Whole-exome sequencing identifies recurrent AKT1mutations in sclerosing hemangioma of lungSeung-Hyun Junga,b, Min Sung Kimc, Sung-Hak Leed, Hyun-Chun Parka,b, Hyun Joo Choid, Leeso Maengd, Ki Ouk Mind,Jeana Kimd, Tae In Parke, Ok Ran Shind, Tae-Jung Kimd, Haidong Xuf, Kyo Young Leed, Tae-Min Kimg, Sang Yong Songh,Charles Leei,j, Yeun-Jun Chunga,b,1, and Sug Hyung Leec,1

aDepartment of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea; bDepartment of Integrated Research Center forGenome Polymorphism, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea; cDepartment of Pathology, College of Medicine, TheCatholic University of Korea, Seoul 06591, Korea; dHospital Pathology, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea;eDepartment of Pathology, Kyungpook National University School of Medicine, Daegu 41944, Korea; fCenter of Laboratory, Yanbian University Hospital,Yanji 133000, China; gMedical Informatics, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea; hDepartment of Pathology,Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon 16419, Korea; iThe Jackson Laboratory for Genomic Medicine, Farmington,CT 06032; and jDepartment of Life Sciences, Ewha Woman’s University, Seoul 03760, Korea

Edited by John D. Minna, University of Texas Southwestern Medical Center, Dallas, TX, and accepted by Editorial Board Member Tak W. Mak August 4, 2016(received for review May 2, 2016)

Pulmonary sclerosing hemangioma (PSH) is a benign tumor withtwo cell populations (epithelial and stromal cells), for which genomicprofiles remain unknown. We conducted exome sequencing of 44PSHs and identified recurrent somatic mutations of AKT1 (43.2%)and β-catenin (4.5%). We used a second subset of 24 PSHs to confirmthe high frequency of AKT1 mutations (overall 31/68, 45.6%; p.E17K,33.8%) and recurrent β-catenin mutations (overall 3 of 68, 4.4%). Ofthe PSHs without AKT1 mutations, two exhibited AKT1 copy gain.AKT1 mutations existed in both epithelial and stromal cells. In twoseparate PSHs from one patient, we observed two different AKT1mutations, indicating they were not disseminated but independentarising tumors. Because the AKT1 mutations were not found to co-occur with β-cateninmutations (or any other known driver alterations)in any of the PSHs studied, we speculate that this may be the single-most common driver alteration to develop PSHs. Our study revealedgenomic differences between PSHs and lung adenocarcinomas, includ-ing a high rate of AKT1 mutation in PSHs. These genomic features ofPSH identified in the present study provide clues to understanding thebiology of PSH and for differential genomic diagnosis of lung tumors.

pulmonary sclerosing hemangioma | whole-exome sequencing |AKT1 mutation | copy number alteration

Pulmonary sclerosing hemangioma (PSH) is a benign tumorthat usually presents as a solitary, well-defined mass in the

lung (1). PSH predominantly affects females (1:5) with a higherincidence in the Far East (2). As the name indicates, PSH is oftenhemorrhagic and sclerotic. Histologically, the tumor cells in PSHconsist of two cell types (cuboidal epithelial and polygonal stromalcells) (3). Immunohistochemical and ultrastructural studies haveidentified that both cells are derived from undifferentiated re-spiratory epithelium that is the histologic origin of lung adeno-carcinoma as well. Previous studies have shown that PSH andadenocarcinoma in the lung share some immunohistochemical andgenetic features. For example, expression of TTF-1, which plays acrucial role in normal lung function and morphogenesis, is commonto PSH and lung adenocarcinoma (3). In addition, allelic imbalanceand CpG island methylation in some loci have been reported inthese two tumors (4, 5). However, whereas many driver genes forlung adenocarcinomas have been identified, for somatic mutations,there have not been any candidate driver mutations identified inPSHs, except for low-frequency mutations in β-catenin and TP53(6, 7). Frequent somatic mutations identified in lung adenocarci-nomas, such as KRAS and EGFR, have not been detected in PSH,suggesting that genomic alterations of these two lung tumors mightbe different from each other. Furthermore, there is no evidence ofPSH progression to lung cancers. These earlier data indicate thatdespite the common cellular origin of PSH and lung adenocarci-noma, genetic mechanisms for their development may be different.

Somatic genetic alteration is a driving force for the development oftumor. Even a benign tumor contains somatic mutations, albeit lesscommon than in a malignant tumor. For example, uterine leiomyomaand breast fibroadenoma, common benign tumors, harbor recurrentmutations in MED12 (8, 9). Based on the established concept thatPSH is a true tumor, we hypothesize that it may harbor somaticmutations. For a comprehensive elucidation of genetic alterations incancers, genomes of many tumors have been studied by using whole-exome (WES) or whole-genome sequencing analysis (10–12). How-ever, to date, such high-throughput sequencing data on PSH islacking. In this study we analyzed genomes of the PSH by WES.

