Oncotarget

Case Reports:

Somatic mutations and increased lymphangiogenesis observed in a rare case of intramucosal gastric carcinoma with lymph node metastasis

PDF |  HTML  |  Supplementary Files  |  How to cite  |  Order a Reprint

Oncotarget. 2018; 9:10808-10817. https://doi.org/10.18632/oncotarget.24289

Metrics: PDF 826 views  |   HTML 1202 views  |   ?  

Naoki Ikari, Shota Aoyama, Akiyoshi Seshimo, Yuji Suehiro, Tomoko Motohashi, Shohei Mitani, Sawako Yoshina, Etsuko Tanji, Akiko Serizawa, Takuji Yamada, Kiyoaki Taniguchi, Masakazu Yamamoto and Toru Furukawa _

Abstract

Naoki Ikari1,2,*, Shota Aoyama1,*, Akiyoshi Seshimo1, Yuji Suehiro3, Tomoko Motohashi3, Shohei Mitani3, Sawako Yoshina3, Etsuko Tanji2, Akiko Serizawa1, Takuji Yamada1, Kiyoaki Taniguchi1, Masakazu Yamamoto1 and Toru Furukawa2,4,5

1Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Tokyo, Japan

2Institute for Integrated Medical Sciences, Tokyo Women’s Medical University, Tokyo, Japan

3Department of Physiology, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan

4Department of Surgical Pathology, Tokyo Women’s Medical University Hospital, Tokyo, Japan

5Department of Histopathology, Tohoku University Graduate School of Medicine, Sendai, Japan

*These authors have contributed equally to this work

Correspondence to:

Toru Furukawa, email: toru.furukawa@med.tohoku.ac.jp

Keywords: early gastric cancer; lymph node metastasis; lymphangiogenesis; NBN; PAX8

Received: May 18, 2017    Accepted: January 13, 2018    Published: January 22, 2018

ABSTRACT

Background and aim: Intramucosal gastric adenocarcinoma of the well-moderately differentiated type only exhibits lymph node metastasis in extremely rare cases. We encountered such case and investigated both the lymphangiogenic properties and somatic mutations in the cancer to understand the prometastatic features of early-stage gastric cancer.

Methods: We quantitatively measured the density of lymphatic vessels and identified mutations in 412 cancer-associated genes through next-generation target resequencing of DNA extracted from tumor cells in a formalin-fixed and paraffin-embedded tissue. Functional consequence of the identified mutation was examined in vitro by means of gene transfection, immunoblot, and the quantitative real-time polymerase chain reaction assay.

Results: The intramucosal carcinoma was accompanied by abundant lymphatic vessels. The metastatic tumor harbored somatic mutations in NBN, p.P6S, and PAX8, p.R49H. The PAX8R49H showed significantly higher transactivation activity toward E2F1 than the wild-type PAX8 (P< 0.001).

Conclusions: Our data suggest that increased lymphangiogenesis and somatic mutations of NBN and/or PAX8 could facilitate lymph node metastasis from an intramucosal gastric carcinoma. These findings may potentially inform evaluations of the risk of developing lymph node metastasis in patients with intramucosal gastric cancer.


INTRODUCTION

An endoscopic resection is recommended as a standard treatment (absolute indication) for early gastric carcinomas that fulfill the following criteria: a differentiated-type adenocarcinoma without ulcerative findings, of which the depth of invasion is clinically diagnosed as cT1a and the diameter is ≤ 2 cm [1]. This recommendation is based on the rare occurrence of lymph node (LN) metastasis, which is reported to be 0.12% (3/2402) or 0% (0/6456), in patients with early gastric cancer fulfilling the above criteria [13]. Despite the rare occurrence of LN metastasis, published reports have emphasized the importance of careful evaluation of LN state by using computed tomography (CT) because if a LN metastasis is found, a surgical resection with lymphadenectomy should be performed instead of the endoscopic resection [2, 4]. Hence, mechanistic insights into LN metastasis from early gastric cancer could provide clues for improving the present criteria for the absolute indication of endoscopic resection for early gastric cancer. We obtained a rare opportunity to explore a case of intramucosal gastric adenocarcinoma with synchronized LN metastasis. In this case, we performed a quantitative lymphatic vessel density evaluation, targeted resequencing of 412 cancer-associated genes by next-generation sequencing technology, and a subsequent functional analysis for a mutated gene.

RESULTS

Patient characteristics and clinical course

A 68-year-old woman, with a history of eradicated Helicobacter pylori infection, suffered from transient epigastralgia. The patient underwent upper gastrointestinal endoscopy, which elucidated an irregular mucosal lesion in the gastric angle (Figure 1A). A biopsy revealed a tubular adenocarcinoma of the well-differentiated type. Carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) levels were 8.5 ng/ml and 50 U/ml, respectively. Although the lesion seemed to be confined within the gastric mucosa and to fulfill the absolute indication of endoscopic resection, a suspicious LN metastatic lesion (23 mm diameter) beside the left gastric artery was noted on a CT scan (Figure 1B). A laparoscopic tumor biopsy revealed that the tumor was an enlarged LN with tubular adenocarcinoma that was histologically similar to the gastric tumor (Figure 1C). Positron emission tomography/CT showed no apparent uptake except in the LN tumor. Thus, the patient was diagnosed to have an early gastric cancer with a regional LN metastasis and underwent the standard distal gastrectomy with D2 lymph node dissection. After surgery, CEA and CA19-9 levels normalized. The patient underwent no adjuvant chemotherapy and has remained in good health without any signs of recurrence or other malignant tumors for 39 months (most recent follow-up).

