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15-Lipoxygenase-2 Expression inBenign and Neoplastic Lung: AnImmunohistochemical Study andCorrelation With Tumor Gradeand Proliferation

ADRIANA L. GONZALEZ, MD, RICHARD L. ROBERTS, MD, PHD,PIERRE P. MASSION, MD, SANDRA J. OLSON, MS, YU SHYR, PHD,

AND SCOTT B. SHAPPELL, MD, PHD

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15-Lipoxygenase-2 (15-LOX-2) is an arachidonic acid–metaboliz-ng enzyme expressed in prostate, lung, skin, esophagus, and cornea.n the benign prostate, it is expressed in differentiated secretorypithelial cells, where its enzymatic product 15-HETE may regulateranscription by activating the nuclear receptor peroxisome prolif-rator–activated receptor gamma (PPAR�). 15-LOX-2 and 15-HETEormation are reduced in prostate carcinoma. The distribution of5-LOX-2 in the normal lung and its expression in lung carcinomasas not been reported and was investigated in the current study bysing immunohistochemistry and tissue microarrays (TMAs). In be-ign lung, 15-LOX-2 immunostaining was noted exclusively in type IIneumocytes, which are known to express PPAR�. Of 160 lungarcinomas, 15-LOX-2 was expressed in non-small cell carcinomasNSCLC), including 33 of 69 (48%) adenocarcinomas, with 10 of 1663%) bronchioloalveolar carcinomas immunopositive. Fourteen of5 (25%) squamous cell carcinomas and 2 of 14 (14%) large cell

arcinomas showed weak immunostaining. All 19 neuroendocrine

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umors were negative. Better differentiated NSCLCs showed greater5-LOX-2 expression, with a significant inverse correlation between5-LOX-2 immunostaining and tumor grade (P < 0.03). A significantnverse correlation was also noted between 15-LOX-2 immuno-taining and tumor cell proliferation (Ki-67 immunostaining; P <.0001). These findings suggest a possible role of 15-LOX-2 inegulating secretory differentiation and proliferation in benign lungnd NSCLCs, particularly adenocarcinomas. HUM PATHOL 35:40-849. © 2004 Elsevier Inc. All rights reserved.

Key words: lipoxygenase, lung cancer, arachidonic acid, peroxi-ome proliferator–activated receptor.

Abbreviations: LOX, lipoxygenase; AA, arachidonic acid; 15-OX-2, second human 15-lipoxygenase; HPETE, hydroperoxyeicosa-

etraenoic acid; HETE, hydroxyeicosatetraenoic acid; PPAR�, perox-some proliferator–activated receptor gamma; TMAs, tissue

icroarrays; SCC, squamous cell carcinoma.

Cyclooxygenase and lipoxygenase (LOX) metabo-ites of membrane phospholipid-derived arachidoniccid (AA) are important mediators of inflammatoryesponses and may play a role in neoplastic processes.1-3

uman 5-LOX, 12-LOX, and 15-LOX-1 are most char-cteristically expressed in blood cells2 but may also haveltered expression in epithelial-derived malignancies,ncluding colon, breast, lung, bladder, and prostateancer.3 15-LOX-1 is expressed in bronchial epithelialells, and increased expression has been noted in lungancer.4,5 Increased expression has also been reportedn prostate cancer,3,6 whereas both increased and de-

From Department of Pathology, Department of Medicine, De-artment of Preventative Medicine, Vanderbilt Ingram Cancer Cen-er, and Department of Urologic Surgery, Vanderbilt University Med-cal Center, Nashville, TN. Accepted for publication March 25, 2004.

Supported in part by National Institutes of Health Specialized Pro-ram of Research Excellence (SPORE) for Lung Cancer, 1 P50A090949 (A.L.G., P.P.M.), and by a Discovery Grant from the Vander-ilt Ingram Cancer Center (S.B.S.). P.M. is supported by a Merit Reviewntry Program Award from the Veterans Administration.

Address correspondence and reprint requests to Adriana L.onzalez, MD, Department of Pathology, C3321 Medical Centerorth, Vanderbilt University Medical Center, Nashville, TN 37232-561.

0046-8177/$—see front matter© 2004 Elsevier Inc. All rights reserved.

reased expression have been reported in colon cancerompared with in benign colon mucosa.7,8

