JP2012163332A - Kit for diagnosing malignancy of stomach cancer - Google Patents
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Abstract
Description
本発明は胃癌の悪性度を決めるために使用するキットである。詳しくは抗LAT1抗体を使用した免疫組織学的染色法によって、胃癌の悪性度を決めるためのキットである。 The present invention is a kit used for determining the malignancy of gastric cancer. Specifically, it is a kit for determining the malignancy of gastric cancer by immunohistological staining using anti-LAT1 antibody.
細胞が増殖や成長のために細胞膜を介してアミノ酸を細胞内に取り込むためには正常細胞のみならず癌細胞でもアミノ酸トランスポーターが必須である。大型の中性アミノ酸の輸送にはL型アミノ酸トランスポーターが必要であり、多くはL型アミノ酸トランスポーター(LAT1, SLC7A5)に依存しており、このLAT1は金井らによって最初にクローニングされた1。続いて、２番目のタイプとして機能的にも分子的にも別個のLAT2 が分離された。このLAT2は大型のみならず小型のアミノ酸も輸送する2。 An amino acid transporter is essential not only for normal cells but also for cancer cells in order for cells to take up amino acids into cells through the cell membrane for proliferation and growth. 1 The transport of large neutral amino acids is required L-type amino acid transporter, many rely on L-type amino acid transporter (LAT1, SLC7A5), the LAT1 is was first cloned by Kanai et al. Subsequently, LAT2 was isolated as a second type, functionally and molecularly distinct. This LAT2 transports not only large but also small amino acids 2 .
LAT1は主としてヒトの正常脳、脾臓、胸腺、精巣、胎盤組織や前立腺、食道、肺などの癌組織に発現している3-5。本発明者らは前立腺癌の予後の判定には従来のGleason の組織学的グレイドシステムとともにLAT1の発現が有効なマーカーであることを以前に示した。さらに幾つかの癌細胞株でもLAT1 mRNAの発現が高いことも認めている6。 LAT1 is expressed mainly in normal human brain, spleen, thymus, testis, placental tissue, and cancer tissues such as prostate, esophagus, and lung 3-5 . The inventors have previously shown that LAT1 expression is an effective marker in conjunction with the conventional Gleason histological grade system for prognosis of prostate cancer. Furthermore, we have observed that LAT1 mRNA is highly expressed in several cancer cell lines 6 .
しかしながら胃癌においてはこれまで不明であった。その為、胃癌におけるLAT1の免疫組織学的発現を本研究で検索した。すなわち我々が最近発明した抗LAT1モノクローナル抗体を使用して、非腫瘍性胃粘膜、腺腫、スキルス胃癌、非スキルス胃癌でLAT1発現を比較した。 However, it was unknown until now in gastric cancer. Therefore, the immunohistological expression of LAT1 in gastric cancer was searched in this study. That is, we used the recently invented anti-LAT1 monoclonal antibody to compare LAT1 expression in non-neoplastic gastric mucosa, adenoma, Skills gastric cancer, and non-Skills gastric cancer.
