Research Paper

The Combination Of Weak Expression Of PRDX4 And Very High MIB-1 Labelling
Index Independently Predicts Shorter Disease-free Survival In Stage I Lung
Adenocarcinoma

Akihiro Shioya,1 Xin Guo,1 Nozomu Motono,2 Seiya Mizuguchi,3 Nozomu Kurose,1,3
Satoko Nakada,1,3 Akane Aikawa,1,3 Yoshitaka Ikeda,4 Hidetaka Uramoto,2 Sohsuke
Yamada1,3*

Short running head: PRDX4/MIB-1 predicts recurrence in stage I-LUAD

Departments of 1Pathology and Laboratory Medicine & 2Thoracic Surgery, Kanazawa
Medical University, Ishikawa; 3Department of Pathology, Kanazawa Medical University
Hospital, Ishikawa; and 4Division of Molecular Cell Biology, Department of Biomolecular
Sciences, Saga University Faculty of Medicine, Saga, Japan.
*Corresponding author: Sohsuke Yamada, M.D., Ph.D., Department of Pathology andLaboratory Medicine, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa,1

920-0293,

Japan.

Tel:

81-76-218-8264;

Fax:

81-76-286-1207;

andsohsuke@kanazawa-med.ac.jp
Word count: 5,857 words, Abstract 218 words; references: 34; figures and tables: 7.

2

E-mail:ABSTRACT
Background: Oxidative stress plays pivotal roles in the progression of lung adenocarcinoma(LUAD) through cell signaling related closely to cancer growth. We previously reported thatperoxiredoxin 4 (PRDX4), a secretory-type antioxidant enzyme, can protect against thedevelopment of various diseases, including potential malignancies. Since many patients withearly-stage LUAD develop recurrence, even after curative complete resection, weinvestigated the association of the PRDX4 expression with the clinicopathological featuresand recurrence/prognosis using post-surgical samples of stage I-LUAD.
Methods: The expression of PRDX4 and MIB-1, a widely accepted Ki67 protein, wasimmunohistochemically analysed in 206 paraffin-embedded tumour specimens of patientswith stage I-LUAD. The PRDX4 expression was considered to be weak when less than 25%of the adenocarcinoma cells showed positive staining.
Results: A weak PRDX4+ expression demonstrated a significantly close relationship withpathologically poor differentiation, highly invasive characteristics and recurrence. The
decrease in PRDX4-positivity potentially induced cell growth in LUAD, which wascorrelated

significantly

with

a

very

high

MIB-1

labellingindex(≥17.3%).

Univariate/multivariate analyses revealed that the subjects with both weak PRDX4+3

expression and a very high MIB-1 index had significantly worse disease-free survival rates
than other subjects.
Conclusions: The combination of weak PRDX4 expression and a very high MIB-1 index can
predict high proliferating activity and recurrence with a potential poor prognosis, especiallyin post-operative stage I-LUAD patients.

Keywords: lung adenocarcinoma (LUAD); stage I; PRDX4; MIB-1; recurrence.

4

Introduction
Lung cancer is one of the most common fatal malignancies in developed countries [1,2] and it
has been the number-one cause of cancer-related deaths among Japanese for two decades. Up
to 105,000 new cases of lung cancer were diagnosed in 2013, and in 2016, more than 50,000
patients died of it in Japan alone (http://ganjoho.jp/reg_stat/statistics/stat/summary.html).
More than 85% of lung cancer cases are classiﬁed as non-small cell lung cancer (NSCLC),
and lung adenocarcinoma (LUAD) is the most well-known histopathological subtype of
NSCLC in Japan [3]. The 5-year overall survival rate is reportedly less than 20% for NSCLC,
including LUAD [4], and surprisingly, up to 30% of patients develop recurrence within 5
years, even in cases of stage I-LUAD after curative complete surgical resection [5,6]. The
potential cell growth of LUAD, regardless occult metastases at the time of operation, issuggested to be primarily responsible for its recurrence with a subsequent poor prognosis [7].
Therefore, predicting which patients are prone to develop recurrence after surgery is critical,
even with early-stage LUAD. Indeed, clinicopathological elements, such as the differentiationor tumour-node-metastasis (TNM) stage of LUAD, can strongly suggest the risk ofrecurrence and/or the prognosis [8,9], but no molecular or genetic factors have yet been
identified, and the clinical significance of such biological markers is still under evaluation.

