Cell-Cycle and DNA-Damage Response Pathway Is Involved In

Published OnlineFirst October 13, 2017; DOI: 10.1158/1078-0432.CCR-17-1582

Biology of Human Tumors Clinical Cancer Research Cell-Cycle and DNA-Damage Response Pathway Is Involved in Leptomeningeal Metastasis of Non–Small Cell Lung Cancer Yun Fan1, Xuehua Zhu2, Yan Xu3, Xuesong Lu4, Yanjun Xu1, Mengzhao Wang3, Haiyan Xu4, Jingyan Ding2, Xin Ye2, Luo Fang5, Zhiyu Huang5, Lei Gong5, Hongyang Lu1, Weimin Mao1, and Min Hu2

Abstract

Purpose: Leptomeningeal metastasis (LM) is a detrimental risk. Intriguingly, low overlapping of somatic protein-changing complication of non–small cell lung cancer (NSCLC) and asso- variants was observed between paired CSF and primary lesions, ciated with poor prognosis. However, the underlying mechanisms exhibiting tumor heterogeneity and genetic divergence. Moreover, of the metastasis process are still poorly understood. genes with CSF-recurrent genomic alterations were predominant- Experimental Design: We performed next-generation panel ly involved in cell-cycle regulation and DNA-damage response sequencing of primary tumor tissue, cerebrospinal ﬂuid (CSF), (DDR), suggesting a role of the pathway in LM development. and matched normal controls from epidermal growth factor Conclusions: Our study has shed light on the genomic varia- receptor (EGFR) mutation-positive NSCLC patients with LM. tions of NSCLC-LM, demonstrated genetic heterogeneity and Results: The status of EGFR-activating mutations was highly divergence, uncovered involvement of cell-cycle and DDR path- concordant between primary tumor and CSF. PIK3CA aberrations way, and paved the way for potential therapeutic approaches to were high in these patients, implicating an association with LM this unmet medical need. Clin Cancer Res; 24(1); 209–16. 2017 AACR.

Introduction LM is defined as the spread of malignant cells to the leptomeninges and throughout the subarachnoid space, and dissemina- Approximately 10% to 15% of patients with non–small cell tion of tumors cells into the cerebrospinal fluid (CSF) compart- lung cancer (NSCLC) in Western countries and 40% to 55% in ment. About 9% to 10% of patients with NSCLC with EGFR- Asia harbor epidermal growth factor receptor (EGFR)-activating activating mutations will develop LM either at initial diagnosis or mutations such as L858R and exon 19 deletion (19Del). EGFR during TKI treatment (9, 10). LM is often associated with poor tyrosine kinase inhibitors (TKIs) have opened up an era of target prognosis and represents a terminal event of NSCLC. The median therapy in NSCLC and markedly extended progression-free sur- overall survival (OS) after LM diagnosis is 3.1 to 11 months, with vival (PFS) of patients with EGFR-activating mutations (1–6). 60% death due to LM or LM together with systemic lesions (9– However, there have been reports of cumulative incidence of 13). So far, there is no effective treatment, though preliminary central nervous system (CNS) metastases over time (7, 8). Diag- clinical activities of osimertinib and AZD3759 in patients with nosis and treatment of leptomeningeal metastases (LM), a form of NSCLC with LM have been reported (14, 15), and other CNS- CNS metastases, remain particularly challenging. penetrant TKIs such as tesevatinib are being developed. Moreover, the mechanisms underlying the metastasis process are not elucidated yet. Further research is warranted to understand the genetic 1Key Laboratory Diagnosis and Treatment Technology on Thoracic Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang, China. 2IMED Asia, AstraZeneca, alternations driving the metastasis and to develop treatment Shanghai, China. 3Department of Respiratory Medicine, Peking Union Medical strategies that improve the survival of patients with NSCLC-LM. College Hospital, Chinese Academy of Medical Sciences, Beijing, China. Plasma circulating tumor DNA (ctDNA) has been shown to 4Research & Development Information, AstraZeneca, Shanghai, China. 5Zhejiang effectively reflect tumor-specific genomic alterations and has been Cancer Hospital, Hangzhou, Zhejiang, China. used to dynamically monitor tumor progression, response, and Note: Supplementary data for this article are available at Clinical Cancer relapse (16). However, due to blood–brain barrier (BBB), patients Research Online (http://clincancerres.aacrjournals.org/). with brain tumors do not present with or present with only low Y. Fan, X. Zhu, and Y. Xu contributed equally to this article. amounts of ctDNA in plasma (17). Recently, a few studies have Corresponding Authors: Min Hu, AstraZeneca, No. 199 Liangjing Road, Zhang- demonstrated that tumor mutations are detectable in the CSF of jiang Hi-tech Park, Pudong New Area, Shanghai 201203, China. Phone: 86-021- patients with various primary and metastatic brain tumors, and 61097820; Fax: 86-021-58387337; E-mail: [email protected]; and ctDNA in CSF recapitulates the genomic landscape of brain Yun Fan, Zhejiang Cancer Hospital, No. 1 Banshan East Road, Hangzhou, Zhejiang tumors better than plasma (18–20). Therefore, CSF may serve as 310022, China. Phone: 86-0571-88122396; Fax: 86-0571-88122396; E-mail: liquid biopsy to monitor brain tumor evolution. In this study, we [email protected] performed next-generation panel sequencing of matched primary doi: 10.1158/1078-0432.CCR-17-1582 tumor tissues, CSF samples and normal controls from 11 patients 2017 American Association for Cancer Research. with EGFR mutation-positive NSCLC to characterize genomic