Preoperative findings.

Figure 1: Preoperative findings. (A) Endoscopy showed irregular mucosa in the lesser curvature of the gastric angle with ill-defined margins from surrounding atrophic mucosa. (B) Computed tomography showed an irregular oval tumor of 23 mm diameter beside the left gastric artery (arrow). (C) Laparoscopic biopsy of the intra-abdominal tumor revealed a metastatic tubular adenocarcinoma in the lymph node. Hematoxylin and eosin (H&E) staining, original magnification, ×100.

Pathological findings

A pathological examination identified 4 independent lesions, 1–4 mm in diameter, in close vicinity to each other at the gastric angle (Figure 2A). The lesions consisted of well-differentiated tubular adenocarcinomas confined within the mucosal layer without any apparent ulceration (Figure 2B, Supplementary Figure 1). There was no scar formation that could suggest segregation of an originally existing tumor into 4 cancerous lesions. The histological features were identical to those of the metastatic adenocarcinoma in the LN along the lesser curvature (Figure 1C, 2B).

Gross and microscopic pathology of the surgically resected section of the stomach.

Figure 2: Gross and microscopic pathology of the surgically resected section of the stomach. (A) Four irregular mucosal lesions, each 1-4 mm in diameter, were located side by side in the lesser curvature (arrowheads). (B) The primary cancer consisted of tubular adenocarcinoma of the well-differentiated type confined within the mucosal layer without ulceration. H&E staining, original magnification, ×100. (C-E) Images of immunohistochemical staining with anti-D2-40 antibody showed abundant lymphatic vessels in an intratumoral area (C) in contrast with scanty lymphatic vessels in a peritumoral area (D) and a portion of normal mucosa (E). All histological images were taken at the original magnification of ×200.

Evaluation of lymphatic vessels

Formalin-fixed and paraffin-embedded (FFPE) tissue sections were immunohistochemically stained with anti-D2-40 antibody. D2-40-positive lymphatic vessels were particularly dense in the lamina propria of the intramucosal tubular adenocarcinoma in the primary tumor. The dense lymphatic vessels showed irregular shapes with collapsing compared to those in normal mucosa (Figure 2C2E). Lymphatic vessel density was evaluated semi-quantitatively as described by Pak et al [5]. Intratumoral lymphatic vessel density (I-LVD), peritumoral lymphatic vessel density (P-LVD), and control lymphatic vessel density (C-LVD) were 72.3 ± 4.5, 10.7 ± 3.8, and 10.7 ± 8.3, respectively. The I-LVD was significantly higher than P-LVD (P< 0.001) and C-LVD (P< 0.001). Moreover, I-LVD in this patient was strikingly higher than the mean values reported by Pak et al. for node-negative cases, 11.26± 3.84, and N3 cases, 14.16 ± 5.00 [5].

Somatic mutations

We prepared DNA from microdissected FFPE tissue samples of the primary intramucosal gastric carcinoma, the metastatic LN carcinoma, and a normal tissue, and performed target resequencing using an Ion Proton System (Thermo Fisher Scientific, Carlsbad, CA, USA). We employed two panels of target genes to cover the known commonly mutated genes in gastric cancer (Table 1) [68]. One panel was the IonAmpliSeq™ Comprehensive Cancer Panel (Thermo Fisher Scientific) that covered the coding exons of 409 cancer-associated genes, and the other was an Ion AmpliSeq™ Custom DNA Panel (Thermo Fisher Scientific) that was designed to cover the coding exons of RHOA and its regulatory molecules, AKAP13 and DLC1 (Table 1). The mean coverage of each panel was 241.4 and 4816.3 fold per amplicon, respectively. We identified somatic mutations in NBN, p.P6S, and PAX8, p.R49H, in the LN metastasis; however, we did not identify these mutations in the primary tumor (Table 2). These mutations were confirmed by Sanger sequencing (Figure 3A, 3B).

