Sequential Molecular Changes during Multistage Pathogenesis of Small Peripheral Adenocarcinomas of the Lung

Sequential Molecular Changes during Multistage Pathogenesis ofSmall Peripheral Adenocarcinomas of the Lung

Junichi Soh, MD*, Shinichi Toyooka, MD*, Shuji Ichihara, MD*, Hiroaki Asano, MD*, NaruyukiKobayashi, MD*, Hiroshi Suehisa, MD*, Hiroki Otani, MD*, Hiromasa Yamamoto, MD*, KouichiIchimura, MD†, Katsuyuki Kiura, MD‡, Adi F. Gazdar, MD§, and Hiroshi Date, MD**Department of Cancer and Thoracic Surgery, Graduate School of Medicine, Dentistry andPharmaceutical Sciences, Okayama University, Okayama, Japan†Department of Pathology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,Okayama University, Okayama, Japan‡Department of Hematology, Oncology and Respiratory Medicine, Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan§Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern MedicalCenter, Dallas, Texas

AbstractIntroduction—We investigated EGFR and KRAS alterations among atypical adenomatoushyperplasia and small lung adenocarcinomas with bronchioloalveolar features to understand theirrole during multistage pathogenesis.

Methods—Sixty lesions measuring 2 cm or less were studied, including 38 noninvasive lesions (4atypical adenomatous hyperplasias, 19 Noguchi type A and 15 type B) and 22 invasive lesions (typeC) based on the World Health Organization classification and Noguchi’s criteria. EGFR and KRASmutations were examined using PCR-based assays. EGFR copy number was evaluated usingfluorescence in situ hybridization.

Results—EGFR and KRAS mutations were found in 26 (43.3%) and 5 (8.3%) lesions, respectively.Increased EGFR copy number status was identified in 10 lesions (16.7%), both mutant and wild type.EGFR or KRAS mutations were present in 39.5% and 7.9% (respectively) of noninvasive lesions and50% or 9.1% (respectively) of invasive lesions. EGFR copy number was increased in 7.9% and 31.8%of noninvasive and invasive lesions (P = 0.029). Multivariate analysis revealed that increasedEGFR copy number was the only significant factor to associate with invasive lesions (P = 0.035).

Conclusions—EGFR and KRAS mutations occur early during the multistage pathogenesis ofperipheral lung adenocarcinomas. By contrast, increased EGFR copy number is a late event duringtumor development and plays a role in the progression of lung adenocarcinoma independent of theinitiating molecular events.

KeywordsMultistage pathogenesis; EGFR; KRAS; Mutation; Amplification

Copyright © 2008 by the International Association for the Study of Lung CancerAddress for correspondence: Shinichi Toyooka, MD, Department of Cancer and Thoracic Surgery, Okayama University Graduate Schoolof Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. [email protected]: The authors declare no conflict of interest.

NIH Public AccessAuthor ManuscriptJ Thorac Oncol. Author manuscript; available in PMC 2009 October 6.

Published in final edited form as:J Thorac Oncol. 2008 April ; 3(4): 340–347. doi:10.1097/JTO.0b013e318168d20a.