ResultsWhole-Exome Sequencing. We conducted a comprehensive ex-amination of genetic alterations (somatic mutations and copynumber alterations, CNAs) in 44 cases of PSH: 8 fresh-frozenand 36 formalin-fixed paraffin-embedded (FFPE), from 43 pa-tients (two separate PSHs were from one patient), with matchednormal tissues using WES. Most of the patients were female(91%) and the median age was 52 y (range 12–74 y) (Table S1).Coverages of the sequencing depth were mean of 156× forPSHs and 152× for matched normal, with an average of 95% ofbases covered by at least 20 reads in each sample, respectively(Table S2).

Significance

This report is an in-depth genetic profiling of pulmonary scle-rosing hemangioma (PSH). We have discovered that PSH harborrecurrent AKT1 mutations (45.6%), most of which were AKT1p.E17K mutations. This mutation may be the single-most com-mon driver alteration to develop PSHs. In contrast to lung ade-nocarcinoma, PSH genomes harbor only a single driver mutation(AKT1 or β-catenin), which may provide clues to understandingthe benign biology of PSH and for differential genomic diagnosisof lung tumors.

Author contributions: Y.-J.C. and S.H.L. designed research; Y.-J.C. and S.H.L. performed re-search; S.-H.J., M.S.K., S.-H.L., H.-C.P., H.J.C., L.M., K.O.M., J.K., T.I.P., O.R.S., T.-J.K., H.X.,K.Y.L., T.-M.K., and S.Y.S. analyzed data; and S.-H.J., C.L., Y.-J.C., and S.H.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.D.M. is a Guest Editor invited by the EditorialBoard.

Freely available online through the PNAS open access option.

Data deposition: The sequences have been deposited in the NCBI Sequence Read Archivedatabase, www.ncbi.nlm.nih.gov/sra (Project ID: PRJNA297066).1To whom correspondence may be addressed. Email: yejun@catholic.ac.kr or suhulee@catholic.ac.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1606946113/-/DCSupplemental.

10672–10677 | PNAS | September 20, 2016 | vol. 113 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1606946113

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 1,

202

1

A total of 672 nonsilent mutations were identified in the 44PSH genomes (12 mutations per genome, range 0–76) (Fig. 1Aand Dataset S1), corresponding to a mean rate of 0.3 somaticmutations per megabase. This finding is similar to the rates ofother benign tumors [e.g., leiomyoma (13) (0.24 per megabase)and fibroadenoma (9) (0.11 per megabase)] but much lower thanthose observed in lung adenocarcinoma (10) (8.9 per megabase),lung squamous carcinoma (11) (8.1 per megabase), and othercancers (14) (Fig. 1B and Fig. S1). C > T substitutions are themost common mutation type (49.3%; 27.8% at CpG and 21.5%at non-CpG context) in PSHs (Fig. 1A). In contrast, C > Asubstitutions, the most common in lung cancer (26.8%), is lesscommon in the PSHs (14.1%), suggesting that tumorigenic eventsin PSH may be different from those in lung cancer.

AKT1 and β-Catenin Mutations in PSH Genomes. In the PSHs, non-silent, recurrent mutations were found in AKT1 (19 of 44 PSHs:43.2%), CTNNB1 (β-catenin) (2 of 44), and ARID1B (2 of 44)(Fig. 2A). We also identified nonsilent mutations in an additionaleight genes, which overlapped with both the Cancer GeneCensus (15) and Cancer Drivers Database (16): APC, BLM,PLCG1, FAS, ATRX, EP300, KMT2D, and ATM (Fig. 2A), but noTP53 mutations were observed. Among these mutations, threetruncating mutations were identified in tumor-suppressor genes(ARID1B, KMT2D, and ATM). To address whether these muta-tions could be causally implicated in the PSH development, weperformed MutSigCV (14) and OncodriveFM (17) analyses andfound that only AKT1 was significantly mutated in PSH genomes(q-value < 0.01). Key mutations were validated using Sanger se-quencing or digital PCR (Fig. S2 and Table S3). Clinical andhistopathological parameters could not distinguish AKT1 muta-tion (+) and (−) cases of PSHs (P > 0.05).