Table 1: The list of target genes examined in panel sequencing

ABL1

CASC5

EGFR

G6PD

KLF6

MYCN

PIM1

SH2D1A

USP9X

ABL2

CBL

EML4

GATA1

KRAS

MYD88

PKHD1

SMAD2

VHL

ACVR2A

CCND1

EP300

GATA2

LAMP1

MYH11

PLAG1

SMAD4

WAS

ADAMTS20

CCND2

EP400

GATA3

LCK

MYH9

PLCG1

SMARCA4

WHSC1

AFF1

CCNE1

EPHA3

GDNF

LIFR

NBN

PLEKHG5

SMARCB1

WRN

AFF3

CD79A

EPHA7

GNA11

LPHN3

NCOA1

PML

SMO

WT1

AKAP9

CD79B

EPHB1

GNAQ

LPP

NCOA2

PMS1

SMUG1

XPA

AKAP13

CDC73

EPHB4

GNAS

LRP1B

NCOA4

PMS2

SOCS1

XPC

AKT1

CDH1

EPHB6

GPR124

LTF

NF1

POT1

SOX11

XPO1

AKT2

CDH11

ERBB2

GRM8

LTK

NF2

POU5F1

SOX2

XRCC2

AKT3

CDH2

ERBB3

GUCY1A2

MAF

NFE2L2

PPARG

SRC

ZNF384

ALK

CDH20

ERBB4

HCAR1

MAFB

NFKB1

PPP2R1A

SSX1

ZNF521

APC

CDH5

ERCC1

HIF1A

MAGEA1

NFKB2

PRDM1

STK11

AR

CDK12

ERCC2

HLF

MAGI1

NIN

PRKAR1A

STK36

ARID1A

CDK4

ERCC3

HNF1A

MALT1

NKX2-1

PRKDC

SUFU

ARID2

CDK6

ERCC4

HOOK3

MAML2

NLRP1

PSIP1

SYK

ARNT

CDK8

ERCC5

HRAS

MAP2K1

NOTCH1

PTCH1

SYNE1

ASXL1

CDKN2A

ERG

HSP90AA1

MAP2K2

NOTCH2

PTEN

TAF1

ATF1

CDKN2B

ESR1

HSP90AB1

MAP2K4

NOTCH4

PTGS2

TAF1L

ATM

CDKN2C

ETS1

ICK

MAP3K7

NPM1

PTPN11

TAL1

ATR

CEBPA

ETV1

IDH1

MAPK1

NRAS

PTPRD

TBX22

ATRX

CHEK1

ETV4

IDH2

MAPK8

NSD1

PTPRT

TCF12

AURKA

CHEK2

EXT1

IGF1R

MARK1

NTRK1

RAD50

TCF3

AURKB

CIC

EXT2

IGF2

MARK4

NTRK3

RAF1

TCF7L1

AURKC

CKS1B

EZH2

IGF2R

MBD1

NUMA1

RALGDS

TCF7L2

AXL

CMPK1

FAM123B

IKBKB

MCL1

NUP214

RARA

TCL1A

BAI3

COL1A1

FANCA

IKBKE

MDM2

NUP98

RB1

TET1

BAP1

CRBN

FANCC

IKZF1

MDM4

PAK3

RECQL4

TET2

BCL10

CREB1

FANCD2

IL2

MEN1

PALB2

REL

TFE3

BCL11A

CREBBP

FANCF

IL21R

MET

PARP1

RET

TGFBR2

BCL11B

CRKL

FANCG

IL6ST

MITF

PAX3

RHOA

TGM7

BCL2

CRTC1

FAS

IL7R

MLH1

PAX5

RHOH

THBS1

BCL2L1

CSF1R

FBXW7

ING4

MLL

PAX7

RNASEL

TIMP3

BCL2L2

CSMD3

FGFR1

IRF4

MLL2

PAX8

RNF2

TLR4

BCL3

CTNNA1

FGFR2

IRS2

MLL3

PBRM1

RNF213

TLX1

BCL6

CTNNB1

FGFR3

ITGA10

MLLT10

PBX1

ROS1

TNFAIP3

BCL9

CYLD

FGFR4

ITGA9

MMP2

PDE4DIP

RPS6KA2

TNFRSF14

BCR

CYP2C19

FH

ITGB2

MN1

PDGFB

RRM1

TNK2

BIRC2

CYP2D6

FLCN

ITGB3

MPL

PDGFRA

RUNX1

TOP1

BIRC3

DAXX

FLI1

JAK1

MRE11A

PDGFRB

RUNX1T1

TP53

BIRC5

DCC

FLT1

JAK2

MSH2

PER1

SAMD9

TPR

BLM

DDB2

FLT3

JAK3

MSH6

PGAP3

SBDS

TRIM24

BLNK

DDIT3

FLT4

JUN

MTOR

PHOX2B

SDHA

TRIM33

BMPR1A

DDR2

FN1

KAT6A

MTR

PIK3C2B

SDHB

TRIP11

BRAF

DEK

FOXL2

KAT6B

MTRR

PIK3CA

SDHC

TRRAP

BRD3

DICER1

FOXO1

KDM5C

MUC1

PIK3CB

SDHD

TSC1

BRIP1

DLC1

FOXO3

KDM6A

MUTYH

PIK3CD

SEPT9

TSC2

BTK

DNMT3A

FOXP1

KDR

MYB

PIK3CG

SETD2

TSHR

BUB1B

DPYD

FOXP4

KEAP1

MYC

PIK3R1

SF3B1

UBR5

CARD11

DST

FZR1

KIT

MYCL1

PIK3R2

SGK1

UGT1A1

Table 2: Annotations for somatic mutations of NBN and PAX8

Gene

Position

Exon

Coding DNA

Amino acid

COSMIC ID

SIFT

Polyphen2 HDIV

Mutation Taster

GQ

DP

AF

NBN

Chr8:90996774

1

c.16C>T

p.P6S

1102345

tolerated (0.13)

benign (0.429)

disease causing

58

122

0.11

PAX8

Chr2:114004376

3

c.146G>A

p.R49H

-

damaging (0)

Probably damaging (0.999)

disease causing

114

179

0.14

AF, allele frequency based on flow evaluator observation counts; DP, total read depth at the locus; GQ, genotype quality.

