Cancer Sci | January 2007 | vol. 98 | no. 1 | 50–57 doi: 10.1111/j.1349-7006.2006.00346.x © 2006 Japanese Cancer Association

Blackwell Publishing Asia

OCIA domain containing 2 is highly expressed in adenocarcinoma mixed subtype with bronchioloalveolar carcinoma component and is associated with better prognosisTadashi Ishiyama,1 Junko Kano,1 Yoichi Anami,1 Takuya Onuki,1 Tatsuo Iijima,1 Yukio Morisita,2 Jun Yokota3 and Masayuki Noguchi1,4

1Department of Pathology, Institute of Basic Medical Science, and 2Department of Clinical Pathology, Institute of Clinical Medicine, Graduate School of Comprehensive Human Science, University of Tsukuba, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki-shi, Ibaraki 305-8575; 3Biology Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan

(Received July 23, 2006/Revised September 1, 2006/Accepted September 6, 2006/Online publication October 20, 2006)

Although lung adenocarcinoma is a major cause of cancer deathworldwide, details of its molecular carcinogenesis and stepwiseprogression are still unclear. To characterize the sequential progressionfrom bronchioloalveolar adenocarcinoma of the lung (BAC, in situcarcinoma) to adenocarcinoma mixed subtype with BAC component,polymerase chain reaction-based cDNA suppression subtractivehybridization (SSH) was carried out using two representative casesof BAC (non-invasive tumors) and adenocarcinoma mixed subtypewith BAC (invasive tumors). Through differential screening, virtualreverse northern hybridization and quantitative real-time reverse-transcription–polymerase chain reaction (qRT-PCR) we selected fivegenes (TncRNA, OCIAD2, ANXA2, TMED4 and LGALS4) that wereexpressed at significantly higher levels in invasive adenocarcinomamixed subtype with BAC than in BAC. After in situ hybridizationand qRT-PCR analyses, we confirmed that only the OCIAD2 geneshowed significantly higher expression in the tumor cells ofinvasive adenocarcinoma mixed subtype with BAC than in BAC(P = 0.026). We then carried out in situ hybridization of OCIAD2 in 56adenocarcinoma mixed subtype with BAC component and assessedthe correlation between OCIAD2 expression and clinicopathologicalfeatures. In contrast to our expectation, the patients with OCIAD2expression showed a better clinical outcome than those withoutOCIAD2 expression, and OCIAD2 expression showed an inversecorrelation with lymphatic invasion, blood vessel invasion andlymph node metastasis. These results suggest that OCIAD2 begins toexpress at the progression from in situ to invasive carcinoma, andis associated with the favorable prognosis of adenocarcinomamixed subtype with BAC component. (Cancer Sci 2007; 98: 50–57)

L ung cancer is the most common fatal malignancy worldwide,including North America, Europe and Japan.(1) Its histology

can be divided into four major subtypes: squamous cell carcinoma,adenocarcinoma, large cell carcinoma and small cell carcinoma.Among the major subtypes of non-small cell carcinomas(NSCLC), squamous cell carcinoma is directly attributable tocigarette smoking and its sequence of histological changesfrom dysplasia and in situ carcinoma to invasive carcinomahas been well established and accepted.(2) However, because ofits histological and cytological heterogeneity, the sequentialprogression of adenocarcinoma has been very difficult tocharacterize on the basis of morphology.

In 1999, the World Health Organization revised the histologicalclassification of lung tumors, and bronchioloalveolar carcinoma(BAC) was added as one of the major histological subtypes ofadenocarcinoma. Atypical adenomatous hyperplasia (AAH) wasalso newly added as a preinvasive lesion of adenocarcinoma.

BAC is defined as adenocarcinoma that shows no invasivegrowth. Therefore, both AAH and BAC are thought to representthe very early stage of adenocarcinoma without invasion.Histologically, both entities show pure lepidic growth of existingalveolar structures and are sometimes difficult to distinguishfrom each other.