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the LungPlasma panel (Burning Rock Biotech) covering 168 Translational Relevance cancer-associated genes (Supplementary Table S2). Enriched Approximately 10% of patients with non–small cell lung libraries were sequenced on NextSeq 500 sequencing system cancer (NSCLC) with epidermal growth factor receptor (Illumina) with 150-bp pair-end reads. For specimens from (EGFR)-activating mutations will develop leptomeningeal patients P4-11, targeted panel sequencing was performed using metastases (LMs) either at initial diagnosis or during treatment SeqCap EZ System (Roche Nimblegen) and captured by the of tyrosine kinase inhibitors (TKIs). LMs are often associated customized SeqCap EZ Choice Library (Roche Nimblegen) cov- with poor prognosis and represent a terminal event of NSCLC. ering 403 cancer-associated genes (Supplementary Table S3). So far, there is no effective treatment, and the mechanisms Enriched libraries were sequenced on HiSeq X10 sequencing underlying the metastasis process are not elucidated yet. Here, system (Illumina) with 150-bp pair-end reads. On-target average we performed next-generation panel sequencing of matched sequence depth (before deduplication): FFPE tissues >500Â primary tumors and cerebrospinal fluid (CSF) samples from except 300Â in P6T; blood >2,700Â; CSF >4,000Â except patients with EGFR mutation-positive NSCLC-LM, to charac- 1,880Â in P8C. terize genomic alterations during LM development and to understand signaling pathways implicated in this metastasis Bioinformatics data analysis process. Our results demonstrated the value of CSF as liquid BWA (21) was used for mapping the pair-end reads to human biopsy for monitoring LM evolution, the association of reference genome hg19, samblaster (22) for marking duplicate PIK3CA aberrations with LM risk, and the potential role of reads, and VarDict (23) for detection of single-nucleotide variants the cell-cycle and DNA-damage response pathway in LM (SNV) and insertions and deletions (indels). Information on development. microsatellite instability (MSI) was extracted from the output of VarDict. Analysis of structural rearrangements was carried out using ArrayStudio (OmicSoft). SNVs and indels were called with aberrations during LM development and to understand signaling the criteria of (i) variant allele frequency (VAF) 5% or 1% if pathways implicated in this metastasis process. associated with a COSMIC ID; and (ii) 3 supporting reads and 10 total reads after deduplication. Variant calls of hotspot Materials and Methods mutations and structural rearrangements were manually inspected and rescued. Patients and sample collection For tumor and CSF samples with patient-matched normal The 11 patients with NSCLC-LM were diagnosed at Zhejiang DNA, the somatic status was inferred by removing variants with Cancer Hospital and Peking Union Medical College Hospital VAF 10% in the normal control. For tumor and CSF samples during 2009 to 2015. EGFR mutation status of their primary without paired normal DNA, variants with VAF 10% in any of tumors was initially determined by amplification-refractory muta- the normal samples from other patients were filtered. tion system (ARMS) assay at the hospitals, as one of the inclusion Variants with 1% population prevalence in the Single Nucle- criteria. The study was approved by the hospital research ethics otide Polymorphism Database (dbSNP; https://www.ncbi.nlm. committees, and all patients had signed the informed consent nih.gov/snp/) were removed as common SNPs. form. Formalin-fixed paraffin-embedded (FFPE) tumor and nor- Protein-changing variants refer to SNVs and indels that fall into mal tissues and freshly frozen CSF and whole blood samples were functional categories of frameshift, nonsense, start/stop lost, collected for genomic profiling (Supplementary Table S1). All splice acceptor/donor, missense, and inframe insertion/deletion, FFPE tissues were reviewed by histopathologic assessment. Tumor with the first six types as likely deleterious. tissues contained 30% tumor percentage, and normal tissues had no tumor cell contamination under light microscopy. Results Clinical characteristics of patients with NSCLC-LM DNA extraction The clinical characteristics of the 11 patients with lung adeno- Genomic DNA from FFPE tumor and normal tissues was carcinoma with LM were detailed in Table 1. There were five males isolated using GeneRead DNA FFPE Kit (Qiagen) according to and six females, and the median age was 58 years old (range, 32– the manufacturer's protocol. Total DNA from freshly frozen CSF 67). Their primary tumors carried either EGFR L858R or 19Del as was extracted by QIAamp Circulating Nucleic Acid Kit (Qiagen). determined by ARMS assay, and they received TKI treatment Genomic DNA from whole blood was isolated using QIAamp (gefitinib, erlotinib, or icotinib) 0.1 to 16.8 months (median, DNA Mini kit (Qiagen). 2100 Bioanalyzer (Agilent) spectra and 1.8 months) after NSCLC diagnosis. One of the patients had LM at quantification of total CSF DNA were provided in Supplementary initial diagnosis, and 10 developed LM after 1 to 23.1 months Fig. S1. (median, 7.3 months) of TKI treatment. Three patients had brain metastases (BMs) before they developed LM, and one was diag- Next-generation panel sequencing nosed with LM and BM simultaneously. The patients underwent DNA samples were fragmented to approximately 200-bp peak systemic chemotherapy, intrathecal chemotherapy, or whole- size by sonication before library construction. The Illumina brain radiotherapy (WBRT) during their disease courses. libraries were constructed with KAPA Hyper Prep Kit (Kapa Biosystems) according to the manufacturer's protocol. For sam- Profiling of primary tumor and LM-CSF by next-generation ples from patients P1 to 3, targeted panel sequencing was per- panel sequencing formed using SureSelect XT Target Enrichment System for Illu- To understand the mechanisms underlying LM, we collected mina Pair-end Multiplex Sequencing (Agilent), and captured by paired primary tumor and CSF samples from the 11 patients. The