Somatic mutations in NBN and PAX8 identified in the present case.

Figure 3: Somatic mutations in NBN and PAX8 identified in the present case. (A) Validation by Sanger sequencing showed single nucleotide substitution in NBN and PAX8 in the lymph node metastasis (arrow) but not in the primary tumor. (B) Functional domains (colored boxes) and mutated residues (arrows) in nibrin (NBN) and paired box 8 (PAX8).

Functional analysis of PAX8, p.R49H

To determine a functional phenotype of the PAX8R49H, we compared transactivation activities between the wild type PAX8 and the mutant PAX8, i.e., PAX8R49H, toward E2F1 that was known to be a transcriptional target of paired box 8 (PAX8) [9]. We constructed expression vectors harboring the wild type PAX8 or the mutant PAX8, and transfected them into 293T cells. The transfection induced an equivalent level of exogenous expression of encoded proteins as indicated by immunoblots in Figure 4A. Then, we measured transcriptions of E2F1 in the cells by the quantitative real time PCR assay. The result showed that the mutant PAX8 induced a significantly increased level of transcription of E2F1 compared to the wild-type PAX8 as shown in Figure 4B (P < 0.001). These results indicate that the PAX8R49H may exhibit a gain-of-function phenotype compared to the wild-type PAX8.

Transactivation of E2F1 by PAX8.

Figure 4: Transactivation of E2F1 by PAX8. (A) Immunoblot of 293T cells transfected with the PAX8R49H-V5-His (PAX8 R49H), PAX8wild-type-V5-His (PAX8 WT), and pcDNA3.1/V5-His vector (Empty vector) probed with antibodies of anti-PAX8, anti-V5, and anti-beta actin. (B) The relative expression of E2F1 in each transfected 293T cells measured by the quantitative real-time PCR assay. The expression of E2F1 was normalized to the expression of GAPDH and analyzed by means of the 2−ΔΔCT method. An asterisk indicated P < 0.001.

DISCUSSION

Taking a molecular pathologic approach, we examined a peculiar case of gastric intramucosal adenocarcinoma with LN metastasis. The primary tumor seemed to fulfill the criteria for the absolute indication of ER; however, because of the LN metastasis, we performed a surgical resection with lymphadenectomy. In pathological examination, the primary tumor was accompanied by dense lymphatic vessels, and I-LVD proved to be particularly high. This finding suggests that the elevated level of lymphangiogenesis accompanying the adenocarcinoma could have increased the chances of primary cancer cells intravasating lymphatic vessels. LVD is known to be associated with LN metastasis in various human cancers [10]. As for gastric cancer, although differences in the pathological roles of I-LVD and P-LVD are still controversial, several reports showed that I-LVD was higher in tumors associated with LN metastasis [5, 11, 12]. To the best of our knowledge, our case is the first to show that high I-LVD may facilitate LN metastasis even in a case of an intramucosal gastric carcinoma that seemed to meet the criteria for the absolute indication of ER. In the present case, the gastric cancer and metastatic LN were surgically resected because the swollen LN detected by CT was proven to be a metastasis by laparoscopic biopsy before surgery. This demonstrates that careful evaluation of LN state using CT is important, as was previously reported [4]. When LN metastasis is not identifiable on CT, I-LVD could potentially be measured to assess the risk of metastasis after ER because I-LVD can be evaluated in the ER specimen.

Next, we investigated the coding exons of 412 cancer-associated genes by next-generation sequencing. As the primary cancer was uncommonly multicentric localized adjacent to each other, which suggests the possibility of a pre-existing mutational accumulation in the atrophic gastric mucosa due to a history of H. pylori infection as previously reported [13], we avoided using noncancerous mucosa as a normal control sample for this analysis. The results indicated that the present prometastatic intramucosal gastric cancer with extraordinary LN metastasis did not harbor any common mutations for gastric cancer such as TP53, ARID1A, PIK3CA, CDH1, SMAD4, APC, KRAS, or RHOA or its regulatory molecules, AKAP13 and DLC1. Instead, the present case harbored somatic mutations in the LN metastasis: NBN, p.P6S, and PAX8, p.R49H. The identification of only a few mutated genes could be explained by the early stage of the cancer. Molecular pathologic information of such an early-stage cancer with an aberrant prometastatic nature can be a valuable source to help elucidate the mechanism of metastasis because analyzing such cases may lead to the identification of molecular alterations associated with metastasis in a small number of mutated genes.