5

Oxidative stress, induced by reactive oxygen species (ROS), can function as a crucial
and diverse pathophysiological regulator of cellular signalling pathways, such as the response
to inflammatory and growth factor stimulation [10]. Accumulating evidence also suggeststhat the dysregulation of oxidant and antioxidant redox signalling might cause or accelerate ahost of various human diseases, including malignancies [11]. In this vein, the aberrant
expressions of oxidative stressors and antioxidant properties play pivotal roles in theinitiation of the progression of LUAD through cell signalling pathways related closely tocancer growth [12].
Peroxiredoxin 4 (PRDX4) is a member of the PRDX antioxidant enzyme family, which
consists of at least six distinct PRDX genes, expressed in mammals (PRDX1–6) [13]. In
contrast to the merely intracellular localization of other family members, PRDX4 is the only
secretory form, and significant levels of this enzyme have been noted, particularly in cultured
medium [14]. According to our serial in vivo studies, the elevated expression of PRDX4 has
been recognized in not only endoplasmic reticulum but serum and various tissues of mice andhuman with chronic inflammatory diseases, manifesting as metabolic syndrome and potential
malignancies [15,16]. The overexpression of PRDX4 in mice can markedly suppress the local
and systemic levels of ROS and protect various tissues against oxidative damage by reducing
the inflammatory response and apoptosis and/or growth factor stimulation in the6intra-/extra-cellular space [17]. Furthermore, a growing body of evidence suggests that
apoptotic and/or proliferative activities might be significantly correlated with the PRDX4
expression [18,19].
Given the above, we hypothesize that PRDX4 not only regulates basic cellularfunctions of LUAD but is a parameter of cell growth, similar to the widely-accepted Ki67
(MIB-1) protein [20,21]. Furthermore, PRDX4 might be a promising clinical biomarker forthe recurrence/prognosis of LUAD and be a target for early diagnoses and therapies for
LUAD. However, no studies have explored possible associations between the PRDX4
expression, especially in early-stage LUAD, and the clinicopathological characteristics of alesion, including its differentiation and invasiveness or patients’ recurrence/prognosis.
In the current study, using an original, specific rabbit polyclonal PRDX4 antibody
generated against the recombinant PRDX4 protein [22], we evaluated the expression of
PRDX4 in post-surgical specimens using stage I-LUAD patients’ clinicopathological data,
demonstrating that PRDX4 was weakly expressed in most invasive human LUAD specimens,
especially those with poor differentiation, pleural involvement, recurrence, and an MIB-1
labelling index exceeding 17.3% (i.e. very high proliferating activity). These findings suggestthat the combination of weak PRDX4+ expression and a very high MIB-1 index is7

significantly correlated with a poor disease-free survival (DFS; i.e. recurrence) of stage
I-LUAD.

8

Materials and methods
Patients and tissue specimens
Surgically resected stage I-LUAD tissues were evaluated in the present study. Pathologicalreports were reviewed to identify patients who underwent lobectomy (170 patients), partial
resection (4 patients), or segmentectomy (32 patients) for LUAD between January 2005 andDecember 2015 at the hospital of Kanazawa Medical University. All materials in this article
were approved by the Ethical Committee of Kanazawa Medical University (I159). Patients
who suffered perioperative deaths, defined as death during the patient’s initial hospitalization
or within 30 days of surgery, were excluded. A total of 206 patients with available follow-up
data comprised the cohort of this retrospective study after further excluding those with thefollowing characteristics: (a) other prior or concomitant malignant tumours, (b) coexisting
medical problems of sufficient severity to shorten the life expectancy, and (c) adjuvant
chemotherapies or radiotherapies prior to the surgery.
Three pathologists examined all resected specimens to confirm their histopathologicalfeatures, including the differentiation. Revisions in the International System for Staging Lung
Cancer was used for the final staging [23], and all lung adenocarcinomas were further
classified based on the histological classification system from the International Association
for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European
9Respiratory