アミノ酸トランスポーターは正常細胞、腫瘍細胞の維持・増殖にとって不可欠である。本研究では、我々が最近開発したモノクローナル抗体を使って、胃癌におけるL型アミノ酸トランスポーター1 (LAT1) の免疫組織化学的発現を胃腺腫及び非腫瘍性胃粘膜と比較して検索した。進行胃癌87症例を検索して、胃スキルス癌よりも非スキルス癌の方で癌細胞の細胞膜に有意に高いLAT1発現をみとめた。また、リンパ節転移のある胃癌症例では転移のない症例よりもLAT1発現が有意に高かった。LAT1発現と細胞増殖能のマーカーであるKi-67標識率（LI）とは正の相関がみとめられ、非スキルス胃癌では高LAT1発現症例が低発現症例に比較して、有意に予後が不良であった。Cox hazard test によって、非スキルス胃癌ではTNM stage (腫瘍の大きさ、リンパ節転移、血行性転移による進行度)とLAT1発現は互いに独立した予後因子になることを明らかにした。さらにスキルス胃癌を除いた未分化型胃癌では、高LAT1発現群で有意に予後が悪いことも示した。胃癌に比較して胃腺腫ではLAT1発現が有意に低かった。結論として、LAT1発現は胃癌の細胞増殖及び予後とリンクしており、LAT1を標的とした抑制剤が抗癌剤として将来役に立つ可能性があげられる。 Amino acid transporters are essential for the maintenance and growth of normal cells and tumor cells. In this study, we investigated the immunohistochemical expression of L-amino acid transporter 1 (LAT1) in gastric cancer in comparison with gastric adenoma and non-tumor gastric mucosa using our recently developed monoclonal antibody. We searched 87 cases of advanced gastric cancer and found significantly higher LAT1 expression in the cell membrane of non-Skills cancer than in gastric Skills cancer. In addition, LAT1 expression was significantly higher in gastric cancer patients with lymph node metastasis than in those without metastasis. A positive correlation was found between LAT1 expression and Ki-67 labeling rate (LI), which is a marker of cell proliferation ability. In non-Skirs gastric cancer, patients with high LAT1 expression had a significantly worse prognosis than those with low expression. there were. Cox hazard test revealed that TNM stage (tumor size, progression by lymph node metastasis, hematogenous metastasis) and LAT1 expression are prognostic factors independent of each other in non-Skirs gastric cancer. Furthermore, undifferentiated gastric cancer excluding Skills gastric cancer also showed a significantly worse prognosis in the high LAT1-expressing group. Compared with gastric cancer, LAT1 expression was significantly lower in gastric adenoma. In conclusion, LAT1 expression is linked to cell proliferation and prognosis of gastric cancer, and inhibitors that target LAT1 may be useful in the future as anticancer agents.
このように本発明は、
[1]抗LAT1モノクローナル抗体を使用した免疫組織化学染色キットは胃癌の悪性度の判定に使用できる。
[2]本モノクローナル抗体はヒトLAT1のN末端1-52のペプチドを特異的に認識することから、ヒト癌の悪性度を評価できる。
Thus, the present invention
[1] An immunohistochemical staining kit using an anti-LAT1 monoclonal antibody can be used to determine the malignancy of gastric cancer.
[2] Since this monoclonal antibody specifically recognizes the N-terminal 1-52 peptide of human LAT1, the malignancy of human cancer can be evaluated.
本発明の目的は、ヒト胃癌におけるLAT1発現が正常胃粘膜や非癌性病変とでは違うことを証明することである。本発明者は、既に報告されている脳グリオーマ(神経膠腫)、肺癌、食道癌と同様に、胃癌においてもLAT1高発現が悪性度を示すことを確認した4,5,12。しかし、スキルス型胃癌は非スキルス型胃癌に比較してLAT1発現スコアが低く、この型の癌細胞はアミノ酸輸送においてLAT1依存度が低いことが示唆される。この現象は、今後real time PCR等の他の方法で確認する必要がある。 The purpose of the present invention is to demonstrate that LAT1 expression in human gastric cancer is different from normal gastric mucosa and non-cancerous lesions. The present inventor confirmed that high LAT1 expression shows malignancy in gastric cancer as well as brain glioma (glioma), lung cancer, and esophageal cancer that have already been reported 4,5,12 . However, Skills gastric cancer has a lower LAT1 expression score than non-Skills gastric cancer, suggesting that this type of cancer cell is less LAT1-dependent in amino acid transport. This phenomenon needs to be confirmed by other methods such as real time PCR in the future.