AKT1 is a serine/threonine kinase that stimulates many cancer-related processes, including cell proliferation, survival, andgrowth (18). All 19 AKT1 mutations identified (11 p.E17K,1 p.E17K/p.I19L, 4 p.Q79K/p.W80R, 1 p.Q79K/p.W80G, 1 p.E49K/p.N53H, and 1 p.L52R/p.F55Y) were localized to the pleckstrinhomology domain (Fig. 2B), which is crucial for membrane locali-zation and downstream activation of AKT1 (19). Not only AKT1p.E17K, but also p.N53-, p.F55-, p.Q79-, and p.W80-alteringmutations are known to promote growth factor-independentcell proliferation compared with wild-type AKT1 (20). Tofurther assess the recurrence of AKT1 mutations in PSHs,we performed Sanger sequencing of AKT1 in an independentvalidation set (24 FFPE PSHs), which confirmed the high fre-quency of AKT1 mutations in PSHs [12 of 24 (50%): 11 p.E17Kand 1 p.Q79K/p.W80R] (Table 1 and Fig. 2B). Among PSHswith p.E17K mutations, all but one harbored a single AKT1mutation. Those PSHs with non-p.E17K mutations harbored ad-ditional AKT1 mutations (“double” AKT1 mutations). All doubleAKT1 mutations were detected in the identical sequencing readswith similar variant allele frequency (VAF) (Fig. S3), suggestingthat the double AKT1 mutations occurred in cis configuration inthe same cells. The overall AKT1mutation prevalence in PSHs was45.6% (31 of 68), most of which were p.E17K (23 of 68, 33.8%).Recurrent AKT1mutations, especially p.E17K, have been detectedin breast cancers (4–8%) (20, 21) but rarely in the other cancers,including lung cancers (0–0.9%) (10–12).As for β-catenin mutations, p.S37F was identified in two dif-

ferent PSHs in the discovery set and p.G38C in a PSH in thevalidation set (3 of 68, 4.4%). β-catenin p.S37F is a hotspotmutation reported in many tumors (22, 23) and β-catenin mu-tations have been previously reported in PSHs (1 of 37, 2.7%,p.S33C) (6). AKT1 and β-cateninmutations did not co-occur in anyof the PSHs we studied here.

Fig. 1. Landscape of somatic alterations in the PSH genomes. (A) The numbers of somatic nonsilent mutations (Upper) and types of base pair substitutions(Lower) are shown. (B) Mutation frequency in PSHs compared with those in other tumor types. Black bars represent the median values. Lung adenocarcinoma(LUAD) and lung squamous cell carcinoma (LUSC) data are from The Cancer Genome Atlas (TCGA) by Lawrence at al. (14). Fibroadenoma and leiomyoma dataare from reports by Lim et al. (9) and Mehine et al. (13), respectively.

Fig. 2. Cancer-related mutations in the PSH genomes, and a schematic diagram of AKT1 mutations. (A) Cancer-related mutations according to WES in the44 PSH cases (discovery set). (B) Schematic diagram of the AKT1mutations in PSHs (Upper). The x axis represents amino acid positions. The y axis represents thenumber of mutations. Distribution of AKT1 mutations in the discovery (44 cases) and validation (24 cases) sets are indicated.

Jung et al. PNAS | September 20, 2016 | vol. 113 | no. 38 | 10673

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 1,

202

1

Mutation Profiles in Multiple PSHs in One Patient. Although multiplePSHs is a rare entity (24), one patient (SH06) in our study pre-sented with two separate masses in the upper (tumor 1, T1) andlower (tumor 2, T2) lobes. To investigate their clonal origins, wecompared the tumors’ germ-line and somatic variants. Almost all ofthe germ-line variants in the two PSHmasses were shared with theirmatched normal (96.1% for T1 and 93.0% for T2), whereas somaticvariants identified in the two PSH masses were mutually exclusive,indicating distinct clonal origins (i.e., double primary tumors) (Fig.3). Interestingly, ATK1 mutations were identified in both lesions,but the variants were different from each other (p.L52R/p.F55Y inT1 and p.E17K in T2).

AKT1 Mutations in PSHs from Histologically Different Cells. Evidencehas shown, by analyzing microsatellite patterns, that both cu-boidal epithelial and polygonal stromal cells in PSHs could bemonoclonal (25), but this hypothesis needs more evidence. Inaddition, there are still doubts about the true neoplastic natureof the components (entrapped normal epithelial cells and non-neoplastic stromal cells). We performed microdissections toprocure two cell types in three PSHs and analyzed somatic andgerm-line variant status, including the AKT1 mutation in the twotypes of cells. We found AKT1 p.E17K mutations in both celltypes in the PSHs (Fig. 4), suggesting their uniclonal origin. Tofurther validate this finding, we performed Sanger sequencing ofsix somatic and two germ-line variants in both epithelial andstromal cells. Regardless of somatic and germ-line event, all ofthe variants were detected in both cell types (Fig. S4).