Society(ERS)/International

Multidisciplinary

Classification

of

Lung

Adenocarcinoma [24].
In accordance with this IASLC/ATS/ERS classification system [24], adenocarcinoma insitu (AIS) cases were selected using haematoxylin and eosin (H&E)-stained sections
according to the following criteria: localized lesion (≤3 cm) with growth of neoplastic cells
along pre-existing alveolar structures, lack of stromal invasion, absence of papillary or
micropapillary patterns, and absence of intra-alveolar tumour cells. Tumours were
subclassified as minimally invasive adenocarcinoma (MIA) in cases with a solitaryadenocarcinoma (≤3 cm) with a predominantly lepidic growth pattern and ≤5 mm invasion in
the greatest dimension of any one focus. The invasive component to be measured in MIA wasdefined as follows: histological subtypes other than a lepidic pattern (i.e. acinar, papillary,
micropapillary, or solid) or tumour cells infiltrating myofibroblastic stroma. The invasive
component was measured morphometrically, and a 5-mm cut-off value was used to
distinguish MIA from lepidic-predominant invasive adenocarcinoma (LPA). For cases that
contained multiple tumour foci, only the largest focus was examined. Elastica van Gieson(EVG) stains were also performed if necessary. MIA was excluded if the tumour invaded thelymphatics, blood vessels, pleura, or contained tumour necrosis. LPA and non-lepidic
adenocarcinomas with invasion that were >5 mm in diameter were classified as invasive10

adenocarcinoma and divided further into acinar (APA), papillary (PPA), solid (SPA),
mucinous adenocarcinoma (MA), and micropapillary (MPA) based on their predominantinvasive pattern in H&E sections.
Clinical information was gathered from patients’ records. The disease-free survival(DFS) and disease-specific survival (DSS) were defined as the interval from the date ofsurgery to recurrence and from the date of surgery to death, except for patients who died fromcauses other than LUAD, or the most recent clinic visit, respectively. Patients were followed
up and prospectively evaluated every month within the first postoperative year and at
approximately two- to four-month intervals thereafter using chest X-ray, thoracic andabdominal computed tomography (CT), brain magnetic resonance imaging (MRI), serum
biochemistry, or measurements of tumour markers. CT, MRI, and bone scintigraphy were
performed every six months for three years after surgery. Additional examinations were
performed if any symptoms or signs of recurrence were recognized. Formalin-fixed,
paraffin-embedded tissue blocks came from our Department of Pathology & laboratory
medicine. EVG and immnohistochemical D2-40 (Nichirei Bioscience Co., Tokyo, Japan,
diluted 1:1) staining very clearly revealed pleural involvement (pl) and vascular invasion (v)in the former, and lymphatic invasion (ly) in the latter, respectively.