NBN encodes nibrin (NBN), a member of the MRE11/RAD50 double-strand break repair complex [14]. A truncating mutation in NBN causes a defective response to DNA double-strand breaks, which results in an unstable genome and a predisposition to malignancies [15]. The mutated P6 residue of NBN is not located within any functional domains. However, the P6S mutation of NBN was identified in a patient with uterine corpus endometrioid carcinoma (COSM1102345 in the COSMIC database, http://cancer.sanger.ac.uk/cosmic). NBN, p.K653fs is reported to be identified in peritoneal metastasis of gastric cancer by whole-exome sequencing elsewhere [16]. Although functional impacts of NBNP6S are not known, the mutation could cause genomic instability or copy number variations.

PAX8 encodes paired box 8 (PAX8), a transcription factor required for the formation of thyroxine-producing follicular cells, of endodermal origin [17]. PAX8R49C has been identified as a somatic mutation in gastric cancer according to the COSMIC database (COSM4084322); however, PAX8R49H has not been reported. The R49 residue of PAX8 is located within the paired box domain (Figure 3B), which may be the reason that PAX8R49H was predicted to be functionally damaging by some prediction programs, namely Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), and MutationTaster (http://www.mutationtaster.org/). Missense mutations within the paired box domain, such as p.Q40P, p.S54G, p.C57Y, p.L62R, are known to cause congenital hypothyroidism, thyroid hypoplasia and aplasia, and/or kidney agenesis due to the loss of its transactivation effect [1821]. PAX8 is expressed highly in the thyroid and kidney as well as slightly but evidently in gastric mucosa and gastric cancer (GEO10420251 and GEO95672775 in Gene Expression Omnibus Profiles, https://www.ncbi.nlm.nih.gov/geoprofiles). PAX8 is reported to be expressed in metastatic non-small cell lung cancers and to promote cell migration via interaction with MET and RON [22].

By our experiments testing a transactivating function of PAX8 toward E2F1, we found that the PAX8R49H was able to show a gain-of-function phenotype in transactivation of E2F1 compared to the wild type PAX8. E2F transcription factor 1 (E2F1) encoded by E2F1 is well known for its tumor suppressive role in conjunction with retinoblastoma protein 1 (RB1), however, it is also known to play some promoting roles in cancer as reported to promote an epithelial-mesenchymal transition (EMT) by transactivating FOXL2 in gastric cancer cells, which may be associated with increased LN metastasis in patients with gastric cancer [23]. Thus, our result and these compelling evidences suggest that PAX8R49H could have promoted EMT and subsequent LN metastasis via E2F1 in the present case.

In the present case, the somatic mutations were identified only in the metastatic LN tissue and not in the primary tumor. To explain this crucial observation, one can argue that the LN metastasis was not derived from the gastric adenocarcinoma but from elsewhere in other organs. However, we determined that the LN tumor was likely to be a metastasis of the intramucosal gastric carcinoma, which was cured by the surgical resection, through the following evidence: identical histology between the gastric tumor and the metastatic LN tumor, anatomical location of the metastatic LN, and the concerted decrease of elevated tumor markers. Moreover, the patient has not manifested any other malignant neoplasms for more than 3 years after surgery and no adjuvant chemotherapy. Alternatively, the failure of finding mutations in the intramucosal gastric carcinoma could be due to tumor heterogeneity, i.e., a small number of prometastatic clones in the primary tumor. A previous report indicated that a lethal metastatic clone of prostate cancer was derived from only a single small lesion in 36 sectioned blocks of the primary cancer [24]. Thus, it is likely that cells with a metastatic ability may have existed as just a tiny fraction in the primary tumor, which could not be detected. On the other hand, we could not exclude the possibility that the mutations occurred after metastasizing to the LN. Postmetastatic mutations could give advantages of survival and growth of cells in the metastatic site. Fractions of mutated calls were 11% and 14% in NBN and PAX8, respectively. This relatively minor fraction of mutated alleles may be due to 1) heterogeneity of cancer cells, 2) wild cancer cells derived from the collective dissemination of tumor clusters [25], or 3) normal lymphocytes with density drastically higher than that of the cancer cells, as shown in Figure 1C, meaning the ratio of cancer-derived DNA should be substantially lowered by contamination of even a small volume of peripheral lymphocytes. This could encourage the assumption that these mutations contributed to facilitation and/or development of LN metastasis, since they are more selected in the metastatic LN.

In conclusion, intramucosal gastric carcinoma that seemed to fulfill the criteria for the absolute indication of ER had a LN metastasis and thus was resected surgically. Increased lymphangiogenesis was observed in the primary tumor. Moreover, somatic mutations of NBN, p.P6S, and PAX8, p.R49H were observed in the metastatic tumor. The PAX8R49H showed a gain-of-function phenotype in transactivation of E2F1. These findings may serve not only to develop biomarkers and/or molecular therapeutic targets but also to revise current recommendation for ER resection of early gastric cancer.