CNA and Loss of Heterozygosity. To characterize PSH genomicalterations at a copy number level, we analyzed the same 44 PSHsusing the WES data (Table S4). Arm-level somatic CNAs weredetected on 1q, 5p, 5q, 8p, 8q, 14q, 19p, and 19q (gains) and 13q,15q, 16q, and 19p (losses) (7 of 44, 15.9%) (Fig. 5A), which is farfewer than that in lung cancers (10, 11), but only 1q and 14q gainswere recurrent in the PSHs. The most common CNA was 14q gains(3 of 44, 6.8%), where AKT1 resides (Fig. 5B). One of the threePSHs with 14q gains also harbored AKT1 mutations, whereas the

other two did not. In total, 33 PSHs (48.5%) harbored either anAKT1 mutation or 14q copy gain.We also assessed the B-allele profiles and identified three copy-

neutral loss-of-heterozygosity (LOH) events (Fig. 6). Two of theseevents (SH48 and SH55) harbored LOH on 14q and co-occurredwith AKT1 p.E17K mutation. Mean VAF of AKT1 mutations inthe two PSHs with 14q LOHwas 2.5 times higher than that without14p LOH (65.4% vs. 25.6%, respectively) (Fig. 6B), suggesting thatthe 14q LOH might have occurred after AKT1 mutation events inthese cases. Wide distribution of AKT1 VAFs may be a result ofsubclonal nature of AKT1 mutations in some samples or nonneo-plastic cell contamination. When looking at the histology of PSHs,we observed that nonneoplastic stromal components (for example,inflammatory cells) besides the neoplastic polygonal stromal cellswere embedded in the analyzed tissues for WES at various degrees(Fig. S5 and Table S3). Adjusted VAFs considering tumor cellcontents in AKT1-mutated cases were 25–49% in non-LOH casesand 88–97% in LOH cases (Table S3), suggesting that the AKT1mutations in PSH may be clonal. The other one (SH18) harbored59-megabase–sized LOH on chromosome 10 (10q22.2-q26.3),which has been observed in PSHs previously (4). Tumor-suppressorgenes PTEN and TCF7L2 reside in this area.

Pathway Analysis. To further gain insights into the role of genomicalterations underlying PSH development, we analyzed the genomedata through pathway analysis using the Kyoto Encyclopedia ofGenes and Genomes (KEGG) database. Several mutated genesin PSHs were significantly enriched for cell survival, proliferation,growth, and angiogenesis activities (Table S5). Of note, AKT1 acti-vation is closely connected to these pathways (Fig. 7A). We identifiedmutations in multiple components of the AKT/GSK3/β-catenin sig-nalings, including CTNNB1, APC, and TCF7L1. In addition, a copyloss of STK11 and mutations in mammalian target of rapamycin(MTOR) and EIF4E2 in PI3K/AKT/mTOR signaling pathways, aswell as mutations in MYT1 and FOXO1 responsible for cell cycleregulation and cell survival, were detected in the PSH genomes (Fig.7B). Interestingly, several gene alterations are related to the VEGFsignaling pathway that activates angiogenesis, possibly suggestingtheir roles in the vascular phenotype (“hemangioma”) of PSH. AKT1has been engaged as a target in ongoing clinical trials (26), suggestingthat AKT1 inhibitors may have potential therapeutic value in PSH.

DiscussionIn this study, we attempted to identify the genetic alterations ofPSH genomes by WES. First, we observed highly frequent AKT1gene alterations (somatic mutations and copy gains) in the PSHgenomes. Based on the current knowledge of AKT1 mutations(oncogenic activities and recurrent hotspot mutations) (20, 27,28), our data suggest that AKT1 gene alterations are the mostcommon genetic driver that might contribute to the PSH de-velopment. Recurrent AKT1 mutation was first reported inbreast cancers and subsequently in other tumors (29). In these

Table 1. Somatic mutations in AKT1 and CTNNB1

Gene

Discovery set (n = 44) Validation set (n = 24) Combined set (n = 68)

Mut WT Mut rate (%) Mut WT Mut rate (%) Mut WT Mut rate (%)

AKT1 19 25 43.2 12 12 50.0 31 37 45.6p.E17K 11 33 25.0 11 13 45.8 22 46 32.4p.E17K and p.I19L 1 43 2.3 0 24 0 1 67 1.5p.E49K and p.N53H 1 43 2.3 0 24 0 1 67 1.5p.L52R and p.F55Y 1 43 2.3 0 24 0 1 67 1.5p.Q79K and p.W80R 4 40 9.1 1 23 4.2 5 63 7.4p.Q79K and p.W80G 1 43 2.3 0 24 0 1 67 1.5

CTNNB1 2 42 4.5 1 23 4.2 3 65 4.4p.S37F 2 42 4.5 0 24 0 2 66 2.9p.G38C 0 44 0 1 23 4.2 1 67 1.5

Mut, mutation; WT, wild type.

Fig. 3. Mutation profiles of two separate PSHs from one patient. (Left)Correlation heat map of germ-line variants in the two PSHs with thematched normal. (Right) Somatic mutations identified in the two separatePSHs (SH06-T1 and SH06-T2) with the three functional categories.