11

Preparationofantibodies

against

PRDX4

and

secondaryantibodies,andimmunohistochemistry of tissue samples
A rabbit anti-PRDX4 IgG was produced as previously described [22]. Immunohistochemical
staining was performed by the antibody-linked dextran polymer method for antibody-bridgelabelling, with haematoxylin counterstaining (EnVision; Dako Cytomation, Co., Glostrup,
Denmark). Deparaffinized and rehydrated 4-µm sections were incubated in 10% H2O2 for 5
min to block the endogenous peroxidase activity. The sections were then rinsed and incubatedwith rabbit polyclonal anti-PRDX4 (diluted 1:1000) and mouse monoclonal MIB-1 (Ki67;
Dako Cytomation, Co., diluted 1:50) antibodies for 2 h and 30 min, respectively [19,21]. The
second antibody-peroxidase-linked polymers were then applied, and the sections wereincubated with a solution consisting of 20 mg of 3.3’-diaminobenzidine tetrahydrochloride,
65 mg of sodium azide, and 20 ml of 30% H2O2 in 100 ml of Tris-HCL (50 mM, pH7.6).
After counterstaining with Meyer’s haematoxylin, the sections were observed under a lightmicroscope. The sections were first scanned at a low power for all fields (original
magnification: × 40) with tumour and non-tumour tissues to account for the heterogeneity of
distribution. The number of cells showing positive staining and the pattern of staining wererecorded. Necrotic tissues, stromal cells, and lymphoid cells were not included in therecording.
12The evaluation of the immunohistochemical results by scoring
The immunoreactivity for PRDX4 in each case was assessed semi-quantitatively by
evaluating the proportion of positive cells compared to the total neoplastic LUAD cells. We
selected and validated the immunohistochemical cut-off scores for PRDX4 positivity (25%)
and the MIB-1 labelling index (17.3%) based on the performance of a receiver operating
characteristic (ROC) curve analysis [25]. All patients were divided into two groups based on
the PRDX4 expression as follows: strong when the PRDX4 staining was ≥25% and weak
when the staining was <25%.
All histological and immunohistochemical slides were evaluated by two independent
observers (certified surgical pathologists in our department; A.S. and N.K.) using a blind
protocol design (observers blinded to the clinicopathological data). The agreement between
the observers was excellent (more than 90%) for all antibodies investigated, as measured by
the interclass correlation coefficient. For the few (less than 1%) instances of disagreement, aconsensus score was determined by a third board-certified pathologist (S.Y.) in our
department [21,26,27].

Statistical analyses
13

The significance of correlations was determined using Fisher’s exact test or χ2 test, whereappropriate, in order to assess the relationships between the immunohistochemical expression
and the clinicopathological features [27]. Survival curves were plotted with the Kaplan-Meiermethod and compared with the log-rank test. Hazard ratios and 95% confidence intervals(95% CIs) were estimated using univariate or multivariate Cox proportional hazard models[21,26-29]. All statistical tests were two-tailed, with values of P < 0.05 considered to besignificant.
All of the above statistical analyses were performed with the EZR (Saitama Medical
Center, Jichi Medical University, Japan) graphical user interface for the R software program(The R Foundation for Statistical Computing, version 2.13.0) [27,28,30]. More precisely, it isa modified version of R commander (version 1.6-3) that incorporates the statistical functions
frequently used in biostatistics.

14

Results
Patient characteristics
The clinicopathological features of the 206 patients with stage I-LUAD who were able to beevaluated are summarized in Table 1. The range of age at surgery was 33–83 years (averageand median were 67 and 68 years, respectively). More than half of patients (128/206) had a
Brinkman index (BI) under 400; the remaining patients (78/206) were ≥ 400 BI. The mediantumour size was 22 mm (range: 6–50 mm). The tumour grading included 112
well-differentiated (54.4%), 78 moderately differentiated (37.9%), and 16 poorly
differentiated adenocarcinoma (7.8%). According to further histopathological analyses with
the IASLC/ATS/ERS classification system (Travis et al., 2011), 19 (9.2%) patients had AIS,
38 (18.4%) MIA, 52 (25.2%) LPA, 29 (14.1%) APA, 49 (23.8%) PPA, 3 (1.5%) MA, 3(1.5%) MPA, and 13 (6.3%) SPA. Postoperative follow-up was available for all 206 patients(average: 51 months; range: 2–145 months). The median postoperative DFS was 42 months
with a 1-year recurrence rate of 2.9%, 2-year recurrence rate of 11.2%, 5-year recurrence rateof 17%, and total recurrence rate of 20%.