MATERIALS AND METHODS

Ethics and informed consent

This study was approved by the ethical committee of Tokyo Women’s Medical University (protocol #272). Written informed consent was obtained from the patient for research and publication.

Quantitative analysis of lymphatic vessel density

FFPE tissue sections were immunohistochemically stained by using anti-D2-40 antibody (Covance Antibody Products, San Diego, CA, USA) and Autostainer Link 48 (Dako, Glostrup, Denmark). Quantitative analysis of lymphatic vessel density (LVD) was performed by counting the D2-40 stained lymphatic vessels according to Pak et al [5]. Intratumoral (I)-LVD, Peritumoral (P)-LVD, and LVD in the normal control tissues (C-LVD) were counted.

Panel design for the next-generation sequencing

The Ion AmpliSeq™ Comprehensive Cancer Panel covering all coding exons of 409 cancer-associated genes and an Ion AmpliSeq™ Custom DNA Panel covering all coding exons of RHOA and its regulatory molecules, AKAP13 and DLC1, were used. In total, all coding exons of 412 genes were examined (Table 1).

Tissue dissection and DNA preparation

FFPE tissue samples from the primary gastric tumor, the metastatic LN tumor, and a normal portion of the stomach (submucosa or deeper area) were used for genetic analysis. Areas of adenocarcinoma found by microscopic observation were manually dissected. DNA was prepared with a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). DNA from the metastatic LN tumor and normal gastric tissue were analyzed by next-generation sequencing. For the primary cancer, since each lesion was small, we mixed the DNA together and only analyzed it by Sanger sequencing.

Next-generation sequencing

Sequencing libraries were prepared using Ion AmpliSeq™ Library Kit 2.0 (Thermo Fisher Scientific) according to the manufacturer’s instruction. The quantity of DNA amplicons was evaluated using a High Sensitivity DNA kit (Agilent Technologies, Waldbronn, Germany). Sequencing was performed using an Ion Proton™ System (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Variant calling and annotation

Data analyses were performed using the Ion Torrent Suite Software (version 5.0.3). After base calling, the reads were aligned against the reference human genome (hg19) using the TMAP algorithm within the Torrent Suite. Variants with Genome quality > 50 and an allele frequency > 10% were considered. For further single nucleotide polymorphism (SNP) analysis, only non-synonymous nucleotide exchanges were considered. SNPs reported to be > 1% in 1000G, ExAC or ESP6500si were dismissed. SNPs detected only in tumor tissues were counted. All somatic variations annotated were validated by Sanger sequencing.

Sanger sequencing

Genomic portions of somatic mutations were amplified by using paired primers of 5′-GGTTACGCGGTTGCACGTCG-3′ and 5′-TCTGCCC TTACCTCCTGCCG-3′ for NBN and 5′-CTTTGTGAA TGGCAGACCTC-3′ and 5′-AAGGATCTTGCTGACGCA GC-3′ for PAX8. The amplified products were analyzed by Sanger sequencing, as described previously [26].

Cell culture

The human embryonic kidney 293T was obtained from the European Collection of Authenticated Cell Cultures (ECACC 12022001). The cells were cultured using Dulbecco’s Modified Eagle’s Medium (Sigma, St. Louis, MO, USA) with 10% fetal bovine serum in a humidified incubator at 37°C with 5% CO2.

Plasmid vectors

The wild-type PAX8 cDNA was amplified from a fetal kidney cDNA library (Agilent Technologies) by PCR using the KOD Plus DNA polymerase system (TOYOBO, Osaka, Japan). The paired primers used were as follows: forward 5′-TTTAAGCTT/CCCCGGCGATGCCTCACAAC-3′, and reverse 5′-TTTGAATTC/CAGATGGTCAAAGGCCGTGGC-3′. The amplified product was separated by agarose gel electrophoresis. A band corresponding to an equivalent molecular weight of PAX8 (NM_003466.3) was extracted, purified, and cloned into the pcDNA3.1/V5-His vector (Invitrogen, San Diego, CA, USA) at the HindIII and EcoRI sites to generate the wild-type PAX8-V5-His vector. The mutant PAX8 (p.R49H)-V5-His vector was generated by means of a site-directed mutagenesis technique using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) with following primers: 5′- GACGCGGAGCTGGTGAGAGATGTCGCAG-3′ and 5′-CTGCGACATCTCTCACCAGCTCCGCGTC-3′ according to the manufacture’s instruction. Nucleotide sequences of the created plasmid vectors were confirmed by Sanger sequencing.

Cell transfection

293T cells were seeded at a concentration of 5×105 cells/well in 6-well plates 24 h before transfection. Transfection of each created plasmid vectors or the control pcDNA3.1/V5-His vector was performed using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instruction. 24 h after transfection, cells were collected and proceeded to following immunoblotting and the quantitative real-time PCR assay. The experiment was performed twice in duplicate.