10674 | www.pnas.org/cgi/doi/10.1073/pnas.1606946113 Jung et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 1,

202

1

tumors, however, the frequency of AKT1 mutations was muchlower (∼8%) than that of the PSHs in our study. When combiningAKT1mutations with AKT1 copy gain, AKT1 genetic alterations inPSH reached 48.5%, which is the highest incidence of AKT1 al-terations in human tumors (the COSMIC database). We also ob-served β-catenin mutation in 4.4% of the PSHs that was mutuallyexclusive with AKT1 mutation in PSHs. In addition, several tumor-related genes, including ARID1B, APC, BLM, PLCG1, FAS, ATRX,EP300, KMT2D, and ATM were detected in the PSHs at low inci-dences (2.3–4.5%). As for CNAs, 15.9% of the PSHs harboredalterations, whereas most PSHs (84.1%) did not exhibit any no-ticeable CNAs. Such a high incidence of oncogenic AKT1 alter-ations and a low incidence of other mutations, as well as few CNAsin the PSHs, suggest that AKT1 alterations are likely key players inPSH development. Other mutations and CNAs might possiblycontribute to PSH pathogenesis, but their causal roles or the roles inconjunction with AKT1 signaling remain to be clarified.Of note, either AKT1 or β-cateninmutation in malignant tumors

usually co-occurs with other driver genomic alterations (22, 30,31). However, AKT1 mutations in benign tumors, such as me-ningiomas and PSHs, and β-catenin mutations in pilomatricomado not usually co-occur with each other or other known drivermutations (32–34). It is possible that AKT1 or β-catenin mutationalone is able to produce a benign tumor but not a malignant tu-mor. AKT1 mutation itself has a weak transforming activity (35),which might make the cells proliferate and change morphologybut not acquire the capability to further progress to malignancy.The histogenesis of the dual cells present in PSHs has long puzzled

scientists. One theory suggests that both cuboidal epithelial and po-lygonal stromal cells represent a neoplastic origin by identifyingmonoclonality of these two cells (3). The other theory suggests thatthe polygonal cell population may represent neoplastic cells, whereasthe cuboidal cells represent entrapped epithelial cells from immu-nohistochemical analysis (25). Our study identified that both of thecell components in PSHs, cuboidal epithelial and polygonal stromal,harbored the same somatic and germ-line variants, supporting theformer theory. Our data suggest that both cells are a true neoplasticcomponent and that they may be histologically different but notgenetically. Some nonneoplastic respiratory epithelial cells maybe entrapped in the PSHs, but many of the cuboidal epithelial cells,especially deep inside of the PSHs, may represent neoplastic cells. Itcould be possible that the microdissection was not specific enoughand that both components were present in the preparations. How-ever, this possibility, if any, with a minor contamination of stromalcells into epithelial cells, may be low because the height of mutantpeaks in the sequencing (Fig. 4 and Fig. S4) are similar (even higher

in epithelial cells in some cases) between the two cells. Furthermore,we discovered that multinodular PSHs in a patient harbored twodifferent AKT1mutation variants, indicating that the multinodularityin PSH may not result from metastasis.Tumor-suppressor genes constitute the largest group of heredi-

tary cancer genes, whereas only a few oncogenes—including RET,MET, KIT, and AKT1—are associated with hereditary tumor de-velopment (36). For example, Proteus syndrome, a congenital dis-ease involving overgrowth of skin, muscles, fatty tissues, and bloodvessels, as well as pulmonary cysts and venous dilatation, is causedby somatic mosaic mutations of AKT1 p.E17K (37). Afflicted in-dividuals are at increased risk for developing benign tumors of theovary, meninx, and the parotid gland. However, the risk of malig-nant tumor is not increased (38). Congenital AKT1 p.E17K muta-tions associated with benign tumors and vascular lesions mightexplain the role of somatic AKT1 mutation in the PSH phenotype.Despite the predominant prevalence of AKT1mutations, ∼40%

of PSHs exhibited neither driver mutation nor CNA, suggestingepigenetic or not yet identified genetic origin of this disease. Fur-ther genetic or epigenetic studies with AKT1 mutation-negativePSHs will be required to discover other driver alterations of PSH.In conclusion, our study has revealed genomic differences

between PSHs and lung adenocarcinomas, including a high rateof AKT1 mutation and few CNAs in PSH. These genomic fea-tures of PSH identified in the present study provide clues tounderstanding the benign biology of PSH and for differentialgenomic diagnosis of lung tumors.

Materials and MethodsSclerosing Hemangioma Tissues. Approval for this study was obtained fromthe Institutional Review Board at the Catholic University of Korea, College ofMedicine. Clinicopathologic features of the patients are summarized in TableS1. The PSH patients in our study did not show any congenital abnormalitiesof Proteus syndrome. All of the patients were Korean. Fresh-frozen tissues

Fig. 4. Presence of AKT1 E17K mutation in both epithelial and stromal cells inrepresented PSH cases. (A) A sclerosing hemangioma shows both epithelial (ar-rows) and stromal (arrowheads) components before the microdissection. (B) Themicrodissection procures the epithelial cells and leaves holes behind. (C) Separatelymicrodissected epithelial and stromal cells in three PSHs show identical mutations.