More recently, a second human 15-lipoxygenase15-LOX-2) was described that has only approximately0% sequence identity to human 5-LOX, 12-LOX, and5-LOX-1.9 15-LOX-2 expression occurs in epithelialrgans, including lung, prostate, skin, cornea, andsophagus.10 In addition to differences in tissue distri-ution, 15-LOX-2 has distinct enzymatic propertiesompared with 15-LOX-1. 15-LOX-1 catalyzes relativelyonselective oxygenations, including the formation ofmixture of 15-hydroperoxyeicosatetraenoic acid (15-PETE) and 12-HPETE from AA and formation of

dditional peroxidation products from bis-dioxygen-tions.2 In contrast, 15-LOX-2 metabolizes free AA ex-lusively to 15-HPETE,9 which has led to a postulatedigand function of the ultimate 15-hydroxyeicosatetra-noic acid (15-HETE) product.2,11,12 The 15-LOX-2etabolite 15-HETE has been shown to activate

eroxisome proliferator–activated receptor gammaPPAR�),12-15 a nuclear transcription factor involved indipocyte and possibly epithelial differentiation.16 Inrostate, for example, 15-LOX-2 expression and 15-OX-2 mRNA are reduced in carcinoma,11,17,18 and5-LOX-2 and 15-HETE have been shown to negativelyegulate cell cycle progression in epithelial cells

12,19

ells, further suggesting a possible contribution of

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15-LIPOXYGENASE-2 IN LUNG CANCER (Gonzalez et al)

educed expression of 15-LOX-2 to altered prolifera-ion and differentiation in carcinoma.

15-LOX-2 mRNA has been identified in lung tis-ue.9 However, no studies have yet reported the cellularistribution of 15-LOX-2 in benign lung or its possiblexpression in lung carcinomas. The objectives of thistudy were to characterize the distribution of 15-LOX-2n benign and neoplastic lung and to investigate aossible correlation of 15-LOX-2 expression with lungarcinoma cell differentiation and proliferation.

ATERIALS AND METHODSase Selection and Tissue Microarrayonstruction

Paraffin-embedded tissues from 35 non-tumor surgicalathology and autopsy cases (including normal, chronic in-ammation, granulomatous inflammation, bronchopneumo-ia, diffuse alveolar damage [DAD], atelectasis, emphysema,brosis, and infarction) were used to assess distribution of5-LOX-2 immunostaining in normal and non-neoplastic dis-ased lung with conventional (non-TMA) slides. A total of 160araffin-embedded lung tumors were investigated by usingoth conventional and TMA slides. Forty-two of these 160umor cases were immunostained on conventional slides, in-luding 31 not included in the TMA blocks. Sections of tumorepresentative of the case and adjacent benign lung weresed for conventional slide immunohistochemistry. An addi-ional 129 lung tumor cases were used for construction of 2MA blocks. Hematoxylin and eosin–stained slides were re-iewed to ensure correct histological tumor typing. Tumorsere classified and grade was assessed according to the 1999orld Health Organization (WHO) classification.20 In the

ase of several poorly differentiated carcinomas, more spe-ific categorization according to the 1999 WHO classificationf lung tumors was accomplished by using TTF-1 (Cell Marque,ot Springs, AR) and CK7 immunostains and mucicarmine

tain (DakoCytomation Artisan mucicarmine kit; Dako, Carpin-eria, CA) as adjuncts to histology. This resulted in revised diag-oses for 11 poorly differentiated non-small cell carcinomas, as

arge cell carcinoma (n � 6) and as poorly differentiated ade-ocarcinoma (n � 5).

For TMA analysis, representative areas of tumor werehosen by a surgical pathologist with special interest in lungathology (A.L.G.). The 2 TMA blocks were constructed withBeecher Instruments Tissue Arrayer (Silver Spring, MD),

sing 2 or 3 representative 0.6-mm areas of each tumor fromhe original paraffin blocks. The 160 tumor cases consisted of9 adenocarcinomas, of which 16 were bronchioloalveolararcinomas (BACs), 2 mucinous and 14 non-mucinous); 55quamous cell carcinomas (SCCs); 14 large cell carcinomas;nd a variety of neuroendocrine tumors, including 5 smallell carcinomas, 9 typical carcinoid tumors, and 5 large celleuroendocrine carcinomas. The invasive adenocarcinomashowed several patterns including solid (n � 9), acinar (n �), mucinous (n � 2), papillary (n � 2), and mixtures ofcinar, bronchioloalveolar, papillary, and solid patterns (n �8). Additionally, 1 monophasic synovial sarcoma, 1 high-rade sarcoma with chondroid differentiation, and 1 carcino-arcoma (all thought to be primary pulmonary sarcomas)ere included on the TMA.

Clinical survival data and tumor stage21 were collectedrom the Tumor Registry and Bioinformatics Core of the

anderbilt Ingram Cancer Center. Survival analysis was cal- c

841

ulated from the date of diagnosis to date of death or last datef contact for live patients.