Immunoblotting

Collected cells were lysed in modified RIPA buffer containing 1×complete mini protease inhibitor cocktail (Sigma) and 1×PhosSTOP phosphatase inhibitor cocktail (Sigma). Cell extracts containing equal amounts of proteins were boiled in loading buffer, applied to 10-20% polyacrylamide gradient gel, and separated by electrophoresis. Then the proteins were blotted onto a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan). After blocking using the ECL Blocking Agent (Amersham Biosciences, Buckinghamshire, UK) for 1 h, the membrane was incubated with primary antibodies overnight. Primary antibodies used were the mouse monoclonal anti-PAX8 antibody (1:200 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA), the mouse monoclonal anti-V5 antibody (1:5000 dilution) (Thermo Fisher Scientific), and the mouse monoclonal anti-β-actin antibody (1:1000 dilution) (Sigma). The membrane was incubated with a secondary antibody for 1 h. The secondary antibody used was horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody (1:10000 dilution) (GE Healthcare, Buckinghamshire, UK). Signals were visualized using the ECL Prime Western Blotting Detection Reagent (Amersham Biosciences) and LAS 4000 Mini system (Fujifilm, Tokyo, Japan).

Quantitative real-time PCR assay

Total RNA was isolated from collected cells using RNeasy Mini kit (Qiagen, Hilden, Germany). cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Pre-designed primer/probe sets for human PAX8 (Hs00247586_m1) and E2F1 (Hs00153451_m1) in TaqMan gene expression assay system (Thermo Fisher Scientific) were used in the quantitative real-time PCR assay. GAPDH was used as an endogenous control. The analyses were performed by means of the 2−ΔΔCT method [27] upon the 7500 Real-Time PCR system (Thermo Fisher Scientific) according to the manufacture’s instruction. The experiment was performed twice in triplicate.

Statistical analyses

Continuous data are described as the mean and standard deviation and were compared using Tukey’s method.

Abbreviations

CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; C-LVD, Lymphatic vessel density in the normal control tissues; CT, computed tomography; EMT, epithelial-mesenchymal transition; ER, endoscopic resection; FFPE, Formalin-fixed and paraffin-embedded; H&E, hematoxylin and eosin; I-LVD, Intratumoral-lymphatic vessel density; LN, lymph node; LVD, lymphatic vessel density; NBN, nibrin; PAX8, paired box 8; PCR, polymerase chain reaction; P-LVD, Peritumoral lymphatic vessel density; RB1, retinoblastoma protein 1.

Author contributions

NI, SA, MY, SM, and TF conceived the study and designed the experiments. KT coordinated sample acquisition. NI, SA, KT, TY, and MY contributed in acquisition of clinical data. NI, ET, and TM performed experiments. YS, NI, SY, TM, and TF performed the bioinformatics data analysis. NI, SA, MY, SM, and TF contributed to drafting and critical review of manuscript.

ACKNOWLEDGMENTS

We are grateful to the patient whose commitment to the research and tissue donation made this study possible.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

FUNDING

This work was supported by JSPS KAKENHI Grant Number JP16K10518.

REFERENCES

1. Japanese gastric cancer association. Japanese gastric cancer treatment guidelines 2014 (ver. 4). Gastric Cancer. 2017; 20:1–19.

2. Lee S, Choi KD, Hong SM, Park SH, Gong EJ, Na HK, Ahn JY, Jung KW, Lee JH, Kim DH, Song HJ, Lee GH, Jung HY, Kim JH. Pattern of extragastric recurrence and the role of abdominal computed tomography in surveillance after endoscopic resection of early gastric cancer: korean experiences. Gastric Cancer. 2017; 20:843–52. https://doi.org/10.1007/s10120-017-0691-z.

3. Tanabe S, Ishido K, Matsumoto T, Kosaka T, Oda I, Suzuki H, Fujisaki J, Ono H, Kawata N, Oyama T, Takahashi A, Doyama H, Kobayashi M, et al. Long-term outcomes of endoscopic submucosal dissection for early gastric cancer: a multicenter collaborative study. Gastric Cancer. 2017; 20:45–52.

4. Kim DJ, Kim W. A case of single lymph node metastasis near the common hepatic artery following a curative endoscopic resection for gastric mucosal cancer. Gastric Cancer. 2014; 17:387–391.

5. Pak KH, Jo A, Choi HJ, Choi Y, Kim H, Cheong JH. The different role of intratumoral and peritumoral lymphangiogenesis in gastric cancer progression and prognosis. BMC Cancer. 2015; 15:498.

6. Bass AJ, Thorsson V, Shmulevich I, Reynolds SM, Miller M, Bernard B, Hinoue T, Laird PW, Curtis C, Shen H, Weisenberger DJ, Schultz N, Shen R, et al, and Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014; 513:202–209.

7. Kakiuchi M, Nishizawa T, Ueda H, Gotoh K, Tanaka A, Hayashi A, Yamamoto S, Tatsuno K, Katoh H, Watanabe Y, Ichimura T, Ushiku T, Funahashi S, et al. Recurrent gain-of-function mutations of RHOA in diffuse-type gastric carcinoma. Nat Genet. 2014; 46:5837.