210 Clin Cancer Res; 24(1) January 1, 2018 Clinical Cancer Research

Table 1. Clinical characteristics of the 11 patients with lung adenocarcinoma with LM

Patient P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 Published OnlineFirstOctober13,2017;DOI:10.1158/1078-0432.CCR-17-1582 clincancerres.aacrjournals.org Sex M M F M M F F F M F F Age 61 65 48 50 61 49 67 58 32 65 45 EGFR mutation type in 19Del L858R L858R L858R L858R 19Del L858R L858R L858R 19Del 19Del primary tumor TNM staging at initial T2N2M1, IIIa T1bN3M1, IV T1bN3M1, IV T4N2M1, IV T4N2M1b, IV T2N2M0, IIIa T4N3M1b, IV T4N3M1b, IV T2aN1M1a, IV T4N3M1b, IV T4N3M1a, IV diagnosis Surgery À0.2 mo / / À0.03 mo / À0.03 mo / / 0 / / BM diagnosis / / / / / 14.43 mo À0.03 mo 5.07 mo / À0.03 mo / LM diagnosis 11.53 mo 8.6 mo 4.47 mo 14 mo À0.23 mo 39.87 mo 8 mo 5.07 mo 21.27 mo 9.1 mo 35.3 mo OS after NSCLC Alive (>44.33 mo) 24.33 mo 5.63 mo 21.63 mo 11.77 mo 58.6 mo 13.97 mo 15.73 mo 23.7 mo 14.1 mo 47.8 mo diagnosis OS after LM diagnosis Alive (>32.8 mo) 15.73 mo 1.17 mo 7.63 mo 12 mo 18.73 mo 5.97 mo 10.67 mo 2.43 mo 5 mo 12.5 mo EGFR-TKI Cmpd Gefitinib Gefitinib Gefitinib Gefitinib Erlotinib Gefitinib, after LM Gefitinib Gefitinib Gefitinib, after Icotinib Gefitinib on September 28, 2021. © 2018American Association for Cancer erlotinib LM erlotinib Research. Start 1.37 mo 1.87 mo 0.2 mo 13 mo 0.1 mo 16.77 mo 0.1 mo 0.23 mo 1.77 mo 2.53 mo 15.03 mo End Till now Lost to Until death Lost to Until death Until death Until death Until death Until death Until death Until death follow-up follow-up PFS 10.17 mo 6.73 mo 4.27 mo 1 mo 9.4 mo 23.1 mo 7.9 mo 4.83 mo 19.5 mo 6.57 mo 20.27 mo Extracranial response Bone metastasis, PR PR SD SD No extracranial lesion PR PR SD SD SD not evaluable after surgery