胃腺腫はLAT1 発現スコアも強さも胃癌と正常腺腺窩上皮との中間の値を示した。本発明者は胃における良性及び悪性上皮性腫瘍のLAT1発現を確認した。他の臓器の良性腫瘍では、口腔の異形成病変、肺の異型腺腫様過形成病変でLAT1発現が報告されている。驚いたことに、胃癌症例の背景胃粘膜の腸上皮化生で胃癌と同様のLAT1高発現がみとめられた13,14。このことは、ヒト大腸の粘膜にLAT1が発現していることと関連しているかもしれない15。このように胃の腸上皮化生における高LAT1発現は粘膜の表現型として説明しうる。しかしながら、I型胃癌（癌細胞の粘液が腸型）とG型胃癌（癌細胞の粘液が胃型）の間にはLAT1発現において有意な差が認められなかった。すなわち、本発明者はG型胃癌でもLAT1高発現を確認した。腸型胃癌を除いた胃癌に限定すると、LAT1発現高スコア群が低スコア群に比較して予後が不良な傾向を認めた。 Gastric adenomas showed intermediate values between LAT1 expression score and strength between gastric cancer and normal adenoid epithelium. The inventor confirmed LAT1 expression in benign and malignant epithelial tumors in the stomach. In benign tumors of other organs, LAT1 expression has been reported in dysplastic lesions of the oral cavity and atypical adenoma-like hyperplastic lesions of the lung. Surprisingly, in the background of gastric cancer cases, intestinal metaplasia of the gastric mucosa showed high LAT1 expression similar to that of gastric cancer 13,14 . This may be related to the expression of LAT1 in the mucosa of the human large intestine 15 . Thus, high LAT1 expression in gastrointestinal metaplasia can be explained as a mucosal phenotype. However, there was no significant difference in LAT1 expression between type I gastric cancer (cancer cell mucus is intestinal type) and G type gastric cancer (cancer cell mucus is gastric type). That is, the present inventor confirmed that LAT1 was highly expressed even in G-type gastric cancer. When limited to gastric cancer excluding intestinal gastric cancer, the LAT1-expressing high score group tended to have a poorer prognosis than the low score group.
胃癌においてKi-67標識率とLAT1発現強さとの弱い相関が認められたことはLAT1が細胞増殖に寄与していることを意味しているかもしれない。この現象は肺の非小細胞癌でも報告されている16。しかしながら、他の幾つかの癌ではそのような相関が認められていないので、この食い違いについてはさらなる研究が必要である。本研究では、胃癌におけるLAT1とp53発現との間で弱い相関がみとめられたことは、LAT1発現がp53によって規制されている可能性がある。しかし、この点についてはやはりさらなる検索による確認が必要である。 The weak correlation between Ki-67 labeling rate and LAT1 expression intensity in gastric cancer may mean that LAT1 contributes to cell proliferation. This phenomenon has also been reported in non-small cell lung cancer 16 . However, since there is no such correlation in some other cancers, further research is needed on this discrepancy. In this study, the weak correlation between LAT1 and p53 expression in gastric cancer may indicate that LAT1 expression is regulated by p53. However, this point still needs to be confirmed by further search.
既報では、肺癌において、LAT1発現が有意な予後規定因子であり、予後不良な結果を示している3,16,17。例えば、Kaira らによると、LAT1陽性肺癌は陰性肺癌に比べてリンパ節転移率が有意に高い16。この報告と同様に、本発明者はリンパ節転移のある胃癌症例ではリンパ節転移のない胃癌症例に比較して、LAT1発現スコアが有意に高いことを示した。しかしながら、我々の研究では、悪性度の高いスキルス癌を含む胃癌症例全体の検索ではLAT1発現と予後との有意な関連を見つけられなかった。一方、非スキルス癌に限定すると、LAT1発現高スコア群は有意に予後が不良であり、多変量解析でもLAT1発現が予後決定因子であることを確認できた。このことはLAT1発現とp53発現とに有意に相関があったことと関連しているかもしれない。また、スキルス胃癌症例の予後が不良であることは癌性腹膜炎を生じやすいなど、他の因子によっている可能性があげられる。 Previously, LAT1 expression is a significant prognostic factor in lung cancer and has shown poor prognosis 3,16,17 . For example, according to Kaira et al., LAT1-positive lung cancer has a significantly higher rate of lymph node metastasis than negative lung cancer 16 . Similar to this report, the present inventor has shown that the LAT1 expression score is significantly higher in gastric cancer cases with lymph node metastasis than in gastric cancer cases without lymph node metastasis. However, our study failed to find a significant association between LAT1 expression and prognosis in the search for whole gastric cancer cases, including highly malignant Skills cancer. On the other hand, when limited to non-skills cancer, the LAT1 high score group had a significantly poor prognosis, and multivariate analysis confirmed that LAT1 expression was a prognostic determinant. This may be related to a significant correlation between LAT1 expression and p53 expression. In addition, the poor prognosis of Skills gastric cancer cases may be caused by other factors such as cancer peritonitis.
