: 557-569
        • Amin M.B.
        • Edge S.B.
        AJCC Cancer Staging Manual.
        Springer, New York, NY2017
        • Cancer Genome Atlas Network
        Comprehensive molecular portraits of human breast tumours.
        Nature. 2012; 490: 61-70
        • Le D.T.
        • Durham J.N.
        • Smith K.N.
        • Wang H.
        • Bartlett B.R.
        • Aulakh L.K.
        • et al.
        Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.
        Science. 2017; 357: 409-413
        • Chan T.A.
        • Yarchoan M.
        • Jaffee E.
        • Swanton C.
        • Quezada S.A.
        • Stenzinger A.
        • Peters S.
        Development of tumor mutation burden as an immunotherapy biomarker: utility for the oncology clinic.
        Ann Oncol. 2019; 30: 44-56
        • Chen Z.
        • Cao Z.
        • Zhang W.
        • Gu M.
        • Zhou Z.D.
        • Li B.
        • Li J.
        • Tan E.K.
        • Zeng L.
        LRRK2 interacts with ATM and regulates Mdm2-p53 cell proliferation axis in response to genotoxic stress.
        Hum Mol Genet. 2017; 26: 4494-4505
        • Sanders L.H.
        • Laganiere J.
        • Cooper O.
        • Mak S.K.
        • Vu B.J.
        • Huang Y.A.
        • Paschon D.E.
        • Vangipuram M.
        • Sundararajan R.
        • Urnov F.D.
        • Langston J.W.
        • Gregory P.D.
        • Zhang H.S.
        • Greenamyre J.T.
        • Isacson O.
        • Schule B.
        LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.
        Neurobiol Dis. 2014; 62: 381-386
        • Ho D.H.
        • Jang J.
        • Joe E.H.
        • Son I.
        • Seo H.
        • Seol W.
        G2385R and I2020T mutations increase LRRK2 GTPase activity.
        Biomed Res Int. 2016; 2016: 7917128
        • Rudenko I.N.
        • Kaganovich A.
        • Hauser D.N.
        • Beylina A.
        • Chia R.
        • Ding J.
        • Maric D.
        • Jaffe H.
        • Cookson M.R.
        The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation.
        Biochem J. 2012; 446: 99-111
        • Greenman C.
        • Stephens P.
        • Smith R.
        • Dalgliesh G.L.
        • Hunter C.
        • Bignell G.
        • et al.
        Patterns of somatic mutation in human cancer genomes.
        Nature. 2007; 446: 153-158
        • Kett L.R.
        • Dauer W.T.
        Leucine-rich repeat kinase 2 for beginners: six key questions.
        Cold Spring Harb Perspect Med. 2012; 2: a009407