Systemic chemo NSCLC of Metastasis Leptomeningeal in DDR and Cycle Cell Start 11.3 mo 0 0.07 mo 0.9 mo / 42.17 mo / / / / 1.17 mo End 15.07 mo 17.7 mo 0.07 mo 17.33 mo / 48.3 mo / / / / 11.17 mo Intrathecal chemo Start 11.53 mo 8.6 mo / / 0.13 mo 42.27 mo 8.03 mo 5.17 mo 21.47 mo 9.27 mo 35.53 mo End 14.43 mo 12.33 mo / / 0.6 mo 43.4 mo 8.23 mo 5.4 mo 23.2 mo 9.73 mo 36.17 mo WBRT Start 11.7 mo / / / 0.87 mo 14.87 mo 0.5 mo / 21.93 mo 0.73 mo / End 12.17 mo / / / 2.07 mo 16.27 mo 1.6 mo / 22.07 mo 2.63 mo / lnCne e;2()Jnay1 2018 1, January 24(1) Res; Cancer Clin Sample collection Primary tumor À0.2 mo 0 0.2 mo À0.03 mo 0 0 0 0 0.9 mo 0 14.43 mo CSF 12 mo 10.7 mo 4.53 mo 14 mo 10.37 mo 56.07 mo 8.3 mo 5.3 mo 22.43 mo 9.1 mo 35.47 mo NOTE: Information on sex, age, EGFR mutation type in primary tumor (by ARMS assay), TNM staging at initial diagnosis, surgery, BM diagnosis (by MRI), LM diagnosis (by cytology), survival, EGFR-TKI treatment, systemic/ intrathecal chemotherapy, WBRT and collection of primary tumor/CSF specimens. The time points of surgery, BM/LM diagnosis, treatment start/end, and sample collection were calculated as months from NSCLC diagnosis (by pathology). Tumor responses were assessed according to Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST 1.1). Progression was deﬁned as the development of a new lesion or relapse of any lesions during the treatment course. OS of patient P1 was calculated by data cutoff date (April 25, 2017). Abbreviations: Cmpd, compound; F, female; M, male; mo, months; OS, overall survival; PFS, progression-free survival; PR, partial response; SD, stable disease. 211 Published OnlineFirst October 13, 2017; DOI: 10.1158/1078-0432.CCR-17-1582