Association of the PRDX4 expression with the clinicopathological variables and DFS

15

Based on the cut-off points for the PRDX4 and MIB-1 expression, all subjects were divided
into two groups for each parameter: a weak and strong PRDX4 group and a low and high
MIB-1 group ( Figure 1). To clarify the association of PRDX4 expression (weak vs. strong
PRDX4+) (Figure 2) with the clinicopathological characteristics of the cohort, the variables
were split as shown in Table 2. There were no significant differences between the patientswith weak and strong PRDX4+ tumour expressions in terms of the age, gender, and BI (P >0.05). The moderately to poorly differentiated tumour rate in the strong PRDX4+ samples
was 30/103 (29.1%), but the rate was 65/103 (63.1%) in weak PRDX4+ samples.
Furthermore, the highly invasive (APA/PPA/MA/MPA/SPA) adenocarcinoma rate was 30/103(29.1%) in strong PRDX4+ samples but 67/103 (65%) in weak PRDX4+ samples. Weak
PRDX4 expression was closely associated with moderate to poor differentiation (P < 0.0001),
highly invasive subtypes (APA/PPA/MA/MPA/SPA) (P < 0.0001), and a high (≥17.3%)
MIB-1 labelling index (P = 0.0018, r = -0.172) but not with the tumour size or presence of v
and ly (P > 0.05). PRDX4 expression was apparently detectable in the adjacentnon-neoplastic bronchioloalveolar epithelium (Figure 2). On immunohistochemistry, PRDX4and MIB-1 displayed intracytoplasmic and nuclear expression patterns, respectively (Figure2). Furthermore, the PRDX4 stain status was significantly correlated with the presence of pl16

(P = 0.017). The rate of PRDX4 expression in an intracytoplasmic pattern was much lower ininvasive LUAD areas, including pl (+), than in non-invasive ones (Figure 3).
In a Kaplan–Meier analysis, lung adenocarcinoma patients with weak PRDX4+
expression had a significantly shorter postoperative DFS than those with strong PRDX4+
expression (P = 0.004, Figure 4A). Lung adenocarcinoma patients with weak PRDX4+ and ahigh MIB-1 index had a markedly shorter postoperative DFS than other patients (P < 0.0001,
Figure 4B). However, the PRDX4 expression was not associated with the postoperative DSS
in the present study.

The combination of weak PRDX4 expression and a high MIB-1 labelling index represents asignificant independent prognostic indicator for lung adenocarcinoma
To assess whether or not the PRDX4 expression was an independent predictor of
postoperative DFS, a Cox proportional-hazards model was created in a forward fashion
including only covariates that had statistically significant correlations with the DFS, using an
inclusion threshold of P < 0.05 (Table 3). A univariate analysis showed that the tumour size(> 2 cm), tumour grade, and presence of pl, ly, and v and both weak PRDX4+ and a high
MIB-1 labelling index status, were significant predictors of a poor survival (P = 0.021, <0.0001, < 0.0001, = 0.0002, < 0.001, and < 0.0001, respectively). Furthermore, a multivariate
17

analysis showed that, after correction for confounding variables, the combination of weak
PRDX4+ expression and a high MIB-1 index remained an independent prognostic indicator
for the DFS (P = 0.013), as well as the tumour grade (P = 0.0009).

18

Discussion
In the present large cohort, we showed that weak PRDX4 expression was closely correlatedwith various critical clinicopathological features of 206 patients with post-surgical LUAD
especially in stage I, using a unique polyclonal antibody raised against the distinctive,
recombinant PRDX4 protein. The current findings have indicated, for the first time, that the
combination of weak PRDX4+ expression and a very high MIB-1 labelling index is noveland powerful independent marker for post-operative recurrence with a potential pooroutcome in stage I-LUAD patients.
Recurrence in LUAD patients after curative surgery remains a significant problem andcan significantly affect the clinical course and survival of these patients [6,31]. Accumulated
data suggest that weak PRDX4+ expression in stage I-LUAD is closely associated withpathological poorly differentiated characteristics and further invasive/aggressive behaviours,
including pleural involvement or recurrence; furthermore, lesions with a weak PRDX4+
expression often co-express a very high MIB-1 labelling index (≥ 17.3%), resulting in
potential cell growth (i.e. high proliferating activity) of even early-stage LUAD. We wereable to prove a critical, key specific antioxidant molecule, PRDX4, which should be poorly
differentiated, invasive/proliferative and recurrent tumour markers or therapeutic targets
especially for stage I-LUAD.
19