結論として、正常胃粘膜に比較して胃腺腫はLAT1発現が高く、胃癌細胞はさらに高い。LAT1発現の強さは胃癌の細胞増殖と予後に相関しているため、近い将来開発されるLAT1抑制物質が画期的な分子標的抗癌治療になりうる可能性を示している。LAT1発現が増強していることは、癌細胞が細胞増殖に必要な栄養物を強く要求していると考えられる。このように、LAT1機能を抑制することはヒトの多くの種類の癌治療法の開発につながる18。今後、癌細胞の増殖を抑制する低分子LAT1抑制物質が胃癌において有意に治療効果を示しうることは、大いに期待できる。 In conclusion, gastric adenomas have higher LAT1 expression and gastric cancer cells are higher than normal gastric mucosa. The strength of LAT1 expression correlates with cell proliferation and prognosis of gastric cancer, suggesting that LAT1 inhibitors developed in the near future can be a revolutionary molecular targeted anticancer therapy. The enhanced expression of LAT1 is thought to strongly demand the nutrients necessary for cell growth by cancer cells. Thus, suppressing the LAT1 function leads to the development of many types of cancer therapy in humans 18. In the future, it is highly expected that a low molecular weight LAT1 inhibitor that suppresses the growth of cancer cells can have a significant therapeutic effect in gastric cancer.
１．モノクローナル抗体の作成
LAT1のN末端の52 アミノ酸（アミノ酸1-52に相当）に対する抗ヒトLAT1モノクローナル抗体をハイスループットプロテオミクス法にて作成した20,21。この方法で、特異的な抗LAT1モノクローナル抗体を作るハイブリドーマを得た。このハイブリドーマ細胞をマウスBALB/c に腹腔内に注射した後に、腹水を回収してこれをアフィ二ティカラム(HiTrap protein G, GE Healthcare Bio-science AB) を通して精製抗体を得た。
1. Creation of monoclonal antibodies
An anti-human LAT1 monoclonal antibody against the 52 amino acids (corresponding to amino acids 1-52) at the N-terminus of LAT1 was prepared by high-throughput proteomics 20,21 . By this method, a hybridoma that produces a specific anti-LAT1 monoclonal antibody was obtained. After this hybridoma cell was injected intraperitoneally into mouse BALB / c, ascites was collected and purified antibody was obtained through affinity column (HiTrap protein G, GE Healthcare Bio-science AB).