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Adenocarcinomas of the lung represent a heterogeneous group of tumors. The major subtypesidentified by the World Health Organization (WHO) consist of bronchioloalveolar carcinoma(BAC), acinar, papillary, or solid with mucin.1,2 However, the majority of tumors consist ofmixtures of two or more subtypes. BAC is defined as a noninvasive tumor that has lepidicspread, i.e., replacement of the bronchiolar or alveolar epithelium by tumor cells. The WHOalso recognized a peripheral lesion, atypical adenomatous hyperplasia (AAH), as a precursorlesion for BAC. These findings indicate a multistage progression model for most peripheraladenocarcinomas—AAHs giving rise to noninvasive BAC tumors, which later becomeinvasive (mixed histology adenocarcinomas having a BAC component). In 1995, Noguchi andcolleagues developed an independent classification for small peripheral adenocarcinomas (≤2cm in size) consisting of six tumor subtypes.3 Types A, B, and C consisted entirely orpredominantly of a BAC component (“replacement tumors”). Types A and B consisted of BACtumors without (type A) or with (type B) areas of alveolar collapse and subsequent fibrosis.Among these two subtypes, nodal metastases were never present, and these tumors had a 100%5-year survival if they were small (≤2 cm) and were completely resected. Type C tumors wereBAC tumors with foci or fibroblastic proliferation. 28% of these tumors had lymph nodemetastases and the 5-year survival was 74.8%, similar to the average survival rate for smalladenocarcinomas as a group. For practical purposes, we can regard the Noguchi type A and Btumors as the equivalent of WHO BAC subtype and Noguchi type C tumors as the equivalentof WHO mixed (i.e., invasive) tumors having a prominent BAC component. Certain molecularchanges are characteristic of adenocarcinomas of the lung. These include mutations within thetyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene, amplificationsof the EGFR gene, and activating mutations of the KRAS gene. EGFR mutations, especiallydeletions in exon 19 and L858R point mutations in exon 21, target lung cancers arising incertain subsets: never smoker status, adenocarcinoma histology, female gender, and East Asianethnicity.4–9 The mutations have been reported to be more frequent in adenocarcinomas havingBAC features.4,6,10 The KRAS gene is a member of the ras family of oncogenes, and is locateddownstream of EGFR in the signaling pathway of the EGFR surface receptor. KRAS mutationsare found in 10 to 30% in non-small cell lung cancer (NSCLC), especially adenocarcinomas,and over 90% of the mutations occurred in codon 12, occasionally in codon 13, and rarely incodon 61.11–15 Although both KRAS and EGFR mutations target lung adenocarcinomas, theformer are associated with ever smoker status, male gender, and non-East Asian ethnicity.16,17 Thus, the two mutations seem to target different subpopulations and their presence mayindicate alternative methods of adenocarcinoma pathogenesis.18 Of interest, EGFR andKRAS mutations may be present in AAH and in nonmalignant peripheral airways adjacent tocancers.19–21 Indeed, KRAS mutations or EGFR mutations in exon 19 or 21 cause multifocalBAC in mouse models.22,23 Increased copy number of EGFR, as assessed by fluorescence insitu hybridization (FISH) assay, was associated with lymph node metastasis, more advancedpathologic stage, and poor prognosis.24–28 This alteration has been described in NSCLC,especially adenocarcinomas, and may occur in mutant- or wild-type tumors.26,27 Furthermore,the three molecular changes discussed above may influence tumor response to small molecularweight tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib. Both activatingEGFR mutations and increased gene copy number are associated with good clinical outcomeof EGFR-TKIs treatment,27,29,30 while KRAS mutations are associated with clinical resistance.30,31

To elucidate the roles of EGFR or KRAS mutations and increased EGFR copy number in themultistage pathogenesis of peripheral adenocarcinomas, we studied these molecular changesin small tumors having BAC features.

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PATIENTS AND METHODSPatients and Materials

During October 1997 to April 2005, 838 patients with NSCLC underwent pulmonaryoperations at Department of Cancer and Thoracic Surgery, Okayama University Hospital. Ofthese, 48 patients had lung adenocarcinoma (or AAH lesions) measuring 2 cm or less in greatestdimension and who had not received chemotherapy, radiotherapy, or targeted therapy beforesurgery. Among them, single lesions were resected from 39 patients. There were 8 patientswho had 19 synchronous lesions in their resected specimens (2–4 lesions per resection) andone patient who had 2 metachronous lesions resected over a 6-year interval. Details of thesynchronous and metachronous lesions, as defined by the criteria of Martini and Melamed,32

are presented in Table 1. Thus, from the 48 patients, a total of 60 lesions were available forstudy. Institutional review board permission and informed consent were obtained from all 48patients for genetic analyses.