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primary tumor tissues were obtained before EGFR-TKI therapy addition, PIK3CA H1047L and PTEN R130Q were identified in and CSF samples during TKI treatment (Table 1). Specimens were the primary tumor of patients P6 and P11, respectively. Although profiled by next-generation panel sequencing on cancer-associ- with a small sample size, the prevalence of PIK3CA mutations in ated genes using patient-matched whole blood or adjacent nor- this NSCLC-LM cohort (five out of 11) is significantly higher than mal tissues as control if available. Somatic variants were called what was reported in general lung adenocarcinoma population— and annotated according to their functional impacts. only five out of 371 and 44 out of 1552 patients with EGFR mutation-positive NSCLC in Western and Asian datasets, respec- Concordance of EGFR-activating mutations in primary tumor tively, carried PIK3CA mutations (P < 0.001; Supplementary Table and LM-CSF S5). These results implicated that aberrations in the PI3K pathway We first investigated whether CSF carried the same EGFR may be associated with NSCLC-LM risk. mutation type(s) as the primary tumor. The status of EGFR- activating mutations was highly concordant between primary Genetic divergence between primary tumors and LM tumor and CSF pairs of all the patients, except in patient P10 To better understand the underlying mechanisms of LM, we whose primary tumor had undetectable EGFR mutation, while performed pair-wise comparison of somatic variants from pri- both L858R and 19Del were detected in CSF (Table 2). We mary tumors and CSF. Among the 11 patients, samples from reasoned that the negative EGFR testing results by panel sequenc- patients P1 to 3 were profiled using a small gene panel with a ing in primary tumor of patient P10 were likely due to low amount 260 kb coverage region (Supplementary Table S2), whereas of input and poor sequence coverage (only 20Â and 29Â effective specimens from patients P4 to 11 were sequenced with a large depth around L858R and 19Del region, respectively). Indeed, panel of 3.6 Mb region (Supplementary Table S3). Hence, to keep 19Del was originally detected in her primary tumor by ARMS the consistency of data interpretation, we focused our analysis on assay (Table 1). Taken together, there were eight patients with the latter batch of results. Interestingly, low overlapping was L858R, one with 19Del, and two with L858R and 19Del dual observed in somatic protein-changing variants between matched mutations. The primary tumor of patient P6 also showed low VAF primary tumor and CSF samples (Fig. 1A). Note that 3.2% to of de novo resistance mutation EGFR T790M (Table 2). No T790M 25.7% (median, 5.8%) of variants from primary tumor were was detected in any of the CSF samples, although the majority of identified in paired CSF, and 5.4% to 32.4% (median, 17.9%) patients developed LM during TKI treatment. of variants in CSF overlapped with those in the primary site. Moreover, characterization of somatic SNVs within protein-cod- PIK3CA Association of mutations with NSCLC-LM ing regions revealed different mutational spectrum of matched EGFR Patients with NSCLC harboring mutations present with primary tumor and CSF (Fig. 1B). There was a consistent trend fi EGFR LM signi cantly more frequently than those with wild-type toward elevated A>G and decreased C>T transitions in CSF P < (9.4% vs. 1.7%, 0.001; ref. 9), suggesting that elevated EGFR samples comparing to paired primary tumors (P < 0.01). Even signaling may promote LM development. We thus screened the more intriguingly, there was a significant difference regarding size presence of selected hotspot mutations and structural rearrange- distribution of indels between primary tumor and CSF (Fig. 1C). ments that are known oncogenic drivers (Supplementary Table CSF harbored more small indels (mostly 1 bp) either in overall or PIK3CA S4). Interestingly, E545K mutation was detected in both MSI regions. By contrast, tumor samples carried substantial num- primary tumor and CSF from four out of 11 patients (Table 2). In bers of large indels (>50 bp). These data suggested a high degree of tumor heterogeneity and genetic divergence during LM Table 2. EGFR mutations and alterations of the PI3K pathway in patients with development. NSCLC-LM Patient Primary tumor LM-CSF Involvement of cell-cycle regulation and DNA-damage P1 EGFR 19Del (41.1%) EGFR 19Del (18.2%) response (DDR) pathway in NSCLC-LM PIK3CA E545K (0.7%) PIK3CA E545K (8.3%) To highlight genomic alternations implicated in the LM pro- P2 EGFR L858R (54%) EGFR L858R (37.3%) PIK3CA PIK3CA cess, we combined the somatic protein-changing variants identi- E545K (16.1%) E545K (4.2%) fi P3 EGFR L858R (63.4%) EGFR L858R (17%) ed either from primary tumor or CSF samples of patients P4 to 11 PIK3CA E545K (9.4%) PIK3CA E545K (17.6%) and grouped them according to their occurrence. In total, there P4 EGFR L858R (30.8%) EGFR L858R (16%) were 4246 unique variants, among which 129 in CSF and 47 in P5 EGFR L858R (35.9%) EGFR L858R (11.3%) tumor showed 3 occurrence in each sample type, respectively PIK3CA E545K (23.7%) PIK3CA E545K (2.8%) (Supplementary Table S6). As the pair-wise comparison indicated EGFR EGFR P6 L858R (28.8%) L858R (63.6%) that primary tumors and LM are highly divergent, we emphasized EGFR T790M (2.1%) PIK3CA H1047L (20%) on the variants prevalent in CSF over primary tumors, or vice P7 EGFR L858R (50%) EGFR L858R (46.9%) versa. Using the cutoff of n 3 in CSF and 1 in tumor, there were P8 EGFR L858R (14%) EGFR L858R (12.5%) 55 CSF-recurrent variants mapping to 40 genes (Supplementary P9 EGFR L858R (34.4%) EGFR L858R (22.4%) Tables S6 and S7). On the contrary, with the same criteria, we only P10 ND EGFR L858R (1.4%) had eight tumor-recurrent variants located to seven genes, among EGFR 19Del (10.4%) EGFR EGFR which four genes overlapped with those of CSF. When we clas- P11 L858R (8.8%) L858R (2.0%) fi EGFR 19Del (13.7%) EGFR 19Del (11.2%) si ed these 43 genes based on existing biological pathway knowl- PTEN R130Q (5.2%) edge, 29 of them involved in cell cycle and DNA damage, NOTE: Mutation status of EGFR, PIK3CA, and PTEN in primary tumor and CSF chromatin remodeling and epigenetics, cytoskeleton remodeling, samples from patients P1–11, as detected by panel sequencing. The VAF of each TGFb, PI3K/AKT, Notch, Wnt or Hedgehog signaling, etc. (Sup- mutation was indicated in parentheses. plementary Table S7). Noteworthy, cell-cycle regulation and DDR Abbreviation: ND, mutation not detected. appeared to be the predominant pathway which harbored