mmunohistochemistry

Paraffin immunoperoxidase was performed by an avidin-iotin method on a Ventana automated immunostainer (Ven-ana Medical Systems, Tucson, AZ) with a 15-LOX-2 poly-lonal antibody made against the purified protein11 and usedt a 1:1000 dilution without pretreatment. This antibody ispecific for human 15-LOX-2, without appreciable cross-reac-ivity to human 5S-LOX, 12S-LOX, and 15S-LOX-1.11 Immu-ostaining correlates with 15-LOX-2 mRNA expression by initu hybridization22 and is eliminated by preincubation of thentibody with purified 15-LOX-2 protein.11 To more defini-ively identify type II pneumocytes for comparison to 15-OX-2 immunolocalization in alveolar-lining cells,23-25 a sub-

et of cases on conventional slides was also immunostained bysing antibodies to surfactant proteins SP-B (1:250) and pro–P-B (1:200; gifts from Dr. Jeff Whittset, Children’s Hospitaledical Center, Cincinnati, OH), and thyroid transcription

actor (TTF-1; 1:100; Cell Marque). CK7 (DakoCytomation)as performed with 1:100 dilution. Immunostaining for theroliferation marker Ki-67 (MIB-1 clone; Immunotech, West-rook, ME) was performed with 1:50 dilution and citratentigen retrieval.

15-LOX-2 immunostaining in tumors on conventionalnd TMA slides was assessed semiquantitatively as 0-3� asollows: 0, no staining or equivocal staining in �5%; 1�,efinite weak-moderate in �20% of cells; 2�, weak to strongtaining in 21%-50% of cells or weak staining in the majorityf cells; 3�, moderate to strong staining in �50% of cells.5-LOX-2, SP-B, pro–SP-B, and TTF-1 immunostaining inontumor areas were assessed in a similar manner. Each

ndividual TMA sample per case was evaluated for 15-LOX-2mmunostaining. All tumor samples were reviewed blindly ateast 3 times, with individual assessments yielding similar re-ults. The highest tumor immunoscore per each TMA caseas used in subsequent statistical analyses, with a score of at

east 1� considered 15-LOX-2 immunopositive. Ki-67 tumormmunostaining in conventional and TMA slides was evalu-ted as an estimated percentage of positive cells (prolifera-ion index), determined in a blinded manner without knowl-dge of the 15-LOX-2 expression status. The proliferationndex was separately determined on each TMA sample perase, and the highest value per case was used in subsequenttatistical analyses.

tatistical Analysis

All data are expressed as means � SD. The relationshipetween 15-LOX-2 immunostaining and tumor cell prolifer-tion (Ki-67 immunostaining) was established by the generalinear model method. The �2 test and the Fisher’s exact testere employed for testing the associations between 15-LOX-2

mmunostaining and tumor grade, tumor stage, and patienturvival status. All tests of significance were 2-sided, and differ-nces were considered statistically significant when P value was0.05. SAS version 8.02 (Cary, NC) was used for all analyses.

ESULTS5-LOX-2 Immunostaining in Benign Lungissue

Positive 15-LOX-2 immunostaining in benign lungas noted in focal alveolar-lining cells, exclusively in

ells with type II pneumocyte morphology (Fig 1). The

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HUMAN PATHOLOGY Volume 35, No. 7 (July 2004)

IGURE 1. 15-Lipoxygenase-2 (15-LOX-2) expression in benign lung. (A) Scattered 15-LOX-2–immunopositive cells with features ofype II pneumocytes in alveoli (arrow) in normal lung. The large airway epithelium including bronchiolar epithelium (arrowhead)s negative (400�). (B) Alveolated lung, showing scattered 15-LOX-2–immunopositive cells with features of type II pneumocytesarrows). 15-LOX-2 immunoreactivity is largely nuclear but is often both cytoplasmic and nuclear (400�). (C and D) Photomicro-raphs of serial sections of normal human lung immunostained for 15-LOX-2 (C) and surfactant protein proSP-B (a marker for type

I pneumocytes, D). Histologically similar cell populations lining the alveoli stain positively for both immunomarkers, supporting thedentity of the 15-LOX-2–positive cell population as type II pneumocytes (400�). (E) 15-LOX-2 immunoreactivity in reactive type IIneumocytes. Photomicrograph of human lung showing increased numbers of 15-LOX-2–positive type II pneumocytes in reactive

issue around a focus of lung adenocarcinoma (asterisk; 100�). (F) 15-LOX-2 immunoreactivity in reactive type II pneumocytes.hotomicrograph of human lung autopsy tissue showing increased numbers of 15-LOX-2 immunoreactive type II pneumocytesarrow) in regions of diffuse alveolar damage with regenerating alveolar epithelium (400�).