The following clinical factors were evaluated: age, sex, smoking status, and tumor size. All 60cases were categorized into four histologic subtypes based on WHO classification andNoguchi’s criteria1,3; AAH (n = 4), type A (n = 19), type B (n = 15), and type C (n = 22).Because AAH is considered as a preinvasive lesion and Noguchi types A and B represent thenoninvasive BAC (as defined by the WHO classification), we combined three categories andcompared their findings with those present in Noguchi type C tumors (mixed subtype invasiveadenocarcinomas with BAC component by the WHO classification). We excluded the othertypes of small adenocarcinomas (Noguchi types D, E, and F, WHO acinar, papillary, or solidsubtypes) from this study as a convincing multistage pathogenesis has not been defined forthese tumors.

DNA Extraction and Mutation Analyses of EGFR and KRAS GenesFor all 60 lesions that were formalin fixed and paraffin embedded, DNAs were isolated byDEXPAT (TaKaRa, Shiga, Japan) following the manufacturer’s instructions. Among them,DNAs of 17 frozen lesions were also isolated by digestion with proteinase K, followed byphenol-chloroform (1:1) extraction and ethanol precipitation from frozen specimen.33 EGFRmutations examination was limited to exon 19 deletions and exon 21 L858R point mutationsby two methods: mutant-enriched or nonenriched PCR assays as described previously.34

Mutant-enriched PCR is a two-step PCR with intermittent restriction digestion to eliminatewild-type genes selectively, thus enriching the mutated genes at high sensitively. NonenrichedPCR is a one-step PCR without intermittent restriction digestion. The product of theamplification in the both methods was analyzed on 12% polyacrylamide gel electrophoresis(PAGE) via ethidium bromide staining. The common deletions of exon 19 were distinguishedfrom the wild type based on PCR product length polymorphisms using 12% PAGE. For exon21, Sau96I digestion, which could specifically digest the mutant type, was done before 12%PAGE. Mutant-enriched PCR assay can detect one mutant out of 2 × 103 wild-type genes. Onthe other hand, nonenriched PCR assay can detect one mutant of 10 wild-type genes. Weanalyzed KRAS mutations using a PCR assay with restriction digestion, followed by 12%PAGE, which was detectable of codon 12 point mutation based on the report by Kahn et al.35

These methods were predicted to detect about 85% of EGFR tyrosine kinase domainmutations8,36 and about 90% of KRAS mutations.11,12,37.

EGFR Copy Number Analysis by FISH AssayParaffin-embedded tissues of all 60 specimens were studied by FISH assay. A dual-color FISHassay was performed using the LSI EGFR spectrumOrange/CEP7 spectrumGreen probe(Vysis, Downers Grove, IL) following manufacturer’s instructions. At least 100

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nonoverlapping inter phase nuclei per slide were scored by two independent observers (I.S.,S.J.). All cases were classified into six FISH strata according to the criteria of Cappuzzo et al.27 Using their criteria, samples scored as having disomy, low trisomy, high trisomy, or lowpolysomy were classified as “FISH negative,” whereas samples scored as high polysomy orgene amplification were categorized as “FISH positive.”

Statistical AnalysesThe differences of significance among categorized groups were compared using χ2 or Fisherexact test tests as appropriate for univariate analyses. Logistic regression models using allvariables were used to further explore observed differences and to identify baseline factors thatmay independently correlate with histologic classification. All data were analyzed withStatView 5.0 Program for Windows (SAS Institute Inc., Cary, NC). All statistical tests weretwo-sided and probability values <0.05 were defined as being statistically significant.

RESULTSPatient Characteristics

The details of patient characteristics are shown in Table 2. Pathologic examination of the 60lesions from 48 patients indicated that each of the 60 lesions was a discreet presumablyindependently arising lesion. Of the 48 patients (9 of whom had multiple lesions), 31 had oneor more noninvasive lesions whereas 17 had only invasive lesion. At the time of analysis, onlyone patient with an invasive cancer (which was EGFR FISH positive, but wild type forEGFR and KRAS mutations) had died of recurrent lung cancer 3 years after diagnosis. Themean follow-up duration of the 47 survivors were 35.0 months (range, 8.4–102.9 months).Thus, the relationship between survival and clinicopathologic or molecular features could notbe analyzed.