In 1995, Noguchi et al. divided small lung adenocarcinomas(i.e. 2 cm in diameter or less) into two major groups, each ofwhich was further subdivided into three types.(3) One of thesetwo major groups is the replacing growth type, which showslepidic growth of pulmonary alveolar structures. The other is thenon-replacing growth type, which shows non-lepidic growth anddestruction of the original alveolar framework. The three sub-types in the former group include localized bronchioloalveolarcarcinoma (LBAC) without active fibroblastic proliferation(types A and B) and LBAC with active fibroblastic proliferation(type C). Types A and B correspond to BAC and have anextremely favorable prognosis. These subgroups show no lymphnode metastasis and have a 5-year survival rate of 100%,although some type C tumors that are mostly diagnosed as theadenocarcinoma mixed subtype with BAC component can showlymph node metastasis and have a less favorable prognosis thantype A and B tumors (5-year survival rate 74.8%). Minami et al.examined small adenocarcinomas morphometrically and reportedthat the ratio of the area of fibroblastic proliferation to that offibrosis is critical for prognosis: if the ratio is less than 10%,then adenocarcinoma mixed subtype with BAC componentshows a very favorable prognosis.(4) Suzuki et al.,(5) Yokose et al.(6)

and Sakurai et al.(7) have also indicated the importance offibrosis and destruction of the alveolar framework for theprognosis of adenocarcinoma. Furthermore, Aoyagi et al. haveinvestigated loss of heterozygosity (LOH) on eight chromosomesin small-sized lung adenocarcinoma showing replacementgrowth,(8) and demonstrated that the multistep carcinogenesisof lung adenocarcinoma progresses in accordance with theaccumulation of multiple allelic losses. Their study also providedsupportive data for the progression of adenocarcinoma fromtypes A and B to type C. These clinicopathological studiessuggest that type C tumors include tumors with favorable andpoor prognosis.

Clinical trials of computed tomography (CT) screening to detectsmall and early stage lung adenocarcinomas have been carriedout in Japan and other countries. One-fourth of the cases detectedshowed pure ground glass opacity (GGO) and half of them showed

4To whom correspondence should be addressed. E-mail: nmasayuk@md.tsukuba.ac.jp

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partial GGO. Small adenocarcinomas that show pure GGO cor-respond to type A tumors (BAC) and have an extremely favorableprognosis. In contrast, cases showing partial GGO have a rela-tively poor prognosis, and correspond to adenocarcinoma mixedsubtype with BAC component. These findings suggest thatadenocarcinoma mixed subtype with BAC component includesearly but invasive tumors showing a poor prognosis. Therefore,it is very important to clarify the molecular mechanism ofstepwise progression from type A tumors to type C tumors andto find molecular markers that can be used to distinguish type Ctumors or adenocarcinoma mixed subtype with BAC componentthat show favorable prognosis from those showing poor prognosis.

Several tumor markers and aberrantly expressed genes, suchas p53, Ki67, K-ras, Survivin, Her-2/Neu and FHIT, have beenreported to accumulate along the progression from BAC to invasivecancer.(9–12) Many molecular profiling studies by cDNA micro-array have been carried out to identify the molecular targets incancer cell for diagnosis and treatment.(13–15) However, definitivemarker genes for the progression of adenocarcinoma mixedsubtype with BAC component have not been identified.

In the present study, using representative type A (BAC) andtype C (small-sized adenocarcinoma mixed subtype with BACcomponent) tumors, we carried out polymerase chain reaction(PCR)-based suppression subtractive hybridization (SSH) toidentify genes related to the sequential progression from in situcarcinoma to invasive carcinoma.

Materials and Methods

Patients and tissue specimens. For subtraction analysis andexpression analysis, frozen materials of 11 BAC (type A and B)and nine small-sized adenocarcinoma mixed subtype withBAC component (type C) were obtained from patients whohad undergone surgical resection at the Department of ThoracicSurgery, Tsukuba University Hospital (Ibaraki, Japan), andthe National Cancer Center Hospital (Tokyo, Japan). A smallamount of each specimen was embedded directly in Tissue-TekOCT Compound (Sakura Finetek Japan, Tokyo, Japan) andfrozen immediately in acetone and dry ice. The specimens werethen stored at −80°C until analysis. Among the 20 specimens,we used one pair of frozen materials that showed representativetype A (BAC) and C (small-sized adenocarcinoma mixed subtypewith BAC component) histology (Fig. 1). For clinicopathologicalanalysis by in situ hybridization, 56 adenocarcinoma mixedsubtype with BAC component were also obtained from patientswho had undergone surgical resection. All specimens were fixedwith 10% formalin and embedded in paraffin. All of the caseswere diagnosed histologically according to the World HealthOrganization classification,(16) and Noguchi’s classification forthe small-sized adenocarcinoma. They were also evaluated by theInternational Union Against Cancer (UICC) staging system.(17)