However, some limitations associated with the present study warrant mention. First,
this is a cohort-based, retrospective study at a single institution, even though we conductedthorough control through the random selection of post-operative stage I-LUAD patients andadherence to strict exclusion criteria. Second, we only conducted immunohistochemical andnot detailed molecular analyses. Further in-depth follow-up in much larger cohorts of stage
I-LUAD patients, along with detailed molecular investigations using LUAD cell culture lines,
will be required to confirm the intriguing correlation of weak PRDX4+ expression and very
high Ki67 expression with recurrence and a subsequent poor survival in post-surgical stage
I-LUAD patients. The mechanism underlying how PRDX4 is involved in cellular signalling
pathways, including the response to growth factor stimulation, should also be examined in a
future research article. However, despite these limitations, the PRDX4 and/or Ki67
expression patterns in both post-/pre-operative tissue and serum samples of LUAD may allow
for improved patient selection of candidates for adjuvant/neoadjuvant systemic therapy aswell as the early prediction of the clinical post-operative course. In addition, sincesecretory-type PRDX4 can appear in body fluids, it might be a quantitative soluble,
tumour-specific marker for LUAD.
We suspected that PRDX4 might have a significant function of inhibiting ROS-related
carcinogenesis of LUAD as a tumour suppressor through oxidant and antioxidant redox
20

signalling pathways associated with cancer growth. Some of our present findings are in linewith those of previous studies of several other human malignancies. For example, acutepromyelocytic leukemia showed significantly reduced PRDX4 expression along with thecontrol of granulocyte colony-stimulating factor (i.e. growth factor) responses [32].
Furthermore, our unpublished data suggest that human hepatocellular carcinoma specimens
with a low expression of PRDX4 tend to have a highly malignant phenotype with a poor
overall survival. However, other findings of ours disagree with those of other groups withregard to PRDX4 immunohistochemistry in squamous cell carcinomas (SCCs) [33,34]. Theseauthors found that patients with an increased PRDX4 expression, which was closely
associated with greater progressive activity, had a significantly shorter post-operative DSS incases of oral cavity SCC [33] and DFS in cases of early-stage lung SCC [34] than those witha low expression. These discrepancies may be due in part to not only the heterogeneity ofmalignancies but also the methodology of assessment in each study, such as the size of thecohort, differences in the antibodies used against each PRDX4, and the arbitrary or strict
selection and validation of the immunohistochemical cut-off scores for PRDX4, which wereoccasionally not based on any ROC curve analyses. Further experiments are necessary toaddress methodology standardization for PRDX4 in clinical specimens after collecting andinvestigating a much larger number of surgical cases.
21

CONCLUSION
Our observations suggest that weak PRDX4+ expression in primary stage I-LUAD isvery closely related to pathological phenotypes with a poor outcome, e.g. those with poordifferentiation, highly invasive characteristics and recurrence, or a very high MIB-1 labelling
index, reflecting a background of marked cancer cell growth/proliferation. Furthermore, the
DFS of LUAD patients with both weak PRDX4+ and a very high MIB-1 index was
significantly shorter than that of other patients. These analyses suggest for the first time thatthe combination of weak PRDX4 and high MIB-1 may be a novel and useful independent
predictor of recurrence with a poor prognosis in patients with primary stage I-LUAD.

List of abbreviations

LUAD, lung adenocarcinoma; PRDX4, peroxiredoxin 4; NSCLC, non-small cell lung cancer;
TNM, tumour-node-metastasis; ROS, reactive oxygen species; EVG, Elastica van Gieson;
AIS, adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma; LPA, invasive
adenocarcinoma, lepidic predominant; APA, invasive adenocarcinoma, acinar predominant;
PPA, invasive adenocarcinoma, papillary predominant; SPA, invasive adenocarcinoma, solid22

predominant; MA, invasive mucinous adenocarcinoma; MPA, invasive adenocarcinoma,
micropapillary predominant; DFS, disease-free survival; DSS, disease-specific survival;

ROC, receiver operating characteristic and SCC, squamous cell carcinoma.