Because some resected samples contained lesions of both noninvasive and invasive categories,we chose to present the data from the 60 individual lesions, rather than from 48 individualpatients.

Somatic Alterations of EGFR and KRAS GenesDetails of genetic and clinical data are shown in Table 3 and Table 4. Among 60 lesions, wedetected EGFR mutations in 26 lesions (43.3%), 15 as exon 19 deletions and 11 as L858Rpoint mutation, with complete concordance of results by nonenriched and mutant-enrichedPCR assays. KRAS codon 12 mutations were detected in 5 of 60 lesions (8.3%). Only onenoninvasive cancer had mutations in both genes. We also confirmed the mutational data ofEGFR and KRAS genes among 17 lesions whose DNAs were extracted from both paraffin-embedded samples and frozen samples. No discrepancies were identified from the analyses ofthese two differently handled DNA specimens.

For EGFR copy number, 50 lesions (83.3%) were scored as FISH negative (disomy in 14lesions, low trisomy in 19, high trisomy in 7, and low polysomy in 10), and 10 lesions (16.7%)were scored as FISH positive (high polysomy in 9 and amplification in 1). Four lesions hadboth EGFR mutation and FISH positivity and one lesion had both KRAS mutation and FISHpositivity. The other 4 lesions as FISH positive were wild type for both genes. Thus, FISHpositivity showed no selectivity for EGFR mutation positive (15.4%), KRAS mutation positive(20.0%), or wild type (16.7%) lesions. Example of FISH results are shown in Figure 1.

Synchronous and Metachronous TumorsTwenty-one synchronous or metachronous lesions were found in nine patients (Table 1),consistent with the field cancer theory.32 One of these patients had metachronous lesions that

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arose in the opposite lungs 6 years apart (patient 1). We regarded these as independent primarytumors. Eight other patients had 2, 3, or 4 synchronous lesions (patients 2–9). Of these, 4patients (patients 2–5) had individual lesions having either noninvasive or invasivecomponents.

Evaluation of Clinical and Genetic Factors According to Histologic DifferenceWe investigated the relationship between clinical and genetic factors by each of the twohistologic subsets (Table 3). By univariate analyses of the clinical factors, larger tumor sizewas significantly associated with invasive tumor (p = 0.027). Of the genetic factors, EGFR orKRAS mutations were present in 15 (39.5%) or 3 (7.9%) of 38 noninvasive lesions and 11(50%) or 2 (9.1%) of 22 invasive lesions; however, these differences were not significant. Bycontrast, increased EGFR copy number was found in 3 (7.9%) of noninvasive lesions and 7(31.8%) of invasive lesions (p = 0.029).

By multivariate analyses, FISH positivity was the only factor that was significantly associatedwith invasive tumors (OR = 6.5, 95% confidence interval: 1.14–37.3, p = 0.035).

DISCUSSIONOur study reveals important differences in the sequential appearance of molecular changesduring multistage pathogenesis of small peripheral adenocarcinomas having a BACcomponent. EGFR mutations were found in all four categories of lesions examined includingan AAH. KRAS mutations, while not detected in the small number of AAH lesions examined,were present in noninvasive and invasive carcinomas of Noguchi types A, B, and C. Therewere no significant differences in the frequencies of these two mutation types between thepathologic types. By contrast, we found a significant difference for EGFR gene FISH status,with FISH positivity being significantly more frequent in the invasive group of lesions (typeC).

Our findings regarding these mutational changes are consistent with the published literature.Both KRAS and EGFR mutations have been described in AAH lesions,20,37,38 and EGFRmutations have been found in the nonmalignant peripheral airways in the vicinity of invasiveperipheral adenocarcinomas,21 indicating that mutations of both genes are early events thatplay a role in tumor initiation.