212 Clin Cancer Res; 24(1) January 1, 2018 Clinical Cancer Research

Cell Cycle and DDR in Leptomeningeal Metastasis of NSCLC

Figure 1. Genetic divergence between primary tumors and LM. A, Low overlapping of somatic protein-changing variants between paired primary tumor and CSF samples. The somatic SNVs and indels affecting protein sequences from each patient were categorized into three groups—tumor unique, CSF unique, or shared. B, Different mutational spectrum of paired primary tumor and CSF samples on somatic SNVs within protein-coding regions. Abbreviations: T, tumor; C, CSF. P values: 0.003906 and 0.007813 by the one-sided Wilcoxon rank-sum test for elevated A>G and decreased C>T in CSF comparing with paired primary tumors, respectively. C, Different size distribution of indels within overall (left) or MSI (right) regions between primary tumor and CSF samples of patients P4 to 11. ÃÃ, P < 0.01; Ã, P < 0.05, by the paired Wilcoxon test.

CSF-recurrent alterations (Fig. 2 and Supplementary Table S7), other (Supplementary Table S8). Therefore, to gain a compre- implicating a role in LM development. Specifically, recurrent hensive view of the genomic landscape of LM, we used total CSF variants in DNA double-strand break (DSB) repair genes BRCA2, DNA for profiling in this study. RAD50, and RAD51AP2 and mismatch repair (MMR) genes MLH3 Among the 11 cases here, T790M was not detected in any of the and MSH3 were enriched in CSF comparing to primary tumors CSF samples, even in patients who developed LM during EGFR- (Fig. 2). Subsets of genes in chromatin remodeling such as TKI treatment. Though we cannot fully exclude the possibility that ARID1B and ATRX also possessed variants more prevalently in concurrent systemic/intrathecal chemotherapy or WBRT may CSF than primary tumor. There was no significant correlation suppress acquiring of the T790M resistance mutation, our results between the presence of recurrent variants in cell-cycle and DDR are consistent with previous reports demonstrating that the emer- pathway and systemic/intrathecal chemotherapy or WBRT (P > gence of T790M in CNS lesions was relatively rare comparing to 0.49 by the two-sided Wilcoxon rank-sum test; Fig. 2). extracranial sites (24–26). This can probably partially be explained by the low exposure of first-generation EGFR-TKIs in CSF due to poor BBB penetration property (26), so that intracra- Discussion nial tumor cells are shielded from the effects of compounds and Our genomic analysis of paired primary tumors and CSF thus will not readily acquire the secondary T790M gatekeeper samples from patients with NSCLC-LM has revealed several mutation. aspects regarding the diagnosis and treatment of this devastating Patients with EGFR mutation-positive NSCLC develop CNS disease. First of all, the EGFR mutation type(s) in CSF were highly metastases more frequently than those with wild-type EGFR (9, concordant with those in primary tumor (100% sensitivity and 27). On the other hand, only about 10% of patients with EGFR 90.9% concordance), suggesting that mutation testing in CSF is a mutation-positive NSCLC develop LM (9, 10), suggesting that potentially powerful molecular diagnostic approach for LM to there are other cofactors contributing to this metastasis process. guide precision medicine. There are two types of DNA sources in We found a high prevalence of PIK3CA mutations in our patient CSF—cell-free DNA (cfDNA) from supernatants and genomic cohort, comparing to the general EGFR mutation-positive lung DNA from cell pellets. It was reported that a higher fraction of adenocarcinoma population, implicating an association with cfDNA is tumor-derived than pellet DNA samples (20). However, LM risk. A similar finding was reported in squamous cell lung in our experience, the EGFR mutation type(s) in CSF cfDNA and cancer (SqCLC) in which patients with PI3K-aberrant tumors pellet DNA sometimes can be different and complement each had more aggressive disease marked by worse survival, higher