Declarations

Ethics approval

All materials including consent to participate in this article were approved by the EthicalCommittee of Kanazawa Medical University (I159).

Consent for publication

Written informed consent was obtained from the patient the patient and his family on

admission for the publication of this case report and any accompanying images.

Availability of data and materials

The dataset supporting the findings and conclusions of this research is included within thearticle.

Competing interests

The authors declare no conflicts of interest in association with this study.
23

Funding

This work was supported in part by Grants-in-Aid for Scientific Research 16K08750 to S.Y.
and 25462202 to H.U.) from the Ministry of Education, Culture, Sports, Science andTechnology, Tokyo, Japan; a grant from the MSD Life Science Foundation, Public Interest
Incorporated Foundation, Japan (to S.Y.); and grants from National Natural Science
Foundation of China (No. 81402490) (to X.G.), Natural Science Foundation of Hebei
Province (No. H2016206170) (to X.G.), and High level talent support project of Hebei
Province (No. CG2015003011) (to X.G.).
Acknowledgment
We would like to thank Yuka Hiramatsu, Mariko Nakano and Manabu Yamashita for theirexpert technical assistance.

24

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31

Table 1. Detailed patients'clinicopathological characteristics
Characteristic

Patients (n=206)

Age (years)
Average

67

Median

68

Range

33-83

>60

166

≤60

40

Sex
Male

104

Female102

Brinkman index (BI)
≥400

78

<400

128

Months after surgery
Average

51

Median

49

Range

2-145

Tumour differentiation
Well

112

Moderately

78

Poorly16

Histopathological subtype
AIS

19

MIA

38

LPA

52

APA

29

PPA

49

MA

3

MPA

3

SPA

13

Tumour size (mm)Average

23.5

Median

22

Range

6-50

32

CEA(μg/L)
≥5

65

<5

141

AIS = adenocarcinoma in situ; MIA = minimally invasive adenocarcinoma; LPA= invasive adenocarcinoma, lepidic predominant; APA = invasive
adenocarcinoma, acinar predominant; PPA = invasive adenocarcinoma, papillary
predominant; SPA = invasive adenocarcinoma, solid predominant; MA =invasive mucinous adenocarcinoma; MPA = invasive adenocarcinoma,
micropapillary predominant

33

Table 2. Detailed correlations between the PRDX4 expression andclinicopathological variables
Strong expression

Weak expression(n=103)(n=103)

Number (%)

Number (%)>60 years

88 (85.4)

81 (78.6)≤60 years

15 (14.6)

22 (21.4)

Male

46 (44.7)

58 (56.3)

Female57 (55.3)

45 (43.7)≥400

36 (35.0)

45 (43.7)<400

67 (65.0)

58 (56.3)

Well

73 (70.9)

38 (36.9)

Moderately

25 (24.3)

54 (52.4)

Poorly5 (4.9)11 (10.7)

AIS

13 (12.6)6 (5.8)MIA

28 (27.2)

10 (9.7)

LPA

32 (31.1)

20 (19.4)

APA

6 (5.8)23 (22.3)

PPA

18 (17.5)

31 (30.1)MA

1 (1.0)2 (1.9)MPA

2 (1.9)1 (1.0)SPA

3 (2.9)10 (9.7)>2cm

52 (50.5)

63 (61.2)≤2cm

51 (49.5)

40 (38.8)(+)

11 (10.7)

25 (24.3)(-)

92 (89.3)

78 (75.7)

P

Age
0.276

Gender0.125

Brinkman index (BI)0.254

Tumour differentiation<0.0001

Histopathologicalsubtype<0.0001

Tumour size
0.161

pl

ly
34

0.017

(+)

32 (31.1)

37 (35.9)(-)

71 (68.9)

66 (64.1)(+)

25 (24.3)

37 (35.9)(-)

78 (75.7)

66 (64.1)≥17.3% (high)

18 (17.5)

39 (37.9)<17.3% (low)85 (82.5)