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ype II cells are those with more abundant and oftenoamy cytoplasm and smooth round nuclear outlines.5-LOX-2 immunostaining in such cells was most oftenoth nuclear and cytoplasmic, similar to patterns ob-erved in other secretory tissues.11,22 Although stainingf alveolar lining cells varied from one area to another,nd not all cells with type II pneumocyte morphologyere positive, most histologically normal lung sections

n areas without inflammation or tumor showed 15-OX-2 immunostaining in 5%-10% of alveolar liningells. In the areas that showed staining, approximately5% of lining cells that had type II pneumocyte mor-hology rather than type I morphology were 15-LOX-2

mmunopositive. In 8 of 18 cases, immunostaining forro–SP-B showed positive immunostaining in cells thatad morphology similar to that of those that were5-LOX-2 immunopositive (Fig 1). Surfactant proteinP-B showed similar but lesser immunopositivity. Inormal areas of tumor cases on conventional slides,5-LOX-2 immunostaining was seen in approximately5% of alveolar cells immunostaining with TTF-1, anntibody known to be positive in type II cells in normalung.26-28

In contrast to positive 15-LOX-2 immunostainingn type II pneumocytes, type I pneumocytes, ciliatedronchial epithelium, goblet cells, and submucosallands in the larger airways (ie, trachea, bronchi, andronchioles) showed no immunostaining with 15-OX-2. 15-LOX-2 immunostaining was also uniformlyegative in stromal and vascular cells. Most inflamma-

ory cells, including lymphocytes and neutrophils, weremmunonegative, whereas macrophages and rare eosin-phils showed weak cytoplasmic immunostaining, notiscernible above background. Increased type II pneu-ocyte 15-LOX-2 staining was noted in conditions of

eactive type II pneumocyte hyperplasia. Strong (3�)taining in a greater percentage of alveolar lining cellsmean, 39%; foci of up to 40%-80%) was seen in areasf reactive type II pneumocyte hyperplasia accompany-

ng reactive processes (n � 20), with the highest ex-ression appreciated in cases of DAD (Fig 1). Interest-

ngly, strong 15-LOX-2 immunostaining was noted ineritumoral type II pneumocytes in 12 of 45 (27%)umor-containing sections (Fig 1). In these peritumoralnd reactive areas, staining for 15-LOX-2 approachedhat of TTF-1, which was also increased in these areasnot shown).

5-LOX-2 Immunostaining in Lungarcinomas

A total of 160 tumors were evaluated by both TMAnd non-TMA methods. 15-LOX-2 immunostaining wasositive in 16 of 42 tumors on conventional slides.mmunostaining in positive cases was patchy in most,ith the percentage of positive tumor cells averaging7% (range, 5%-80%) and the intensity of positivemmunostaining ranging from weak to strong. 15-OX-2 tumor cell immunostaining was noted to beoth nuclear and cytoplasmic (Fig 2). On conventional

lides, 14 of 28 (50%) adenocarcinomas showed posi-

843

ive 15-LOX-2 immunostaining, including 7 of 1258%) BACs and 7 of 16 (44%) non-BAC adenocarci-omas. All 15 poorly differentiated tumors except for 1

arge cell carcinoma were 15-LOX-2 immunonegative.ll neuroendocrine tumors, including small cell carci-omas, were negative.

Possible correlation of 15-LOX-2 expression withumor type was further investigated by extending thenalysis by use of TMAs (Fig 2). With the addition ofhe TMA data, a total of 50 of 160 tumors (31%) wereound to express 15-LOX-2 (Table 1). Forty-eight per-ent of all adenocarcinomas were 15-LOX-2 immu-opositive, including 63% of BACs (Table 1). Because5-LOX-2 immunopositivity was noted most commonlyn lung adenocarcinomas, and particularly BACs, addi-ional preliminary attempts were made to correlate 15-OX-2 expression with histological subtypes of possibleiologic significance in these tumors. Of the positiveACs, only the non-mucinous BACs expressed 15-OX-2. These non-mucinous BACs consisted of pre-ominantly cuboidal to columnar tumor cells. Threeuch tumors showed a Clara cell-like differentiationorphologically, whereas the majority (n � 11) showed

ype II or mixed type II and Clara cell types.20 Only 1 ofhe 3 Clara-cell like tumors was 15-LOX-2 immunopo-itive, whereas 9 of the other 11 non-mucinous BACsere 15-LOX-2 immunopositive. Of the remaining ad-nocarcinomas, 23 of 51 of the tumors with acinar,apillary, solid, and mixed patterns were 15-LOX-2 im-unopositive, whereas 2 pure mucinous tumors were

5-LOX-2 immunonegative. Generally weaker immuno-taining was noted in only 25% of SCCs. All neuroen-ocrine tumors, including small cell carcinomas, were5-LOX-2 immunonegative (Table 1). The immuno-taining intensity and frequency of 15-LOX-2–immu-opositive cells in 15-LOX-2–positive lung tumorsTMA and non-TMA) is summarized in Table 2.

orrelation of 15-LOX-2 Immunostainingith Tumor Grade and Proliferation

15-LOX-2 immunostaining correlated inverselyith tumor differentiation (WHO grade). Consideringll histological subtypes, 15-LOX-2 immunostaining wasoted in 14 of 35 (40%) grade 1 (well differentiated)

umors, 21 of 55 (38%) grade 2 (moderately differen-iated) tumors, and only 15 of 70 (21%) poorly differ-ntiated tumors (Table 3). In comparing 15-LOX-2xpression in better differentiated tumors (grades 1nd 2, n � 90) versus poorly differentiated tumorsgrade 3, n � 70), there was a statistically significantifference, with less immunopositivity in the high-gradeumors (P � 0.03).