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Figure 2. Involvement of cell-cycle regulation and the DDR pathway in LM development. Genes with variants recurrent in CSF (n 3 in CSF and 1in primary tumor) or tumor (n 3in primary tumor and 1 in CSF) were mapped to biological pathways. Cell-cycle and DNA damage presented as the most dominant pathway, followed by chromatin remodeling and epigenetics. The presence of recurrent variants in each gene/sample/patient was shown as light blue (variants of uncertain signiﬁcance) or dark blue (likely deleterious) blocks, and potential exposure of specimens to each treatment regimen was indicated as black blocks. Exposure to treatment was deﬁned as (i) lesion was diagnosed before or during the treatment and (ii) sample was collected during or after the treatment.

metastatic burden, and greater incidence of BM (27% vs. 0% in tumor tissues. We observed more recurrence of variants in CSF others, P < 0.001; ref. 28). It remains to be seen clinically than primary tumors, even though the latter carried a larger whether combination of PI3K inhibitors with EGFR-TKIs can number of total variants. Moreover, when we cataloged genes treat or prevent CNS metastases. with CSF-recurrent variants, the cell-cycle and DDR and chroma- Although LM and their lung primaries carried the same EGFR tin remodeling showed predominance among all the mapped activating mutation(s), we observed a high degree of genetic pathways, implicating that LM may harbor a greater degree of divergence and clonal heterogeneity between the paired samples, genome instability resulting from deficiency in DNA DSB repair, as illustrated by the low proportion of shared variants, different MMR, and chromosome maintenance. Although such genomic mutational spectrum of SNVs and size distribution of indels. abnormalities might be induced by exposure of LM to various Studies of early-stage NSCLC reported a significant amount of treatment regimens, it seemed that there was no significant mutational and copy-number heterogeneity across regions of association between the recurrence of variants in cell-cycle and primary lung tumors (29, 30). Greater heterogeneity was shown DDR pathway and systemic/intrathecal chemotherapy or WBRT. in the metastatic SqCLC, with as little as 15% of mutations shared Whether EGFR-TKI treatment played any role here requires further between primary lung cancer and BM pairs (28). In our hand, study. Nonetheless, even though these treatments may interfere 2.1% to 12.5% (median, 4.1%) of total variants overlapped with cell cycle and cause DNA damage in tumor cells, their between primary tumors and LM-CSF. The sources of genetic immediate mutagenesis effects are likely random across the whole divergence can be multifold, for example, different originating genome, rather than on particular sets of genes and coordinates. leptomeningeal metastatic subclone from the predominant pri- The latter event is more the product of selection and evolution, mary lung cancer clone, additional genomic alterations acquired which requires relatively long time to develop. Therefore, the during the metastasis process, and clonal evolution under tumor chance that enrichment of recurrent variants in DSB and MMR microenvironment and in response to various treatments. genes in CSF is induced directly by treatments per se is low, if any. In the light of the genetic heterogeneity observed in primary Aberrations of ATM, BRCA1 and BRCA2 were observed at sub- tumor-LM pairs, we asked whether CSF samples shared common stantially higher frequencies in metastatic castration-resistant genomic features across patients which differentiated them from prostate cancer (mCRPC) compared with those in primary