２．患者と試料
1993年6月より2003年4月までに北里大学東病院で外科的に切除された進行胃癌（胃壁固有筋層内またはそれより深く浸潤）87症例を収集した。切除された胃は全て、10%フォルマリン固定し、腫瘍病変は5mmの厚さで全割して、パラフィンに包埋した。パラフィンブロックから、4μm厚さの切片を作り、HE染色を施した。全ての症例は日本胃癌取り扱い規約に則り7、組織学的に診断した。87症例中、28例がスキルス型胃癌であった。残りの59症例は非スキルス型胃癌であったが、その内訳は高分化型腺癌11例、中分化型腺癌15例、充実性低分化型腺癌20例、非充実性低分化型腺癌10例、印鑑細胞癌3例であった。
本発明者は、癌病変のLAT1発現に加えて、他に4群におけるLAT1発現を検索した。すなわち、正常胃粘膜20例（gastrointestinal stromal tumor 例や膵癌症例で胃粘膜に病変がないもの）、腸上皮化生のない背景胃粘膜32例(40才以下の胃癌症例の背景胃粘膜)、腸上皮化生のある背景胃粘膜37例(75才以上の胃癌症例で背景に腸上皮化生があるもの)、及び胃腺腫36症例である。
2. Patient and sample
We collected 87 cases of advanced gastric cancer (invasion in or deeper than the gastric wall intrinsic muscle) surgically resected at Kitasato University East Hospital from June 1993 to April 2003. All excised stomachs were fixed with 10% formalin, and the tumor lesions were all divided by 5 mm thickness and embedded in paraffin. A 4 μm-thick section was made from the paraffin block and subjected to HE staining. All of the cases 7 accordance with the Japan gastric cancer handling Terms were histologically diagnosed. Of 87 cases, 28 cases had Skills-type gastric cancer. The remaining 59 cases were non-skilled gastric cancer, which was composed of 11 well-differentiated adenocarcinomas, 15 moderately differentiated adenocarcinomas, 20 solid poorly differentiated adenocarcinomas, and non-solid poorly differentiated adenocarcinomas. There were 10 cancers and 3 seal cell carcinomas.
In addition to LAT1 expression in cancer lesions, the present inventor searched for LAT1 expression in the other 4 groups. 20 cases of normal gastric mucosa (gastrointestinal stromal tumor cases and pancreatic cancer cases without gastric mucosa lesion), 32 cases of background gastric mucosa without intestinal metaplasia (background gastric mucosa of cases of gastric cancer under 40 years old), There are 37 cases of gastric mucosa with epithelial metaplasia (a case of gastric cancer over 75 years old with intestinal metaplasia in the background) and 36 cases of gastric adenoma.
３．免疫組織化学
既に報告されている方法に従って3、外科的に切除されてフォルマリン固定・パラフィン包埋された組織の4μm厚さ切片を使って免疫組織学的染色を行った。使用した一次抗体、希釈、抗原賦活は表１にまとめた。
簡潔に記載すると、組織切片を脱パラフィンし、1%過酸化水素を含むメタノール中で３０分間処理して、内因性peroxidase をブロックした。その後、非特異的反応のブロックの為にProtein block Serum-Free (Dakocytomation, Kyoto, Japan)であらかじめ培養し、切片を一次抗体とともに37℃で１時間培養した。さらにperoxidaseでラベルしたpolymer (Envision, Dakocytomation, Kyoto, Japan), 続いて抗マウスIgG (Gout, Nichirei Bioscience, Tokyo, Japan)抗体で各30分間培養し、3,3’-diaminobenzidine で発色した。核はMeyerのヘマトキシリン溶液で対比染色した。
3. Immunohistochemistry Immunohistological staining was performed using 4 μm thick sections of surgically excised, formalin-fixed, paraffin-embedded tissues according to previously reported methods 3 . The primary antibody used, dilution and antigen activation are summarized in Table 1.
Briefly, tissue sections were deparaffinized and treated in methanol containing 1% hydrogen peroxide for 30 minutes to block endogenous peroxidase. Then, in order to block non-specific reaction, it was cultured in advance with Protein block Serum-Free (Dakocytomation, Kyoto, Japan), and the sections were incubated with the primary antibody at 37 ° C. for 1 hour. The cells were further incubated with peroxidase-labeled polymer (Envision, Dakocytomation, Kyoto, Japan), followed by anti-mouse IgG (Gout, Nichirei Bioscience, Tokyo, Japan) antibody for 30 minutes, and developed with 3,3'-diaminobenzidine. Nuclei were counterstained with Meyer's hematoxylin solution.