Preparation of nuclear extracts. For subtraction analysis, weused two frozen cryostat sections (8 µm) of type A and C(Fig. 1). The tumor cells in each section were collected selectivelyusing a laser capture microdissection system, LM-200 (ArcturusEngineering, Mountain View, CA, USA). Total RNA was isolatedusing TRIzol (Invitrogen, Carlsbad, CA, USA). Using 10 µL ofsample buffer, T7 RNA polymerase promoter-attached, adaptorligation-mediated, and PCR amplification followed by thein vitro T7-transcription (TALPAT) method were performed toobtain enough mRNA to serve as template for suppressionsubtractive hybridization (SSH).(18)

Suppression subtractive hybridization and cloning. Suppressionsubtractive hybridization was performed on TALPAT samplesbetween type A and C tumors using a PCR-Select cDNAsubtraction kit (BD Bioscience Clontech, Palo Alto, CA, USA)with some modifications. After the first-strand and second-strandcDNA had been synthesized in turn, SSH was carried out

according to the manufacturer’s instructions. To clone genes thatwere relatively overexpressed in type C tumors, the forwardsubtracted fragments were then inserted into the T/A cloningvector pCR 2.1 (Invitrogen). Individual transformants carryingsubtracted cDNA fragments were isolated from white colonieson 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside/isopropyl-1-thio-β-D-galactopyranoside agar plates.

Differential screening. A total of 1056 individual recombinantclones were picked randomly. cDNA inserts of the plasmidswere amplified by PCR with secondary PCR primers. The PCRproducts were transferred to a nylon membrane, and fouridentical membranes were made. cDNA from the forward orreverse subtractions were radiolabeled with [32P]dCTP by randompriming. Dot blot hybridization was carried out according to themanufacturer’s protocol (PCR-Select Differential Screeningkit; BD Biosciences Clontech). Visualization was achievedby exposure to Kodak BioMax XAR films (Eastman Kodak,Rochester, NY, USA).

Virtual reverse northern blotting. Dot samples were generatedby PCR using secondary primers from the colonies correspondingto the dots shown to be differentially expressed by dot blot

Fig. 1. Histology of the specimens used for the TALPAT method.(A) type A tumor (bronchioloalveolar carcinoma; BAC), (B) type C tumor(small-sized adenocarcinoma mixed subtype with BAC component).

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differential screening. Then, two identical membranes wereprepared with their PCR products and were hybridized with32P-labeled TALPAT cDNA probes (derived from type C and typeA tumors). The hybridized membranes were exposed to KodakBioMax XAR films (Eastman Kodak) and evaluated relativelyagainst the intensity of glyceraldehyde-3-phosphate dehydrogenase(G3PDH) with an imaging densitometer (Model GS-700 ImagingDensitometer; Bio-Rad Laboratories, Hercules, CA, USA).

Sequencing and homology search. The clones relatively over-expressed in type C tumors compared with type A tumors weresequenced using a BigDye terminator v3.1 cycle sequencingready reaction kit and an ABI PRISM 310 genetic analyzer(both from Applied Biosystems Japan, Tokyo, Japan). Nucleicacid and protein sequence similarity searches were carried outat the National Center for Biotechnology using the basic localalignment search tool (BLAST).