8. Ushiku T, Ishikawa S, Kakiuchi M, Tanaka A, Katoh H, Aburatani H, Lauwers GY, Fukayama M. RHOA mutation in diffuse-type gastric cancer: a comparative clinicopathology analysis of 87 cases. Gastric Cancer. 2016; 19:403–411.

9. Li CG, Nyman JE, Braithwaite AW, Eccles MR. PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein. Oncogene. 2011; 30:4824–4834.

10. Karaman S, Detmar M. Mechanisms of lymphatic metastasis. J Clin Invest. 2014; 124:922–928.

11. Ikeda K, Oki E, Saeki H, Ando K, Morita M, Oda Y, Imamura M, Kakeji Y, Maehara Y. Intratumoral lymphangiogenesis and prognostic significance of VEGFC expression in gastric cancer. Anticancer Res. 2014; 34:3911–3915.

12. Wang XL, Fang JP, Tang RY, Chen XM. Different significance between intratumoral and peritumoral lymphatic vessel density in gastric cancer: a retrospective study of 123 cases. BMC Cancer. 2010; 10:299.

13. Shimizu T, Marusawa H, Matsumoto Y, Inuzuka T, Ikeda A, Fujii Y, Minamiguchi S, Miyamoto S, Kou T, Sakai Y, Crabtree JE, Chiba T. Accumulation of somatic mutations in TP53 in gastric epithelium with Helicobacter pylori infection. Gastroenterology. 2014; 147:407–417.

14. Carney JP, Maser RS, Olivares H, Davis EM, Le Beau M, Yates JR, Hays L, Morgan WF, Petrini JH. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell. 1998; 93:477–486.

15. Chrzanowska KH, Gregorek H, Dembowska-Bagińska B, Kalina MA, Digweed M. Nijmegen breakage syndrome (NBS). Orphanet J Rare Dis. 2012; 7:13.

16. Liu H, Li F, Zhu Y, Li T, Huang H, Lin T, Hu Y, Qi X, Yu J, Li G. Whole–exome sequencing to identify somatic mutations in peritoneal metastatic gastric adenocarcinoma: a preliminary study. Oncotarget. 2016; 7:43894–906. https://doi.org/10.18632/oncotarget.9707.

17. Mansouri A, St-Onge L, Gruss P. Role of Pax genes in endoderm-derived organs. Trends Endocrinol Metab. 1999; 10:164–167.

18. Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, Souabni A, Baserga M, Tassi V, Pinchera A, Fenzi G, Grüters A, Busslinger M, Di Lauro R. PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nat Genet. 1998; 19:83–86.

19. Vilain C, Rydlewski C, Duprez L, Heinrichs C, Abramowicz M, Malvaux P, Renneboog B, Parma J, Costagliola S, Vassart G. Autosomal dominant transmission of congenital thyroid hypoplasia due to loss-of-function mutation of PAX8. J Clin Endocrinol Metab. 2001; 86:234–238.

20. Meeus L, Gilbert B, Rydlewski C, Parma J, Roussie AL, Abramowicz M, Vilain C, Christophe D, Costagliola S, Vassart G. Characterization of a novel loss of function mutation of PAX8 in a familial case of congenital hypothyroidism with in-place, normal-sized thyroid. J Clin Endocrinol Metab. 2004; 89:4285–4291.

21. Congdon T, Nguyen LQ, Nogueira CR, Habiby RL, Medeiros-Neto G, Kopp P. A novel. Mutation (Q40P) in PAX8 associated with congenital hypothyroidism and thyroid hypoplasia: evidence for phenotypic variability in mother and child. J Clin Endocrinol Metab. 2001; 86:3962–3967.

22. Kanteti R, El-Hashani E, Dhanasingh I, Tretiakova M, Husain AN, Sharma S, Sharma J, Vokes EE, Salgia R. Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer. BMC Cancer. 2014; 14:185.

23. Dong J, Wang R, Ren G, Li X, Wang J, Sun Y, Liang J, Nie Y, Wu K, Feng B, Shang Y, Fan D. HMGA2-FOXL2 axis regulates metastases and epithelial-to-mesenchymal transition of chemoresistant gastric cancer. Clin Cancer Res. 2017; 23:3461–3473.

24. Haffner MC, Mosbruger T, Esopi DM, Fedor H, Heaphy CM, Walker DA, Adejola N, Gürel M, Hicks J, Meeker AK, Halushka MK, Simons JW, Isaacs WB, et al. Tracking the clonal origin of lethal prostate cancer. J Clin Invest. 2013; 123:4918–4922.

25. Lambert AW, Pattabiraman DR, Weinberg RA. Emerging biological principles of metastasis. Cell. 2017; 168:670–691.

26. Kuboki Y, Shimizu K, Hatori T, Yamamoto M, Shibata N, Shiratori K, Furukawa T. Molecular biomarkers for progression of intraductal papillary mucinous neoplasm of the pancreas. Pancreas. 2015; 44:227–35.

27. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25:402–08.


Creative Commons License All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.
PII: 24289