Fig. 5. CNAs identified across 44 PSH genomes. (A) The heatmap summarizeschromosomal copy gains (red) and copy losses (blue) in the tumors; each rowrepresents PSH cases. The red boxes denote the arm-level copy gains and the blueboxes denote the arm-level copy losses. Copy number gains on 1q (SH18 and SH66)and 14q (SH42, SH51, and SH53) are identified recurrently. (B) Genome-wide log2ratio plots from three PSHs with copy number gains on 14q. The red arrowsrepresent the 14q copy gains. The x axis represents individual chromosomes andthe y axis represents the relative depth ratio (tumor/matched normal) in log2 scale.

Jung et al. PNAS | September 20, 2016 | vol. 113 | no. 38 | 10675

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 1,

202

1

from eight patients with PSH were obtained from the Tissue Banks of SeoulSt. Mary Hospital of Catholic University (Seoul, Korea), Guro Hospital ofKorea University (Seoul, Korea), and Pusan National University Hospital(Pusan, Korea). Initially, frozen tissues from the tissue banks were cut,stained with H&E, and examined under a microscope by a pathologist, who

identified areas rich in PSH tumor cells. To procure matched normal tissuesfrom each patient, we used lung tissues that were confirmed to be free oftumor cells based on examination under the microscope. The purities of PSHtumor cells were ∼20–80%. In addition, 36 FFPE PSHs from 35 patients (twoPSHs in one patient) were used as the discovery set for WES. Another 24 FFPE

Fig. 6. Copy-neutral LOH events identified across 44PSH genomes. (A) List of copy-neutral LOH eventsidentified in this study. Three PSHs with copy-neutralLOH either on 14q or 10q. (B) VAF of AKT1 mutations.The red circle represents PSHs with 14q LOH. MeanVAF of AKT1mutations in the PSHs with 14q LOH is 2.5times higher than those without 14p LOH (65.4% vs.25.6%, respectively). (C) Genome-wide B allele fre-quency (BAF) plot of the three PSHs with copy-neutralLOH. Red boxes represent copy-neutral LOH events.The x axis represents individual chromosomes and the yaxis represents the BAF.

Fig. 7. Somatically altered pathways in PSH. (A) Schematic representation of possible genetic mechanisms for PSH development. Predicted pathways frommutation data are depicted. Genes are shown along with the percentage of somatic mutations and CNAs. A potential druggable gene is highlighted in greenlightning symbols. (B) Somatic mutations and CNAs in the discovery (44 cases) and validation (24 cases) sets of PSHs.

10676 | www.pnas.org/cgi/doi/10.1073/pnas.1606946113 Jung et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 1,

202

1

PSHs were used for validation of important alterations. The FFPE tissueswere archival tissues collected from Seoul St. Mary Hospital (Seoul, Korea),Incheon St. Mary Hospital (Incheon, Korea), Ujeongbu St. Mary Hospital(Ujeongbu, Korea), St. Vincent Hospital (Suwon, Korea), St. Paul Hospital(Seoul, Korea), Bucheon St. Mary Hospital (Bucheon, Korea), Yeouido St. MaryHospital (Seoul, Korea), and Kyungpook National University Hospital (Daegu,Korea) under the permission of the Institutional Review Board at the CatholicUniversity of Korea, College of Medicine (MC14SISI0002). The tumor cells andnormal cells were separately procured from H&E-stained slides. For genomicDNA extraction, we used the DNeasy Blood & Tissue Kit (Qiagen).

WES. WES was performed for the genomic DNA obtained from tumor andmatched normal specimen using the Agilent SureSelect Human All Exome50Mb kit (Agilent Technologies) and Illumina HiSeq2000 platform, accordingto the manufacturer’s instructions. Acquisition and processing of the se-quencing data were performed as previously described (39). A Burrows–Wheeler aligner was used to align the sequencing reads onto the humanreference genome (UCSC hg19). The aligned sequencing reads were evalu-ated using Qualimap (40), and the sequences were deposited in the Se-quence Read Archive database (Project ID: PRJNA297066).

Identification of Somatic Variants. Somatic variants were identified usingMuTect (41) and SomaticIndelDetector (42) for point mutations and indels,respectively. The ANNOVAR package (43) was used to select somatic variantslocated in the exonic sequences and to predict their functional consequences.To obtain reliable and robust mutation calling, the following somatic variantswere eliminated: (i) read depth fewer than 20 in either the tumor or matchednormal; (ii) polymorphisms referenced in either 1000 Genomes Project, ExomeAggregation Consortium, or Exome Sequencing Project with a minor allelefrequency greater than 2%; and (iii) variants with VAF between 45% and 55%

in a copy-neutral region. Finally, validated variants according to Sanger se-quencing or digital PCR were manually curated.