Real-time quantitative reverse transcription–PCR analysis usingSYBR Green I. Expression of those genes relatively over-expressed in type C tumors was evaluated by real-time reversetranscription–PCR based on the SYBR Green I method. In brief,PCR was carried out with SYBR Premix EX Taq (Perfect RealTime, Takara Bio, Otsu, Shiga, Japan) in an ABI PRISM 5700sequence detector (Applied Biosystems) in a final volume of 25 µLaccording to the manufacturer’s protocol. At the verificationstudy of the quantification by virtual reverse northern, we usedTALPAT-amplified cDNA (type C and A tumors) for the PCRtemplates. And at the quantification study of selected genes byin situ hybridization, we used cDNA reverse-transcribed fromwhole section samples (normal lung, type C and A tumors). ThePCR primers were purchased from Takara Bio, except fortrophoblast-derived non-coding RNA (TncRNA) and G3PDHused as an internal control. The primer sequences of TncRNA andG3PDH were: Tnc RNA, 5′-TTTGGGAGACTGAGGTGGGTG-3′(forward), 5′-AGACAAGTTTTCGCTATGCTGGC-3′ (reverse);and G3PDH, 5′-AATTCCATGGCACCGTCAA-3′ (forward),5′-CCAGCATCGCCCCACTT-3′ (reverse).

In situ hybridization. The overexpressed cDNA, which had beensubcloned into pCR2.1, were amplified by PCR with T7 RNApolymerase promoter-attached primers. The T7 RNA polymerasepromoter-attached PCR products were labeled with digoxigeninusing a DIG-RNA labeling kit (Roche Diagnostics, Penzberg,Germany). The linearized pCR2.1 was used as template, and a senseprobe was made as the negative control. Detection of hybridizedcRNA probes was carried out using horseradish-conjugatedrabbit anti-DIG antibody (DakoCytomation, Carpinteria, CA,USA) and a GenPoint Tyramide Signal Amplification System(DakoCytomation). Specimens were considered positive when>5% of tumor cells showed clear positive staining.

Statistical analysis. Associations between OCIA domain con-taining 2 (OCIAD2) gene expression and clinicopathologicalparameters were evaluated with the Pearson χ2 and Fisher’sexact tests. Survival curves were calculated using the Kaplan–Meier method and then compared using a log-rank test.Statistical analyses were carried out using SPSS Base, version11.0 J for Windows (SPSS Inc., Chicago, IL, USA) at asignificance level of P < 0.05.

Results

Subtractive hybridization and differential screening. To identifygenes that were highly expressed in type C tumors (small-sizedadenocarcinoma mixed subtype with BAC component) incomparison with type A tumors (BAC), we carried out PCR-based subtractive hybridization using cDNA templated byTALPAT-amplified RNA from each representative type C tumorand type A tumor. The amplified subtracted cDNA fragments(type C minus type A) were then subcloned. After transformation,1056 white colonies were selected randomly and cultured. Thedifferential hybridization screening revealed 71 clones thatshowed stronger signals with cDNA probes templated by RNAfrom type C tumors than with cDNA probes from type Atumors. Subsequent semiquantitative screening (virtual reversenorthern) with the TALPAT products from two tissue pairsconfirmed that all 71 clones were highly expressed in type CcDNA compared to type A. Among the 71 highly expressedgenes in type C cDNA, we selected eight clones that wereexpressed at a high level in type C tumors (relative expression> 0.2 compared to G3PDH) and showed a high expression ratio(type C/type A, >5). We performed sequencing and a BLASTsearch of these clones, and the results are shown in Table 1. Forsubsequent analysis, we selected five genes (TncRNA, OCIAD2,ANXA2, TMED4 and LGALS4) as the other cloned genesincluded some that are well known to be cancer-related(CEACAM5, S100A6 and TACSTD1)(19–24) or to play a role in thefundamental metabolism of living cells (ACSL1).(25–27)

Quantitative expression analysis of identified genes in TALPATproducts. To assess differences in the expression levels of thegenes selected by SSH and virtual reverse northern, quantitativereal-time PCR (qRT-PCR) assays were carried out. As shown inFig. 2, qRT-PCR confirmed that all of the selected genes wereexpressed at a significantly higher level in type C tumors than intype A tumors.