Recent reports demonstrate that the KRAS mutations are more frequent in BACs of themucinous subtype whereas EGFR mutations are more frequent in the nonmucinous subtype.10,39 In our 60 lesions, 59 lesions (98.3%) were nonmucinous type of BACs, and only onelesion was a mucinous BAC without genetic alterations. Furthermore, we focused on smalladenocarcinomas with BAC components, which are well-known to correlate with female, neversmoking status.40 KRAS mutations are also associated with ever smoker status, male gender,and non-East Asian ethnicity.16,17 These findings support the lower frequency of KRASmutations in this cohort.

We found one rare case harboring both EGFR and KRAS mutations, who is a 61-year-old manwith heavy smoking history (50 packs per year). This noninvasive and nonmucinous lesion(Noguchi type B) has KRAS codon 12 point mutation and EGFR L858R point mutation withoutEGFR FISH positivity (low trisomy). As mentioned above, KRAS mutations are significantlyfrequent in males and ever smokers whereas EGFR mutations in females and never smokersand BACs, especially in the nonmucinous type.10,16,17,39 However, Shigematsu et al. alsoindicated that EGFR mutations could be detected in 10% of males and 14% of ever smokers.16 These findings might explain this rare case, which has both characteristics of EGFR andKRAS mutations.

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Our current study clearly revealed that increased copy number of the EGFR gene seems to bea progression event independent from the initial alterations, being more than four times morefrequent in invasive lesions than in noninvasive lesions. These differences are also significanteven if the four AAH lesions are removed from the analysis (p = 0.039). Patient number 4(Table 1) is of particular interest. She had synchronous noninvasive AAH and invasive(Noguchi type C) lesions. Although both lesions had identical (L858R) EGFR mutations, theinvasive cancer was FISH positive, whereas the AAH was FISH negative, providing supportfor our concept that the activating mutations occur early in pathogenesis, while FISH positivityis a late event associated with advanced (invasive) cancers. Our findings are consistent withrecent reports that describe EGFR mutations preceding amplification, with amplificationfavoring the mutant allele.41 Our findings clearly demonstrate that FISH positivity occurs asfrequently in mutant- and wild-type tumors, and that these two events associated with responseto tyrosine kinase inhibitors occur independently of each other.26,42

Gazdar et al. have proposed that peripheral lung adenocarcinomas arise via three or morepathways.18 In smokers, activation of the KRAS pathway via mutations occurs, whereas innonsmokers, there is upstream activation of the EGFR pathway via activating mutations. Asthese changes only account for about 50% of lung adenocarcinomas, other initiating eventsmay also contribute to pathogenesis. Based on our finding, a hypothesis of putative molecularpathways to peripheral lung adenocarcinomas is shown in Figure 2. Although EGFR andKRAS mutations are early, possibly initiating events for subsets of adenocarcinomas, increasedcopy number of the EGFR gene seems to be a late or progression event and targetsadenocarcinomas irrespective of the initiating events. However, our sample size is not largeand further investigation is warranted to confirm our hypothesis.

Our findings suggest that increased gene copy number may be a relatively late event in tumorpathogenesis, and that the original molecular status of the tumor may not accurately reflect theresponse to TKIs of a recurrent or metastatic lesion. The recognition that molecular events aredynamic and progressive necessitates retesting advanced or recurrent tumors to determineoptimal, individualized targeted therapies.

ACKNOWLEDGMENTSThe authors thank Mrs. Makiko Tabata, Cancer and Thoracic surgery, Hiroyuki Kugoh, MD, and Kazuhiro Murakami,MD, Division of Molecular and Cell Genetics, Molecular and Cellular biology, Faculty of Medicine, Tottori Universityfor technical support of FISH assay. The authors thank Xian-Jin Xie, PhD, Harold C. Simmons Comprehensive CancerCenter, University of Texas Southwestern Medical Center, Dallas, for support of statistical analyses. MasayukiNoguchi, MD, Department of Pathology, Institute Basic Medical Sciences, University of Tsukuba, kindly providedinformation regarding pathologic classification.

Supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture andTechnology of Japan (18790993 for S.T.) and a grant from the Specialized Program of Research Excellence in LungCancer (P50CA70907) from the National Cancer Institute, Bethesda, Maryland, USA.

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FIGURE 1.Representative examples of fluorescence in situ hybridization (FISH) with epidermal growthfactor receptor (EGFR) (red signals) and chromosome 7 (green signals) probes. Case 1: OneNoguchi type A lesion (left-top, H&E) with EGFR L858R point mutation (left-bottom)indicates EGFR FISH negative (Disomy) (right); Mean values of chromosome 7 and EGFRcopy numbers are 2.19 ± 0.61 and 2.22 ± 0.69 (mean ± SD) per 100 nuclei, respectively. Thepercentage of ≤2 copies of EGFR gene is 90% and the ratio of EGFR gene to chromosome 7is 1.00. Case 2: One Noguchi type C lesion (left-top, H&E) with EGFR exon 19 deletions (left-bottom) indicates EGFR FISH positive (high polysomy) (right); Mean values of chromosome7 and EGFR copy numbers are 3.58 ± 1.30 and 3.36 ± 1.19 (mean ± SD) per 100 nuclei,

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respectively. The percentage of ≥4 copies of EGFR gene is 53% and the ratio of EGFR geneto chromosome 7 is 1.07.

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FIGURE 2.Putative multiple molecular pathways to peripheral lung adenocarcinomas. Adenocarcinomasrepresent a heterogenous mixture, and may arise from peripheral or central airways.Adenocarcinomas arising from the peripheral airways often contain a bronchioloalveolarcomponent. From work presented in this report and from the published literature, it wouldappear that there are at least three early or initiating sets of events. Pathway 1, more frequentlyseen in never or light smokers, involves activating mutations of the epidermal growth factorreceptor (EGFR) gene. Pathway 3, almost limited to ever smokers, involves activatingmutations of the KRAS gene. Pathway 1 targets females and East Asian ethnicity, whereaspathway 3 targets males and non-East Asian ethnicity. However, the combined frequencies ofthese mutations (on a worldwide basis) are about 50% of adenocarcinomas. Thus, one or morealternative pathways (pathway 2) must use other initiating events. From data presented in thisreport, it would appear that increased EGFR copy number is a late (progression) event, andthat targets tumors irrespective of the initiating events. Figure modified from Gazdar AF,Shigematsu H, Herz J, et al. Mutations and addiction to EGFR: the Achilles ‘heal’ of lungcancers? Trends Mol Med 2004;10:481–486; and Wistuba II, Gazdar AF. Lung cancerpreneoplasia. Annu Rev Pathol Mech Dis 2006;1:331–348.

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r)Se

xSm

okin

gSt

atus

Tum

orSi

ze (m

m)

His

tolo

gica

lC

lass

ifica

tion

EGFR

Mut

atio

nEG

FRFI

SHKR

ASM

utat

ion

149

MN

ever

16Ty

pe C

Wt

NW

t

5516

Type

CW

tP

Wt

267

FN

ever

9Ty

pe A

Wt

NW

t

10Ty

pe C

Wt

NW

t

377

FN

ever

9Ty

pe A

L858

RP

Wt

9Ty

pe A

Wt

NW

t

10Ty

pe A

Wt

NW

t

9Ty

pe C

L858

RN

Wt

468

FN

ever

5A

AH

L858

RN

Wt

18Ty

pe C

L858

RP

Wt

572

MEv

er10

Type

BW

tP

Wt

15Ty

pe C

Wt

PW

t

667

FN

ever

15Ty

pe A

Wt

NC

12

20Ty

pe A

ex19

del

sN

Wt

761

FN

ever

4A

AH

Wt

NW

t

10Ty

pe A

Wt

NW

t

865

MEv

er5

AA

HW

tN

Wt

20Ty

pe A

Wt

NW

t

18Ty

pe B

L858

RN

Wt

970

FN

ever

11Ty

pe A

L858

RN

Wt

20Ty

pe B

L858

RN

Wt

EGFR

, epi

derm

al g

row

th fa

ctor

rece

ptor

; FIS

H, f

luor

esce

nce

in si

tu h

ybrid

izat

ion;