The proliferation index was available in 153 of 160ases. The mean Ki-67–labeling index for 15-LOX-2–ositive tumors was 23.2%, whereas the mean Ki-67–

abeling index for 15-LOX-2–negative tumors was0.1%. A statistically significant inverse correlation wasoted between 15-LOX-2 expression and tumor cellroliferation (P � 0.0001).

Possible correlation of 15-LOX-2 expression with

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HUMAN PATHOLOGY Volume 35, No. 7 (July 2004)

IGURE 2. Variable 15-lipoxygenase-2 (15-LOX-2) immunostaining in lung carcinomas. (A) Extensive strong 15-LOX-2 immuno-taining in a bronchioloalveolar carcinoma (BAC) on a conventional slide (400�). (B) Moderate cytoplasmic and nuclear 15-LOX-2mmunostaining in a BAC with septal thickening and scattered inflammation (tissue microarray [TMA] slide; 200�). (C) Diffuse strongytoplasmic and focal nuclear 15-LOX-2 immunostaining in a moderately differentiated adenocarcinoma on a TMA slide (400�).

D) Diffuse strong (predominantly cytoplasmic) 15-LOX-2 immunostaining in a moderately differentiated adenocarcinoma on TMAlide (200�). (E) Absent 15-LOX-2 in poorly differentiated adenocarcinoma on TMA slide (400�). (F) Moderate cytoplasmicmmunostaining in a moderately differentiated squamous cell carcinoma on TMA slide (200�).

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15-LIPOXYGENASE-2 IN LUNG CANCER (Gonzalez et al)

rade and proliferation was analyzed for specific histol-gies, including adenocarcinomas, with and without

nclusion of BACs and SCCs. A similar tendency forigher grade tumors and those with higher prolifera-

ive rates to lack 15-LOX-2 expression was noted, al-hough the results were not statistically significant, pos-ibly because of lower numbers of cases within suchndividual categories.

orrelation of 15-LOX-2 Expression Withumor Stage and Patient Survival

There was no correlation between tumor 15-OX-2 expression and tumor stage, with immunoposi-

ivity noted in 15 of 53 (28%) stage I tumors, 0 of 8tage II tumors, 7 of 19 (37%) stage III tumors, and 9 of5 (60%) stage IV tumors (P � 0.069). One hundredwenty patients had survival data available for analysis,f which 33 patients (27%) had tumors that were 15-OX-2 immunopositive. Estimated median survivalverall was 829 days (95% confidence interval, 525 to268 days). As expected, there was a statistically signif-cant inverse correlation between survival and tumorrade and stage. The estimated median survival foratients with 15-LOX-2–immunopositive tumors was268 days, as opposed to only 669 days for the patientsith 15-LOX-2–negative tumors. Although these find-

ngs are in keeping with the inverse relationship be-ween 15-LOX-2 tumor immunopositivity and tumorrade and proliferation, the survival difference for pa-ients with 15-LOX-2–positive and –negative tumors wasot statistically significant (P � 0.4).

ISCUSSION

The role of 15-LOX-2 in normal and neoplasticpithelial tissue has been intensely studied since theiscovery of this gene in 1997.9-11,17-19,22 In the initialeport of its cloning, 15-LOX-2 mRNA expression wasoted in the lung by using commercially available mul-

itissue Northern blots.9 A subsequent report also de-ected 15-LOX-2 in cultured tracheobronchial epithe-

TABLE 1. 15-LOX-2 Immunostainingin Lung Carcinomas

Histologic Type15-LOX-2

positive, n (%)

denocarcinoma, total 33/69 (48)denocarcinoma, without bronchioloalveolarcarcinoma 23/53 (43)

denocarcinoma, bronchioloalveolarcarcinoma 10/16 (63)

quamous cell carcinoma 14/55 (25)arge cell carcinoma 2/14 (14)arge cell neuroendocrine carcinoma 0/5arcinoid tumor 0/9mall cell carcinoma 0/5ynovial sarcoma, monophasic 0/1igh-grade sarcoma with chondroid features 0/1arcinosarcoma 1/1

ial cells by reverse transcription polymerase chain t

845

eaction (RT-PCR), but only when cultured under con-itions promoting differentiation.29 However, to ournowledge, our current study represents the first reportddressing the normal cellular distribution of 15-OX-2 in benign lung as well as its possible expression

n lung carcinomas.In normal lung, benign lung with reactive pro-

esses, and nontumorous portions of lung specimensrom carcinoma cases, 15-LOX-2 immunostaining wasetected essentially exclusively in alveolar lining cellsith morphological and immunophenotypic character-

stics of type II pneumocytes. The identity of these5-LOX-2–immunopositive alveolar lining cells as typeI pneumocytes was supported by immunostaining inerial sections for surfactant-associated proteins andther antigens expressed in this cell type.23-25 This dis-ribution of 15-LOX-2 in alveolar lining cells in benignung contrasts with the distribution of 15-LOX-1 in thearger airways and inflammatory cells, as also deter-