Expression status of the cloned genes in lung adenocarcinomatissue. To analyze the expression and distribution of the fiveselected genes in small-sized adenocarcinoma mixed subtypewith BAC component, we carried out in situ hybridization using

Table 1. Genes identified as differentially and highly expressed in type C tumor (small-sized adenocarcinoma mixed subtype withbronchioloalveolar carcinoma [BAC] component) than type A tumor (BAC) through subtraction, differential screening and virtual reverse northernscreening

Gene name Official symbolRelative expression vs G3PDH

C/A Ratio Type C Type A

Trophoblast-derived non-coding RNA TncRNA 100.42 3.31 30.37OCIA domain containing 2 OCIAD2 73.38 2.70 27.17S100 calcium binding protein A6 S100A6 36.54 1.84 19.83Annexin A2 ANXA2 64.34 9.24 6.96Carcinoembryonic antigen-related cell adhesion molecule 5 CEACAM5 64.22 2.40 26.78Transmembrane emp24 protein transport domain containing 4 TMED4 45.64 7.57 6.03Acyl-CoA synthetase long chain family member 1 ACSL1 42.24 0.64 65.90Tumor-associated calcium signal transducer 1 TACSTD1 23.27 2.18 10.68Lectin, galactoside-binding, soluble, 4 LGALS4 22.54 0.22 102.77

G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

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10 surgical specimens, including five type A tumors and fivetype C tumors. OCIAD2, ANXA2 and LGALS4 demonstratedpositive expression exclusively in cancerous cells other than innormal epithelial cells in both histological types. Among thethree genes, ANXA2 was positive in both type A and type Ctumors. We were unable to reliably assess the expression statusof TncRNA and TMED4 genes because the TncRNA gene probedid not react with both histological types and the TMED4 geneprobe reacted with the negative control (data not shown).However, LGALS4 and OCIAD2 were expressed only in type Ctumors (small-sized adenocarcinoma mixed subtype withBAC component), and therefore their expression was furtherexamined using large-scale specimens.

Quantitative expression analysis of OCIAD2 and LGALS4 in whole-section RNA samples (type A–C tumors). To compare the expression

levels of the OCIAD2 and LGALS4 genes in type A and Btumors and type C tumors, we first carried out qRT-PCR on totalRNA extracted from 10 tumors (five type A and B, five type C)as a preliminary investigation. This showed that there were nosignificant differences in LGALS4 gene expression between typeA and B tumors and type C tumors (data not shown). However,the OCIAD2 gene tended to show stronger expression in type Ctumors than in either type A or B tumors. In order to examinethe expression of OCIAD2 in type A–C tumors in greater detailand get its expression status in normal cells, we added anothernine adenocarcinoma (five type A and B, four type C) and fivenormal lung mRNA samples and carried out the same analysison 24 samples in all. As shown in Fig. 3, the OCIAD2 genewas expressed significantly more strongly in type C than inboth type A and B (P = 0.026) and there were no significant

Fig. 2. Quantitative real-time reverse transcription–polymerase chain reaction analysis of highlyexpressed genes in type C tumors (small-sizedadenocarcinoma mixed subtype with bron-chioloalveolar carcinoma [BAC] component) incomparison with type A tumors (BAC). All of thegenes selected by both suppression subtractivehybridization and virtual reverse northernscreening were expressed more highly in type Ctumor-derived RNA than in type A-derived RNA.Both RNA samples were identical to those usedin TALPAT. All measurements are shown relativeto the expression level of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene. Barsshow the mean + SD.

Fig. 3. Quantitative real-time real-time reversetranscription–polymerase chain reaction analysisof OCIAD2 expression in small-sized adeno-carcinomas and normal lung. OCIAD2 showedsignificantly higher expression in type C tumors(small-sized adenocarcinoma mixed subtype withbronchioloalveolar carcinoma [BAC] component)than in both type A and B tumors (BAC)(P = 0.026). Lane N1-5, normal lung; A1-3, typeA; B1-5, type B; C1-9, type C tumor-derived RNAsamples extracted from whole section samples.All measurements are shown relative to theexpression level of the glyceraldehyde-3-phosphatedehydrogenase (G3PDH) gene. Bars show themeanmean + SD. *The difference was statisticallysignificant (P < 0.05).