M, m

ale;

F, f

emal

e; A

AH

, aty

pica

l ade

nom

atou

s hyp

erpl

asia

; typ

es A

, B, a

nd C

, cla

ssifi

ed a

ccor

ding

to N

oguc

hi’s

crite

ria; W

t, w

ild ty

pe; L

858R

; exo

n 21

L85

8R p

oint

mut

atio

n; e

x19

dels

, exo

n 19

del

etio

ns; N

, neg

ativ

e; P

, pos

itive

; C12

, cod

on 1

2 po

int m

utat

ion;

pat

ient

num

ber 1

is a

met

achr

onou

s mul

tiple

cas

ean

d pa

tient

s num

ber 2

–7 a

re sy

nchr

onou

s mul

tiple

cas

es.


NIH


NIH


NIH


Soh et al. Page 13

TABLE 2Characteristics of 60 Lesions in 48 Patients

Variables No.

Age (yr) Median 65 (range, 41–84)

<65 28

≥65 32

Sex

Male 23

Female 37

Smoking history

Never 37

Ever 23

Tumor size (mm) Median 12 (range 4–20)

≤12 33

>12 27

Histology

AAH 4

Type A 19

Type B 15

Type C 22

Never, never smokers; Ever, ever smokers; AAH, atypical adenomatous hyperplasia; types A, B and C, histologic subtypes defined based on Noguchi’scriteria.


NIH


NIH


NIH



BLE

3Th

e C

orre

latio

n B

etw

een

His

tolo

gic

Subs

ets a

nd C

linic

al a

nd G

enet

ic F

acto

rs

Var

iabl

esSu

bset

Tot

alN

o. (%

)

Non

inva

sive

Gro

up

Inva

sive

Gro

up

P*

AA

H(n

= 4

)N

o. (%

)

Typ

e A

(n =

19)

No.

(%)

Typ

e B

(n =

15)

No.

(%)

Tot

al(n

= 3

8)N

o. (%

)

Typ

e C

(n =

22)

No.

(%)

Age

(yr)

<

6528

(46.

7)2

(50.

0)9

(47.

4)7

(46.

7)18

(47.

4)10

(45.

5)0.

89

≥

6532

(53.

3)2

(50.

0)10

(52.

6)8

(53.

3)20

(52.

6)12

(54.

5)

Sex

Mal

e23

(38.

3)2

(50.

0)3

(15.

8)9

(60.

0)14

(36.

8) 9

(40.

9)0.

75

Fem

ale

37 (6

1.7)

2 (5

0.0)

16 (8

4.2)

6 (4

0.0)

24 (6

3.2)

13 (5

9.1)

Smok

ing

stat

usN

ever

37 (6

1.7)

2 (5

0.0)

14 (7

3.7)

6 (4

0.0)

22 (5

7.9)

15 (6

8.2)

0.43

Ever

23 (3

8.3)

2 (5

0.0)

5 (2

6.3)

9 (6

0.0)

16 (4

2.1)

7 (3

1.8)

Tum

or si

ze (m

m)

≤

1233

(55.

0)4

(100

)13

(68.

4)8

(53.

3)25

(65.

8) 8

(36.

4)0.

027

>

1227

(45.

0)0

(0)

6 (3

1.6)

7 (4

6.7)

13 (3

4.2)

14 (6

3.6)

EGFR

gen

eM

utan

t26

(43.

3)1

(25.

0)7

(36.

8)7

(46.

7)15

(39.