Cancer-Related Mutations and Validation. We performed MutSigCV (14) andOncodriveFM (17) analyses to identify and validate cancer-related muta-tions. Significantly mutated genes with q-value < 0.01 were considereddriver mutations. Cancer-related genes are also defined as those identifiedin both the Cancer Gene Census (15) and Cancer Drivers Database (16). Wevalidated 51 mutations of 23 genes using either digital-PCR or Sanger se-quencing. Details are summarized in Table S3. Digital-PCR was performedusing the TaqMan Genotyping assay and the QuantStudio 3D digital PCRsystem (Life Technologies) as described elsewhere (44). The digital PCR datawere analyzed using the Relative Quantification module of the QuantStudio3D AnalysisSuite Cloud Software. The confidence level was set to 95%, andthe desired precision value was 10%. In addition, to further assess the re-current mutations in PSHs, we performed Sanger sequencing of AKT1 andCTNNB1 in the independent validation set (24 FFPE PSHs).

DNA Copy Number and LOH Analysis. DNA copy number and LOH were esti-mated usingWES data. The ngCGHmodule and RankSegmentation statisticalalgorithm in NEXUS software v7.5 (Biodiscovery) were used to define CNAs ofeach sample (44). We inferred the LOH events using Sequenza (45). All of theidentified CNAs and LOH events were manually curated in terms of depthratio and B allele frequency.

ACKNOWLEDGMENTS. This study was supported by National Research Founda-tion of Korea (Grant 2012R1A5A2047939), the Korean Health Industry Devel-opment Institute of the Ministry of Health and Welfare of Korea (GrantHI13C2148), and the National Cancer Institute of the NIH (Grant P30CA034196).C.L. is a distinguished Ewha Womans University Professor supported in part bythe Ewha Womans University Research grant of 2015.

1. Chan KW, Gibbs AR, Lo WS, Newman GR (1982) Benign sclerosing pneumocytoma oflung (sclerosing haemangioma). Thorax 37(6):404–412.

2. Kalhor N, Staerkel GA, Moran CA (2010) So-called sclerosing hemangioma of lung:Current concept. Ann Diagn Pathol 14(1):60–67.

3. Devouassoux-Shisheboran M, Hayashi T, Linnoila RI, Koss MN, Travis WD (2000) A clin-icopathologic study of 100 cases of pulmonary sclerosing hemangioma with immuno-histochemical studies: TTF-1 is expressed in both round and surface cells, suggesting anorigin from primitive respiratory epithelium. Am J Surg Pathol 24(7):906–916.

4. Dacic S, et al. (2004) Loss of heterozygosity patterns of sclerosing hemangioma of thelung and bronchioloalveolar carcinoma indicate a similar molecular pathogenesis.Arch Pathol Lab Med 128(8):880–884.

5. Lee HJ, et al. (2011) Comparison of genetic and epigenetic alterations at 11 tumorsuppressor loci in pulmonary sclerosing hemangioma and adenocarcinoma. Exp LungRes 37(6):344–353.

6. Chiang PM, et al. (2008) Frequent nuclear expression of beta-catenin protein but rarebeta-catenin mutation in pulmonary sclerosing haemangioma. J Clin Pathol 61(3):268–271.

7. Wang Y, Dai SD, Qi FJ, Xu HT, Wang EH (2008) p53 protein expression and geneticmutation in two primary cell types in pulmonary sclerosing haemangioma. J ClinPathol 61(2):192–196.

8. Mäkinen N, et al. (2011) MED12, the mediator complex subunit 12 gene, is mutated athigh frequency in uterine leiomyomas. Science 334(6053):252–255.

9. Lim WK, et al. (2014) Exome sequencing identifies highly recurrent MED12 somaticmutations in breast fibroadenoma. Nat Genet 46(8):877–880.

10. Cancer Genome Atlas Research Network (2014) Comprehensive molecular profiling oflung adenocarcinoma. Nature 511(7511):543–550.

11. Cancer Genome Atlas Research Network (2012) Comprehensive genomic character-ization of squamous cell lung cancers. Nature 489(7417):519–525.

12. Imielinski M, et al. (2012) Mapping the hallmarks of lung adenocarcinoma withmassively parallel sequencing. Cell 150(6):1107–1120.

13. Mehine M, et al. (2013) Characterization of uterine leiomyomas by whole-genomesequencing. N Engl J Med 369(1):43–53.

14. Lawrence MS, et al. (2013) Mutational heterogeneity in cancer and the search for newcancer-associated genes. Nature 499(7457):214–218.

15. Futreal PA, et al. (2004) A census of human cancer genes. Nat Rev Cancer 4(3):177–183.

16. Rubio-Perez C, et al. (2015) In silico prescription of anticancer drugs to cohorts of 28tumor types reveals targeting opportunities. Cancer Cell 27(3):382–396.