ined by immunohistochemistry.30-33 The absence of5-LOX-2 immunostaining in normal bronchial epithe-ial cells in actual intact tissue specimens by immuno-istochemistry in the current study versus detection ofRNA by RT-PCR in cultured bronchial epithelial cellsay reflect differences in methodology or culture con-

itions that may not precisely recapitulate bronchialpithelial differentiation in vivo. However, our resultsompared with previous immunostaining studieslearly support differences for 15-LOX-2 and 15-LOX-1n benign lung, a difference also supported by recent initro studies showing differential 15-lipoxygensesozyme activity in bronchial epithelial cells.34 On thether hand, these results do not completely exclude theossibility of up-regulation of 15-LOX-2 in bronchialpithelial cells in inflammatory airway disease in vivo,ecause this was not specifically addressed in the cur-ent study.

The expression of 15-LOX-2 in type II pneumo-ytes is of possible significance in terms of normal lunghysiology. 15-LOX-2 has been implicated in regulatingecretory differentiation and proliferation of differen-iated epithelial cells, functions that may be mediated ateast in part by formation of an endogenous ligand forPAR�.12,19 PPAR� expression in normal lung has been

ocalized to type II pneumocytes, the cell type respon-ible for producing and secreting surfactant.23,25 Al-hough specific co-localization of 15-LOX-2 and PPAR�n type II pneumocytes was not investigated, the ob-erved expression of these 2 genes in a high percentagef type II pneumocytes35 suggests a possible role for a5-LOX-2-PPAR� pathway in regulating secretory dif-erentiation and proliferation of these cells.23,25 15-OX-2 may also regulate cell proliferation in epithelialells in a manner independent of PPAR�,36 such thathe functional significance and mechanism of 15-OX-2 in type II pneumocytes remains to be more fullyxplored. The fact that not all pneumocytes that wereorphologically type II were 15-LOX-2 immunoposi-

ive may reflect some aspect of the normal cycle ofhese cells and transient gene expression. In addition to

heir secretory function, these cells are the regenerative

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HUMAN PATHOLOGY Volume 35, No. 7 (July 2004)

r precursor cells for populating the alveoli and are therecursors for the flatter type I pneumocytes, which didot typically appear to be 15-LOX-2 immunopositive inur study. It is of interest to note how strongly and

TABLE 2. 15-LOX-2

Case No. Diagnosis Grade

MA1-20 BAC, nonmucinous 1MA1-36 BAC, nonmucinous 149 BAC, nonmucinous 1MA1-27/C58 BAC, nonmucinous 1MA2-31/C42 BAC, nonmucinous 210 BAC, nonmucinous 150 BAC, nonmucinous 1MA1-30/C60 BAC, nonmucinous 1MA1-39 BAC, nonmucinous 157 BAC, nonmucinous 2MA1-5 Adenocarcinoma, mixed 3MA2-19 Adenocarcinoma, mixed 2MA2-20/C41 Adenocarcinoma, mixed 2MA2-78 Adenocarcinoma, mixed 3MA2-27 Adenocarcinoma, solid 361 Adenocarcinoma, mixed 2MA2-61 Adenocarcinoma, solid 3MA2-74 Adenocarcinoma, solid 3MA2-39 Adenocarcinoma, mixed 1MA1-24 Adenocarcinoma, mixed 3MA1-46 Adenocarcinoma, mixed 1MA2-71 Adenocarcinoma, mixed 2MA2-10 Adenocarcinoma, mixed 3MA2-51/C30 Adenocarcinoma, mixed 214 Adenocarcinoma, mixed 113 Adenocarcinoma, mixed 254 Adenocarcinoma, papillary 1MA2-11 Adenocarcinoma, mixed 216 Adenocarcinoma, mixed 2MA1-1 Adenocarcinoma, mixed 2MA1-26 Adenocarcinoma, mixed 1MA1-59 Adenocarcinoma, acinar 2MA2-43 Adenocarcinoma, mixed 2MA2-4 Large-cell carcinoma 328 Large-cell carcinoma 3MA1-21 SCC 2MA1-31 SCC 3MA2-13 SCC 3MA2-24 SCC 2MA2-40 SCC 3MA2-47 SCC 2MA2-48 SCC 2MA2-63 SCC 3MA2-65 SCC 279 SCC 2MA1-51 SCC 2MA2-6 SCC 3MA2-73 SCC 1MA1-11 SCC 2MA2-38 Carcinosarcoma 3

Abbreviations: BAC, bronchioloalveolar carcinoma; SCC, squamample.