5)11

(50)

0.43

Wild

34 (5

6.7)

3 (7

5.0)

12 (6

3.2)

8 (5

3.3)

23 (6

0.5)

11 (5

0)

FISH

pos

itive

10 (1

6.7)

0 (0

.0)

1 (5

.3)

2 (1

3.3)

3 (7

.9)

7 (3

1.8)

0.02

9

FISH

neg

ativ

e50

(83.

3)4

(100

)18

(94.

7)13

(86.

7)35

(92.

1)15

(68.

2)

KRA

SM

utan

t5

(8.3

)0

(0.0

)2

(10.

5)1

(6.7

) 3

(7.9

) 2

(9.1

)1

Mut

atio

nW

ild55

(91.

7)4

(100

)17

(89.

5)14

(93.

3)35

(92.

1)20

(90.

9)

AA

H, a

typi

cal a

deno

mat

ous h

yper

plas

ia; t

ypes

A, B

and

C, h

isto

logi

cal s

ubty

pes d

efin

ed b

ased

on

Nog

uchi

’s cr

iteria

; Nev

er; n

ever

smok

ers;

Eve

r; ev

er sm

oker

s; E

GFR

, epi

derm

al g

row

th fa

ctor

rece

ptor

;FI

SH, f

luor

esce

nce

in si

tu h

ybrid

izat

ion.

* P va

lue

was

eva

luat

ed b

y no

n-in

vasi

ve g

roup

ver

sus i

nvas

ive

grou

p.


NIH


NIH


NIH



BLE

4C

orre

latio

n B

etw

een

Gen

etic

Alte

ratio

n an

d C

linic

al F

acto

rs

EGFR

Mut

atio

nEG

FR F

ISH

KRAS

Mut

atio

n

Var

iabl

es

Mut

ant

(n =

26)

n (%

)

Wild

(n =

34)

n (%

)P

Posi

tive

(n =

10)

n (%

)

Neg

ativ

e(n

= 5

0)n

(%)

P

Mut

ant

(n =

5)

n (%

)

Wild

(n =

55)

n (%

)P

Age

(yr)

<65

(n =

28)

12 (4

2.9)

16 (5

7.1)

0.94

2 (7

.1)

26 (9

2.9)

0.08

83

(10.

7)25

(89.

3)0.

66

≥65

(n =

32)

14 (4

3.8)

18 (5

6.2)

8 (2

5.0)

24 (7

5.0)

2 (6

.3)

30 (9

3.7)

Sex

Mal

e (n

= 2

3)7

(30.

4)16

(69.

6)0.

116

(26.

1)17

(73.

9)0.

163

(13.

0)20

(87.

0)0.

36

Fem

ale

(n =

37)

19 (5

1.4)

18 (4

8.6)

4 (1

0.8)

33 (8

9.2)

2 (5

.4)

35 (9

4.6)

Smok

ing

stat

us

Nev

er (n

= 3

7)18

(48.

6)19

(51.

4)0.

295

(13.

5)32

(86.

5)0.

492

(5.4

)35

(94.

6)0.

36

Eve

r (n

= 23

)8

(34.

8)15

(65.

2)5

(21.

7)18

(78.

3)3

(13.

0)20

(87.

0)

Tum

or si

ze

≤12

mm

(n =

33)

10 (3

0.3)

23 (6

9.7)

0.02

45

(15.

2)28

(84.

8)0.

741

(3.0

)32

(97.

0)0.

16

>12

mm

(n =

27)

16 (5

9.3)

11 (4

0.7)

5 (1

8.5)

22 (8

1.5)

4 (1

4.8)

23 (8

5.2)

EGFR

, epi

derm

al g

row

th fa

ctor

rece

ptor

; FIS

H, f

luor

esce

nce

in si

tu h

ybrid

izat

ion;

Nev

er, n

ever

smok

ers;

Eve

r, ev

er sm

oker

s.


Sequential Molecular Changes during Multistage Pathogenesis of Small Peripheral Adenocarcinomas of the Lung

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