４．免疫組織染色の評価
LAT1の免疫反応性の評価はSinicropeの方法3の軽度変法を採用した。すなわち、癌細胞膜の免疫反応性をもとに、4カテゴリを次のように規定した。発現強さ0，反応なし；1, 弱くないしは点状に陽性；2, 中等度に細胞膜全体が陽性；3, 細胞膜全体が強く陽性。各症例で癌組織中における最も高いLAT1発現の強さを採用した。胃癌細胞での代表的なLAT1発現の強さを図１に示した。さらにLAT1発現の領域を全体の胃癌組織を観察してその百分率であらわした。すなわち、0, 陽性なし；1, 局所的 1-10%陽性；2, 部分的 11-30% ; 3, びまん性 30%以上とした。LAT1発現スコアはLAT1発現強さX発現領域の数値であらわした。
p53 発現は核に強く発現している領域を癌組織全体の百分率として、0, 陽性なし；1, 局所的 1-10%陽性；2, 部分的 11-30% ; 3, びまん性 30%以上と評価した。
胃癌組織の粘液表現型の分類は、CD10 (刷子縁), MUC2 (腸杯細胞), MUC5AC (胃腺窩上皮細胞) 及びMUC6 (胃幽門腺)の免疫組織化学的反応性によって行い、4カテゴリ、胃型（G型）、腸型（I型）、胃腸型（GI型）及び分類不可型（U型）を規定した9,10。いずれのマーカーにも陰性な癌はU型とした。
Ki-67陽性細胞は1000以上の有核細胞から陽性細胞を数え、その%をKI-67標識率（LI）とした。
4). Evaluation of immunohistochemical staining
The mild reactivity of Sinicrope's method 3 was adopted to evaluate the immunoreactivity of LAT1. That is, based on the immunoreactivity of cancer cell membranes, the four categories were defined as follows. Intensity 0, no response; 1, weak or punctate positive; 2, moderately positive whole cell membrane; 3, strong whole cell membrane positive. The highest strength of LAT1 expression in cancer tissues was adopted in each case. The typical intensity of LAT1 expression in gastric cancer cells is shown in FIG. Furthermore, the LAT1 expression region was expressed as a percentage of the entire stomach cancer tissue. That is, 0, no positive; 1, local 1-10% positive; 2, partial 11-30%; 3, diffuse 30% or higher. The LAT1 expression score was expressed by the numerical value of the LAT1 expression strength X expression region.
p53 expression is strongly expressed in the nucleus as a percentage of the whole cancer tissue, 0, no positive; 1, local 1-10% positive; 2, partial 11-30%; 3, diffuse 30% or more It was evaluated.
The classification of gastric cancer tissue mucus phenotype is based on the immunohistochemical reactivity of CD10 (brush border), MUC2 (gut goblet cells), MUC5AC (gastric crypt epithelial cells) and MUC6 (gastric pyloric glands). Specified gastric type (G type), intestinal type (I type), gastrointestinal type (GI type) and unclassifiable type (U type) 9,10 . Cancers that were negative for any of the markers were U-shaped.
Ki-67 positive cells were counted from over 1000 nucleated cells, and the percentage was defined as the KI-67 labeling rate (LI).
５．統計学的解析
各グループ間の比較はchi-squared, Mann-Whitney U 或いはKruskal-Wallis test を必要に応じて適用した。患者の生存曲線の比較の有意差はlog-rank testによった。LAT1, Ki-67 LI 及びp53の間での相関はSpearman’s rank correlation coefficient testによって解析した。全ての総計学的解析にはStatView software (Abacus Concepts, Inc. Berkeley, CA, USA)を採用し、p値が0.05以下を統計学的に有意とした。
5. Statistical analysis Chi-squared, Mann-Whitney U or Kruskal-Wallis test was applied as needed for comparison between groups. Significant difference in comparison of patient survival curves was based on log-rank test. The correlation between LAT1, Ki-67 LI and p53 was analyzed by Spearman's rank correlation coefficient test. StatView software (Abacus Concepts, Inc. Berkeley, CA, USA) was employed for all statistical analysis, and a p value of 0.05 or less was considered statistically significant.