17. Gonzalez-Perez A, Lopez-Bigas N (2012) Functional impact bias reveals cancer drivers.Nucleic Acids Res 40(21):e169.

18. Manning BD, Cantley LC (2007) AKT/PKB signaling: Navigating downstream. Cell129(7):1261–1274.

19. Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-Kinase AKT pathway in hu-man cancer. Nat Rev Cancer 2(7):489–501.

20. Parikh C, et al. (2012) Disruption of PH-kinase domain interactions leads to oncogenicactivation of AKT in human cancers. Proc Natl Acad Sci USA 109(47):19368–19373.

21. Banerji S, et al. (2012) Sequence analysis of mutations and translocations across breastcancer subtypes. Nature 486(7403):405–409.

22. Kandoth C, et al.; Cancer Genome Atlas Research Network (2013) Integrated genomiccharacterization of endometrial carcinoma. Nature 497(7447):67–73.

23. Ahn SM, et al. (2014) Genomic portrait of resectable hepatocellular carcinomas: im-plications of RB1 and FGF19 aberrations for patient stratification. Hepatology 60(6):1972–1982.

24. Chen B, et al. (2013) Pulmonary sclerosing hemangioma: A unique epithelial neo-plasm of the lung (report of 26 cases). World J Surg Oncol 11:85.

25. Sartori G, et al. (2007) Microsatellite and EGFR, HER2 and K-RAS analyses in sclerosinghemangioma of the lung. Am J Surg Pathol 31(10):1512–1520.

26. Yap TA, et al. (2011) First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206in patients with advanced solid tumors. J Clin Oncol 29(35):4688–4695.

27. Askham JM, et al. (2010) AKT1 mutations in bladder cancer: Identification of a noveloncogenic mutation that can co-operate with E17K. Oncogene 29(1):150–155.

28. De Marco C, et al. (2015) Mutant AKT1-E17K is oncogenic in lung epithelial cells.Oncotarget 6(37):39634–39650.

29. Carpten JD, et al. (2007) A transforming mutation in the pleckstrin homology domainof AKT1 in cancer. Nature 448(7152):439–444.

30. Cancer Genome Atlas Network (2012) Comprehensive molecular portraits of humanbreast tumours. Nature 490(7418):61–70.

31. Stephens PJ, et al.; Oslo Breast Cancer Consortium (OSBREAC) (2012) The landscape ofcancer genes and mutational processes in breast cancer. Nature 486(7403):400–404.

32. Brastianos PK, et al. (2013) Genomic sequencing of meningiomas identifies oncogenicSMO and AKT1 mutations. Nat Genet 45(3):285–289.

33. Clark VE, et al. (2013) Genomic analysis of non-NF2 meningiomas reveals mutations inTRAF7, KLF4, AKT1, and SMO. Science 339(6123):1077–1080.

34. Chan EF, Gat U, McNiff JM, Fuchs E (1999) A common human skin tumour is caused byactivating mutations in beta-catenin. Nat Genet 21(4):410–413.

35. Lauring J, et al. (2010) Knock in of the AKT1 E17K mutation in human breast epithelialcells does not recapitulate oncogenic PIK3CA mutations. Oncogene 29(16):2337–2345.

36. Knudson AG (2002) Cancer genetics. Am J Med Genet 111(1):96–102.37. Lindhurst MJ, et al. (2011) A mosaic activating mutation in AKT1 associated with the

Proteus syndrome. N Engl J Med 365(7):611–619.38. Cohen MM, Jr (2014) Proteus syndrome review: Molecular, clinical, and pathologic

features. Clin Genet 85(2):111–119.39. Kim TM, et al. (2014) The mutational burdens and evolutionary ages of early gastric

cancers are comparable to those of advanced gastric cancers. J Pathol 234(3):365–374.40. García-Alcalde F, et al. (2012) Qualimap: Evaluating next-generation sequencing

alignment data. Bioinformatics 28(20):2678–2679.41. Cibulskis K, et al. (2013) Sensitive detection of somatic point mutations in impure and

heterogeneous cancer samples. Nat Biotechnol 31(3):213–219.42. DePristo MA, et al. (2011) A framework for variation discovery and genotyping using

next-generation DNA sequencing data. Nat Genet 43(5):491–498.43. Wang K, Li M, Hakonarson H (2010) ANNOVAR: Functional annotation of genetic

variants from high-throughput sequencing data. Nucleic Acids Res 38(16):e164.44. Jung SH, et al. (2016) Genetic progression of high grade prostatic intraepithelial

neoplasia to prostate cancer. Eur Urol 69(5):823–830.45. Favero F, et al. (2015) Sequenza: Allele-specific copy number and mutation profiles

from tumor sequencing data. Ann Oncol 26(1):64–70.

Jung et al. PNAS | September 20, 2016 | vol. 113 | no. 38 | 10677

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 1,

202

1