214 Clin Cancer Res; 24(1) January 1, 2018 Clinical Cancer Research

Cell Cycle and DDR in Leptomeningeal Metastasis of NSCLC

prostate cancers (31–33), suggesting that enrichment of altera- came from recurrent variants (3 in one sample type), which tions in a DDR pathway might be a shared feature of more were then mapped to genes and pathways. Given that artifacts are advanced, metastatic cancer. It has been reported that mutational most likely random, the chance of the same artifact will show up signature comprising of large numbers of small indels (mostly in multiple samples is very low. Moreover, recurrent alternations 1 bp) at nucleotide repeats, whose pattern is often termed MSI, is of cell-cycle and DDR genes were more enriched in CSF, which characteristic of cancers with defective DNA MMR (34). Consis- were freshly frozen samples, further relieving the concern that they tently, we observed significantly more 1-bp size indels within MSI were illusions caused by FFPE artifacts. regions in CSF than primary tumors, supporting the fact that the In conclusion, we have demonstrated the value of CSF as liquid genotype (e.g., aberrations in MMR genes) led to a phenotype biopsy for monitoring LM evolution and directing precision (i.e., genomic instability). medicine. While future study with larger sample size will be Genomic instability, one of the underlying hallmarks of cancers desired to cross-validate our findings, our study is the first explo- (35), provides a platform for genomic alternations driving tumor ration to show that cell-cycle and DDR pathway might play an formation but also represents an Achilles' heel for the therapeutic important role in NSCLC-LM development and point to potential opportunities of synthetic lethality. Recent advancements in new therapeutic strategies targeting this unmet clinical need. PARP inhibitors have proven the clinical utility of DDR-based therapies in ovarian cancer, breast cancer, and mCRPC patients Disclosure of Potential Conflicts of Interest with deleterious BRCA and ATM mutations, or even beyond, No potential conflicts of interest were disclosed. DNA-repair defects in general (36–43). Deficiency of ARID1A, the paralog of ARID1B, has been shown to impair DNA-damage Authors' Contributions checkpoint and sensitize cancer cells to PARP inhibitors in vitro Conception and design: Y. Fan, M. Wang, X. Ye, L. Gong, W. Mao, M. Hu and in vivo (44). Trials in colorectal cancer have illustrated that Development of methodology: Y. Fan, X. Zhu, M. Wang, X. Ye, L. Gong, M. Hu fi Acquisition of data (provided animals, acquired and managed patients, MMR status predicted clinical bene t of immune checkpoint provided facilities, etc.): Y. Fan, X. Zhu, Y. Xu, Y. Xu, J. Ding, X. Ye, Z. Huang, blockade (45), and PD-1 antibody has been approved for the L. Gong, H. Lu treatment of unresectable or metastatic solid tumors having MSI- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, high or MMR defects. Thus, it will be of great interest to see besides computational analysis): Y. Fan, X. Zhu, Y. Xu, X. Lu, M. Wang, H. Xu, L. Fang, BBB-penetrable EGFR-TKIs, whether NSCLC-LM exhibits thera- M. Hu peutic vulnerability to DDR inhibitors or immunotherapy. Writing, review, and/or revision of the manuscript: Y. Fan, X. Zhu, Y. Xu, X. Lu, M. Wang, X. Ye, L. Gong, M. Hu One of the technical challenges in our study is the variant Administrative, technical, or material support (i.e., reporting or organizing calling on FFPE tumor tissues. FFPE specimens tend to have more data, constructing databases): Y. Fan, M. Wang, X. Ye, L. Gong sequencing artifacts than freshly prepared samples due to frag- Study supervision: Y. Fan, M. Wang, W. Mao, M. Hu mentation, cytosine deamination, and oxidative damage. In fact, the mutation load of primary tumors calculated from our panel Acknowledgments sequencing is within the high range of what was reported for lung We thank the patients for providing their samples in this study. We acknowl- adenocarcinoma (34), implicating that some variants are prob- edge Hulin Han (AstraZeneca) for pathology review of FFPE tissues and Changting Liu and Xinying Su (AstraZeneca) for sample management. ably FFPE artifacts. Taking that into consideration, the degree of heterogeneity we observed between primary tumor-LM pairs may The costs of publication of this article were defrayed in part by the payment of be overestimated as the majority of artifact variants are random, page charges. This article must therefore be hereby marked advertisement in and thus there will be low concordance across samples. So far, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. there is no optimal way to fully address this issue. However, we believe that our findings on cell-cycle and DDR pathway are not Received June 2, 2017; revised August 28, 2017; accepted October 10, 2017; compromised by this imperfection. The particular conclusion published OnlineFirst October 13, 2017.

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216 Clin Cancer Res; 24(1) January 1, 2018 Clinical Cancer Research

Cell-Cycle and DNA-Damage Response Pathway Is Involved in Leptomeningeal Metastasis of Non−Small Cell Lung Cancer

Yun Fan, Xuehua Zhu, Yan Xu, et al.

Clin Cancer Res 2018;24:209-216. Published OnlineFirst October 13, 2017.

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