６．結果
(1) 患者の特徴
検索した胃癌患者症例は男性54，女性33からなっていた。年齢は33から85才にわたり、平均は60才であった。TNM分類11による病理学的進行度による症例の内訳は、pT IB 17, pT II 17, pT IIIA 15, pT IIIB 4, pT IV 34症例であった。本研究における胃癌症例の臨床病理学的因子は表２にまとめた。87症例中、56例が胃癌で死亡した。分化型胃癌は12，未分化型胃癌は44例であった。12例の分化型胃癌の中，3例に肝転移(25%)、1例に肺転移(8%)を認めた。一方、44例の未分化型胃癌では、2例に肝転移(4.5%)、11例に腹膜播種(25%),1例に癌性リンパ管症(2.3%)を認めた。
6). result
(1) Patient characteristics The searched cases of gastric cancer consisted of 54 males and 33 females. Age ranged from 33 to 85 years, with an average of 60 years. The breakdown of cases by pathological progression according to TNM classification 11 was pT IB 17, pT II 17, pT IIIA 15, pT IIIB 4, pT IV 34 cases. Clinicopathological factors of gastric cancer cases in this study are summarized in Table 2. Of 87 cases, 56 died of gastric cancer. There were 12 cases of differentiated gastric cancer and 44 cases of undifferentiated gastric cancer. Of 12 differentiated gastric cancers, 3 had liver metastases (25%) and 1 had lung metastases (8%). On the other hand, 44 cases of undifferentiated gastric cancer showed liver metastasis (4.5%) in 2 cases, peritoneal dissemination (25%) in 11 cases, and cancerous lymphangiopathy (2.3%) in 1 case.
(2) LAT1 発現
正常胃粘膜における腺か上皮のLAT1発現はごくわずかで、LAT1発現の強さは低値（1.1 ±1.2, 平均 ± 標準偏差, 図2-a）であった。しかし腸上皮化生のある背景胃粘膜では、LAT1発現強さはより強かった (2.4 ± 0.9)。胃腺腫でもLAT発現は認められ (1.9 ± 1.2)、粘膜固有層の下1/2よりも上1/2の方が強かった。
胃癌では癌細胞膜に強く発現していた (2.6 ± 0.8)。分化型胃癌と未分化型胃癌の間でのLAT1発現に有意な差はなかった。しかし、非スキルス型胃癌はスキルス型胃癌に比べて、LAT1発現スコアが有意に高かった(図2-f)。またLAT1発現スコアは胃腺腫に比較して胃癌でより高かった。さらに胃腺腫や腸上皮化生のある背景胃粘膜におけるLAT1発現の強さは正常粘膜よりも有意に強かった。
正常粘膜、腸上皮化生のある背景胃粘膜、胃腺腫及び胃癌の代表的なLAT1発現を図3に示した。
(2) LAT1 expression LAT1 expression in the gland or epithelium in the normal gastric mucosa was negligible, and the intensity of LAT1 expression was low (1.1 ± 1.2, mean ± standard deviation, Fig. 2-a). However, LAT1 expression was stronger in the background gastric mucosa with intestinal metaplasia (2.4 ± 0.9). LAT expression was also observed in gastric adenoma (1.9 ± 1.2), with the upper half being stronger than the lower half of the lamina propria.
In gastric cancer, it was strongly expressed in the cancer cell membrane (2.6 ± 0.8). There was no significant difference in LAT1 expression between differentiated and undifferentiated gastric cancer. However, non-Skills gastric cancer had a significantly higher LAT1 expression score than Skills gastric cancer (Fig. 2-f). And LAT1 expression score was higher in gastric cancer compared with gastric adenoma. Furthermore, the intensity of LAT1 expression in the background gastric mucosa with gastric adenoma and intestinal metaplasia was significantly stronger than that in the normal mucosa.
Representative LAT1 expression in normal mucosa, background gastric mucosa with intestinal metaplasia, gastric adenoma and gastric cancer is shown in FIG.