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differences in the expression levels of OCIAD2 among normaltissues, type A and type B tumors.

Correlation between OCIAD2 expression and clinicopathologicalfactors in lung adenocarcinoma patients. To assess the correlationbetween OCIAD2 expression and clinicopathological factors,we examined OCIAD2 expression in 56 patients (adenocarcinomamixed subtype with BAC component including type C tumors)by in situ hybridization (Fig. 4). Thirty-four tumors (61%) showedpositive reaction against the antisense probe of OCIAD2. Innormal tissue, some bronchial epithelium also showed positivereaction but alveolar epithelium, vascular endothelium and infiltratinginflammatory cells were negative. As shown in Table 2, loss ofOCIAD2 expression was significantly correlated with lymphaticinvasion, blood vessel invasion and lymph node metastasis(P = 0.011, 0.002 and 0.012, respectively). The correlationbetween OCIAD2 expression and clinical outcome of lungadenocarcinoma was also examined. As shown in Fig. 5A, interms of overall survival, patients who were positive for OCIAD2expression had a significantly better outcome than patients whowere negative for OCIAD2. In particular, for cases with BACcomponent in which the tumor diameter was less than 2 cm,none of the patients who died showed positive OCIAD2expression (Fig. 5B). In terms of event-free survival, OCIAD2-positive patients showed a significantly better outcome thanOCIAD2-negative patients (Fig. 5C). However, there was nocorrelation between OCIAD2 expression and other clinico-pathological factors (Table 2).

Discussion

In the present study, we identified genes that were more highlyexpressed in type C tumors (small-sized adenocarcinoma mixedsubtype with BAC component) than in type A tumors (BAC).Among those genes, we were able to confirm only one (OCIAD2)

Table 2. OCIAD2 gene expression and clinicopathological features inpatients with pulmonary adenocarcinoma

Clinicopathological features

All patients

OCIAD2 expression in in situ hybridization

P-value No. positive

(%)No. negative

(%)

All patients 56 34 22Mean age (years) 65.7 67.53 62.91Sex

Male 31 17 (54.8) 14Female 25 17 (68) 8 0.316

Pathological stageStage I 34 25 (73.5) 9Stage II 3 1 (33.3) 2Stage III 17 7 (41.2) 10Stage IV 2 1 (50.0) 1 0.107

Ly factorNegative 32 24 (75.0) 8Positive 24 10 (41.7) 14 0.011*

V factorNegative 39 29 (74.4) 10Positive 17 5 (29.4) 12 0.002*

Lymph node statusN0 40 29 (72.5) 11N1 and N2 16 5 (31.3) 11 0.012*

Stage I includes IA and IB, stage II includes IIA and IIB, stage III includes IIIA and IIIB. Ly factor, lymphatic vessel invasion; N factor, lymph node involvement; V factor, vascular vessel invasion. *The difference was statistically significant (P < 0.05).

Fig. 4. In situ hybridization analysis of the OCIAD2 gene in lungadenocarcinoma. OCIAD2 was positive in adenocarcinoma mixedsubtype with bronchioloalveolar carcinoma (BAC) component (C) butnegative in BAC (B) and normal lung (A).

Ishiyama et al. Cancer Sci | January 2007 | vol. 98 | no. 1 | 55© 2006 Japanese Cancer Association

that was highly expressed in a large proportion of type Ctumors. The number of genes identified was very limited, duepossibly to individual differences in tumors used for SSH andthe heterogeneity of lung adenocarcinoma. However, although

during the screening process we removed genes that showedonly minimal expression or a very low type C/type A expressionratio, those genes may play important roles in the multistepcarcinogenesis of lung adenocarcinoma.