TABLE 3. 15-LOX-2 Immunostaining and Tumor Grade

Grade 15-LOX-2 Expression, n (%)

(Well differentiated) 14/35 (40)(Moderately differentiated) 21/55 (38)(Poorly differentiated) 15/70 (21)

846

ore uniformly 15-LOX-2 positive the cuboidal, type IIneumocyte alveolar epithelial cells were with reactiveyperplasias accompanying diffuse alveolar damage, asell as the apparent increased expression with hyper-lasia occasionally adjacent to lung carcinomas. In-reased expression with increased alveolar cell prolifer-tion may be part of a reactive process allowing forubsequent reduced proliferation and differentiation.urther studies regarding the biology of 15-LOX-2 inype II pneumocytes will be necessary to explore theseossibilities.

unopositive Tumors

15-LOX-2 Intensity % Tumor Cells Positive Score

1 5 11 10 11 10 12 20 22 30 22 30 22 30 23 50 33 80 33 80 32 5 12 5 12 10 11 10 11 10 11 20 11 20 11 20 12 25 22 30 22 30 22 40 22 50 22 50 22 50 23 55 33 80 33 80 33 80 33 90 33 95 33 95 33 100 31 20 13 30 22 5 12 10 11 20 12 10 11 10 12 10 11 5 11 20 11 20 12 5 12 40 22 50 22 40 23 95 32 5 (sarcoma component) 1

ell carcinoma; TMA, tissue microarray sample; C, conventional slide

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15-LIPOXYGENASE-2 IN LUNG CANCER (Gonzalez et al)

ressed in NSCLCs, with at least focal immunostainingn 31% of all lung carcinomas, including in 48% ofdenocarcinomas. That 15-LOX-2 immunostaining pos-ibly relates to cellular differentiation was indicated byhe significant inverse correlation with tumor grade.5-LOX-2 immunostaining also showed a statisticallyignificant inverse correlation with tumor cell prolifer-tion. These results and the biology of 15-LOX-2trongly support that expression in NSCLCs may bemportant in positively regulating tumor differentiationnd negatively regulating proliferation. Although theseroperties are fundamentally important to the biologicehavior of the tumor, there was no significant corre-

ation between 15-LOX-2 expression and patient sur-ival. This is likely a consequence of the many otheractors determining patient outcome as well as inherentimitations of the study size for this clinically aggressiveumor.

Interestingly, 15-LOX-2 immunostaining was ex-ressed in 63% of BACs. Any clinical or biologic differ-nces between 15-LOX-2–positive and 15-LOX-2–nega-ive BACs remain to be more fully investigated.owever, immunopositivity was noted exclusively in theon-mucinous type of BACs, although the number ofucinous BAC cases examined was small. Nine of the

en 15-LOX-2–immunopositive BACs showed type IIneumocyte-like or mixed type II cell and Clara cellytology, whereas 2 of the 3 BACs with pure Claraell-like cytology were immunonegative. Because someonmucinous BACs are thought to be derived from oresemble type II pneumocytes, one possibility is thathese nonmucinous tumors express 15-LOX-2 in a man-er contributing to secretory differentiation similar to

he 15-LOX-2–immunopositive normal or reactive typeI cells. Many (7 of 10) of the 15-LOX-2–immunoposi-ive BACs also showed septal fibrosis or central scarringnot shown), suggesting the possibility of some type ofpithelial–stromal interaction or paracrine effect of 15-OX-2 metabolites. However, fibrosis was not limited to5-LOX-2–expressing tumors, such that a paracrineunction of 15-HETE in lung carcinomas remains spec-lative. In non-BAC–invasive adenocarcinomas, noeadily apparent correlation was noted between 15-OX-2 expression and histological pattern in 65 non-ucinous adenocarcinomas. That there was a tendency

or more of the solid-type patterns to be immunonega-ive may be related, but this is probably a function ofifferentiation, as more precisely reflected in the statis-

ically significant inverse correlation of 15-LOX-2 im-unostaining and tumor grade. Therefore, the find-

ngs in this study suggest that expression in lungdenocarcinomas may be related to secretory differen-iation in these tumors, and not necessarily a reflectionf origin from or differentiation towards type II pneu-ocytes, because lung adenocarcinomas have many

otential cellular origins.20

The previously known AA-metabolizing 5-LOX, 12-OX, and 15-LOX-1 are expressed highly in peripherallood cells and have been reported primarily as in-reased in a variety of carcinomas.2,3 15-LOX-2 is some-

hat unique, compared with these other genes, in its c

847

igh level of expression in certain benign epithelialells.2 In the prostate, 15-LOX-2 is highly expressed inhe majority of differentiated secretory epithelialells.11 15-LOX-2 is reduced in prostate carcinoma andigh-grade prostatic intraepithelial neoplasia,11,17,18

ith statistically significant retained expression only inower grade tumors,17 which have similar cytoplasmicecretory differentiation as 15-LOX-2–positive benignrostate glands.37 In the skin, 15-LOX-2 expression istrongest in the androgen-regulated sebaceous, apo-rine, and eccrine glands.22 Strong expression in dif-erentiated sebocytes parallels expression of PPAR� inhese cells, and androgens and PPAR� agonists addi-ively promote secretory differentiation in sebocytes.38

n androgen receptor–negative prostate cell cultures,5-LOX-2 is up-regulated by glucocorticoids.39 Al-hough regulation in type II pneumocytes remains toe more fully investigated, the strong expression of5-LOX-2 in these PPAR�-expressing, steroid-respon-ive secretory cells23,25 adds support to the emergingoncept of a role for 15-LOX-2 in regulating prolifera-ion and secretory differentiation in steroid receptornd PPAR�-expressing secretory epithelial cells.