64 (62.1)(+)

9 (8.7)31 (29.2)(-)

94 (91.3)

72 (70.8)

0.555

v
0.095

MIB-1 index
0.0018Recurrence
0.0002

AIS = adenocarcinoma in situ; MIA = minimally invasive adenocarcinoma; LPA =invasive adenocarcinoma, lepidic predominant; APA = invasive adenocarcinoma,
acinar predominant; PPA = invasive adenocarcinoma, papillary predominant; SPA= invasive adenocarcinoma, solid predominant; MA = invasive mucinous
adenocarcinoma; MPA = invasive adenocarcinoma, micropapillary predominant; pl= pleural involvement; ly = lymphatic invasion; v = vascular invasion.

35

Table 3. Univariate and multivariate analyses of the survival in 206 patients with stage I-LUAD, according
to the clinicopathological variables and a low PRDX4 expression and high MIB-1 labelling index
Univariate

Multivariate

Hazard ratio

95% CI

P-value

Hazard ratio

95% CI

P-value

Weak PRDX4+high MIB-1

6

3.16-11.39<0.0001

2.56

1.21-5.42

0.013

Tumour size

2.33

1.14-4.78

0.021

2.14

0.94-4.89

0.068

Differentiation

14.22

5.05-40.01<0.0001

13.1

2.88-59.67

0.0009pl (+)

4.6

2.43-8.73<0.00011.45

0.70-2.99

0.316

ly (+)

3.77

1.88-7.59

0.00021.32

0.61-2.89

0.478

v (+)

3.99

2.02-7.87<0.0001

0.89

0.39-1.99

0.769

36

FIGURE LEGENDS

Figure 1. The results of the receiver operating characteristic (ROC) curve analyses forselecting and validating the immunohistochemical cut-off points for PRDX4 and MIB-1
expression. We selected the cut-off values of PRDX4 and MIB-1 using ROC and the area
under the curve (AUC), as an effective measure of accuracy has been considered ameaningful interpretation. We selected 25 and 17.3, respectively, as the cut-off points for
PRDX4 and MIB-1, since the AUC for recurrence was the highest among allclinicopathological variables.

Figure 2. Representative images of immunohistochemical analyses of PRDX4 and MIB-1 inhuman stage I-LUAD (strong PRDX4 with low MIB-1; weak PRDX4 with high MIB-1).
Intracytoplasmic staining pattern of PRDX4 was confirmed in LUAD cells (inset). (Original
magnification: ×100; inset, ×400). Bar = 200 µm (×100)

Figure3. Representative pictures for H&E, elastica van Gieson (EVG) andimmunohistochemical analyses of PRDX4 in stage I-LUAD tissue with pleural involvement
37(pl). EVG staining very clearly reveals elastic fibres of the visceral pleura (pl(+)). An
intracytoplasmic staining pattern of PRDX4 was confirmed in LUAD cells (inset). (Original
magnification: Bar = 2 mm (×12.5) or 200 µm (×100); inset, ×400).

Figure 4. Kaplan–Meier curves of the disease-free survival (DFS) in patients with lungadenocarcinoma after surgery according to the PRDX4 expression. Weak PRDX4 expression
alone as well as weak PRDX4/high MIB-1 is associated with a significantly shorter
postsurgical DFS in stage I-LUAD patients.

38

Figure 1

PRDX4 (vs. recurrence)

AUC 0.72
95%CI 0.635 - 0.8

MIB-1 (vs. recurrence)

AUC 0.717
95%CI 0.624 - 0.809

Weak PRDX4 expression& very high MIB-1

Strong PRDX4 expression& low MIB-1

Figure 2
PRDX4
MIB-1

Invasive areas (pl+)

Non-invasive areas

High-power view
Low-power view

Figure 3
HE
EVG
PRDX4

Figure 4

BA

P = 0.004

P ＜ 0.0001

Strong PRDX4 expression

The others

Weak PRDX4 expression

Weak PRDX4 expression / MIB-1 ≥ 17.3% (high)