OCIAD2 was originally identified by Strausberg et al. in 2002by its sequential similarity with ovarian carcinoma immunoreactiveantigen 1 through the National Institutes of Health MammalianGene Collection project.(28) OCIAD2 and OCIAD1 form theOCIA domain family. OCIAD1 is a gene that was identified byLuo et al. in 2001 by immunoscreening of an ovarian carcinomacDNA expression library with ascites from ovarian cancerpatients.(29) The function of OCIAD2 has not been elucidated,but the protein may be immunosensitive as an antigen and, likeOCIAD1, could be a cancer-specific protein. The expressionpattern of OCIAD2 in adenocarcinoma mixed subtype withBAC component is of considerable interest. It is not expressedin type A and B tumors (BAC) but is expressed in a proportionof type C tumors (small-sized adenocarcinoma mixed subtypewith BAC component) (Figs 2–4). Therefore, adenocarcinomamixed subtype with BAC component includes tumors both withand without OCIAD2 expression.

At first, we expected that the expression of OCIAD2 might berelated to poor prognosis, as a certain percentage of adenocar-cinoma mixed subtype with BAC component, including type Ctumors, expressed OCIAD2 but pure BAC (type A and Btumors) did not. However, the result was contrary to our expec-tation, and OCAID2 was shown to be a marker for a separategroup of adenocarcinoma mixed subtype with BAC componentthat showed a favorable prognosis. This unexpected result maybe explained as follows. Because only a pair of type A (BAC)and type C (small-sized adenocarcinoma mixed subtype withBAC component) tumors was used for SSH analysis, there wasa possibility that we might have chosen by chance a type Ctumor expressing OCIAD2. The dynamics of OCIAD2 expres-sion in the course of sequential progression of peripheral-typelung adenocarcinoma is still unknown, but two possibilities canbe suggested (Fig. 6). One is that OCIAD2 becomes expressedwhen the tumor progresses from BAC to adenocarcinoma mixedsubtype with BAC component, but the expression decreases inaccordance with malignant progression. The other possibility isthat during malignant progression from BAC to adenocarcinomamixed subtype with BAC component, a proportion of BACbegin to express OCIAD2 and form a group that shows favorableprognosis. In contrast, other tumors that remain negative forOCIAD2 continue to show malignant progression and have apoor prognosis.

Luo et al. have reported that patients who have tumorsexpressing OCIAD1 may develop an antibody against it.(29) Thismeans that OCIAD1 may be a cancer-specific protein and couldbe applicable as a marker for detecting carcinoma. Recently, ithas become possible to detect many small adenocarcinomas byCT screening. Among the carcinomas detected by CT, adenocar-cinoma mixed subtype with BAC component will account forthe major proportion. As adenocarcinoma mixed subtype withBAC component is a mixed population that includes tumorswith an extremely favorable prognosis or a poor prognosis, thereis a need for reliable markers to discriminate less-aggressivetumors or tumors that can be treated by reduction or limitedsurgery, such as wedge resection or segmentectomy. If OCIAD2triggers an immune response like OCAD1, it may be a candidatemarker for distinguishing adenocarcinoma mixed subtype withBAC component and favorable prognosis from tumors with apoor prognosis.

In summary, we have identified an interesting molecule,OCIAD2, that is expressed at a significantly higher level inadenocarcinoma mixed subtype with BAC component but not inBAC (in situ adenocarcinoma). OCIAD2 is significantly associ-ated with a favorable prognosis and may have potential as a

Fig. 5. Survival curves and event-free survival of patients withpulmonary adenocarcinomas. Patients who were positive for OCIAD2expression showed a favorable outcome (A, all cases; B, tumor diameterless than 2 cm) and (C) event-free survival compared with patients whowere negative for OCIAD2 expression.

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molecular marker for selecting tumors that are treatable by limitedsurgery. In order to clarify the molecular mechanism of stepwiseprogression of peripheral-type adenocarcinoma, it will be necessaryto elucidate the function of OCIAD2.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Cancer Research(16-1) from the Ministry of Health, Labor, and Welfare of Japan.

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Fig. 6. Scheme of morphological progression oflung adenocarcinoma with bronchioloalveolaradenocarcinoma (BAC) component. ●, OCIAD2-negative cells; �, OCIAD2-positive cells. ➀ Malignantprogression from BAC to adenocarcinoma mixedsubtype with BAC component, a proportion ofBAC begin to express OCIAD2. ② OCIAD2 becomesexpressed when the tumor progressed from BACto adenocarcinoma mixed subtype with BACcomponent, but the expression decreases inaccordance with malignant progression.

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