The expression of 15-LOX-2 in better differenti-ted lung adenocarcinomas and its significant inverseorrelation with proliferation is in keeping with theypothesis that the enzyme plays a role in regulatingroliferation and secretory differentiation in these bet-er differentiated neoplasms. PPAR� mRNA is ex-ressed in NSCLC, and 50% of primary lung cancershow immunostaining for PPAR�.40 NSCLC cells, in-luding adenocarcinoma-derived A549 cells, are re-ponsive to PPAR� agonists23 that result in growth ar-est, decreased activity of MMP-2, increased expressionf more mature cell markers, induction of apoptosis,nd reduced production of VEGF, suggesting not onlyn anti-proliferative effect but also a possible anti-an-iogenic effect.40,41 Although 15-LOX-2 could exertffects through other mechanisms, expression of a 15-OX-2-PPAR� pathway in NSCLCs may inhibit tumorrowth, invasion, and metastasis, whereas loss of a 15-OX-2-PPAR� pathway may contribute to the malig-ant phenotype, through multiple mechanisms.

In addition to expression in differentiated secre-ory cells and better differentiated adenocarcinomas asn prostate17 and lung (results of current study), 15-OX-2 is expressed in some squamous epithelia. In thepidermis, it appears to be expressed predominantly inhe basal layer of normal skin, whereas it is reduced inasal cell carcinomas.22 In a recent investigation using

mmunohistochemistry with the same antibody as in theurrent study and supported by in situ hybridization,5-LOX-2 was expressed in the squamous epithelium ofhe esophagus and was reduced in esophageal carcino-

as, including adenocarcinomas and SCCs.10 The mu-ine homologue of 15-LOX-2, an 8-LOX, contributes toquamous keratinocyte differentiation by forming a li-and (8-HETE) for PPAR�,42 and human 15-LOX-2 hasecently been shown to also be able to catalyze theormation of a 15-HETE-ester PPAR� agonist.15 In the

urrent study, although lesions such as squamous meta-

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HUMAN PATHOLOGY Volume 35, No. 7 (July 2004)

lasia were not specifically evaluated, variable positive5-LOX-2 immunostaining was noted in some SCCs,nd the inverse correlation with tumor grade washown for analyses including both adenocarcinomasnd SCCs.

Whether all biologic functions of 15-LOX-2 metab-lites are mediated via PPARs remains to be more fullylucidated.36 Indeed, the localization of 15-LOX-2 inhe cytoplasm as well as or instead of the nucleus inenign cells and many tumor cells raises considerationf other mechanisms besides forming ligands for nu-lear receptors.36 All data thus far indicate that suchytoplasmic immunostaining in tissues is specific. Inter-stingly, certain enzymatically less active or inactiveplice variants of 15-LOX-2 do not appear capable ofocalizing to the nucleus yet retain tumor suppressiveunctions.36 These observations suggest that some tu-

or-suppressive functions of 15-LOX-2 may be inde-endent of 15-HETE formation and PPAR� activation.ecause 15-LOX-2 immunostaining in better differen-

iated lung carcinomas is both nuclear and cytoplasmic,uture studies should address possible differential ex-ression of splice variants in the benign and malignant

ung, paralleling ongoing efforts to more fully elucidatehe mechanisms of action of the growth inhibiting func-ions of this gene.

In summary, we have shown some expression of5-LOX-2 in type II pneumocytes in benign lung, in-reased expression in reactive type II pneumocytes, andnzyme expression in almost 50% of lung adenocarci-omas, including 63% of BACs. The finding of an

nverse relationship between 15-LOX-2 and tumorrade and proliferation index suggest an importantole for the 15-LOX-2 pathway in these tumors.hether 15-LOX-2 functions to generate endogenous

igands for PPAR� in type II pneumocytes and in 15-OX-2–positive, better differentiated lung adenocarci-omas or functions through other signaling pathwaysemains to be determined. If functioning at least partlyhrough PPAR�, identification of the 15-LOX-2 expres-ion status of lung tumors may facilitate identificationf patients who possibly are suited for treatment withPAR� agonists.

Acknowledgment. The authors thank Dr. Jeff Whittset,hildren’s Hospital Medical Center, Cincinnati, OH, for theifts of antibodies to surfactant proteins SP-B and pro–SP-B.

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