(3) 胃癌の粘液表現型とLAT1発現
粘液の4マーカー発現に基づいて、87症例を4カテゴリへ分類した。26例はG型、22例はI型、38例はGI型、1例がU型であった。これら4カテゴリの間でLAT1発現を比較したが、有意な違いをみとめなかった。
(3) Mucus phenotype and LAT1 expression of gastric cancer 87 cases were classified into 4 categories based on the expression of 4 markers of mucus. 26 cases were G type, 22 cases were I type, 38 cases were GI type, and 1 case was U type. LAT1 expression was compared among these four categories, but no significant differences were found.
(4) リンパ節転移とLAT1発現との関連
リンパ節転移のある症例は転移のない症例と比較して、癌細胞のLAT1発現の強さが高い傾向を認めた (p=0.0573)。さらにLAT1スコアはリンパ節転移例で転移のない症例と比較して有意に高かった(p=0.0077)。癌細胞のリンパ管や血管侵襲とLAT1発現とは有意な相関がみられなかった（データ省略）。
(4) Relationship between lymph node metastasis and LAT1 expression In patients with lymph node metastasis, LAT1 expression in cancer cells tended to be stronger than those without metastasis (p = 0.0573). Furthermore, the LAT1 score was significantly higher in patients with lymph node metastasis compared with those without metastasis (p = 0.0077). There was no significant correlation between lymphatic and vascular invasion of cancer cells and LAT1 expression (data not shown).
(5) p53 発現とLAT1発現との関連
Spearman’s rank correlation coefficient test によると、胃癌全症例でのp53スコアとLAT1発現の強さとの間には弱い相関を認めた(r=0.459, p<0.0001)。さらに胃癌全症例でのp53発現スコアとLAT1発現スコアとの間にも弱い相関を認めた(r=0.463, p<0.0001)。
(5) Association between p53 expression and LAT1 expression
Spearman's rank correlation coefficient test showed a weak correlation between the p53 score and the intensity of LAT1 expression in all cases of gastric cancer (r = 0.459, p <0.0001). Furthermore, a weak correlation was observed between the p53 expression score and LAT1 expression score in all cases of gastric cancer (r = 0.463, p <0.0001).
(6) Ki-67 LI とLAT1発現との関連
図４に示したように、胃癌におけるKi-67 LIは他の胃病変グループより高かった。胃癌全症例でのKi-67 LI とLAT1発現強さとの間に弱い相関を認めた(r=0.428, p<0.001)。Ki-67 LI とLAT1発現スコアとの間には有意な相関を認めなかった。
(6) Relationship between Ki-67 LI and LAT1 expression As shown in FIG. 4, Ki-67 LI in gastric cancer was higher than other gastric lesion groups. A weak correlation was observed between Ki-67 LI and LAT1 expression intensity in all gastric cancer cases (r = 0.428, p <0.001). There was no significant correlation between Ki-67 LI and LAT1 expression score.
(7) 患者の生存曲線
低及び高LAT1 発現スコア (低スコア0-4、高スコア6-9)で87症例を分けて生存曲線を比較した。全症例では、低LAT1スコア及び高LAT1 スコア群で有意な差がなかった(p=0.0997, 図5a)。しかし、非スキルス型胃癌では、高LAT1発現群は予後が有意に不良であった(p=0.0270, 図5b)。 特にstage IB とIIの非スキルス型胃癌に限定すると、高LAT1は予後が有意に不良であった(p=0.0156, 図5c)。
さらにI型(腸型)を除いた胃癌症例及び未分化癌では、高LAT1 スコア群が予後不良の傾向を示した(それぞれp=0.0570, 図6a；p=0.0558, 図6b)。
さらにスキルス型胃癌を除いた未分化癌（33例）では高LAT1 スコア群が有意に予後不良であった(p=0.0177, 図6c)。
(7) Patient survival curves 87 cases were divided into low and high LAT1 expression scores (low score 0-4, high score 6-9) and the survival curves were compared. In all cases, there was no significant difference between the low LAT1 score group and the high LAT1 score group (p = 0.0997, FIG. 5a). However, in non-Skills-type gastric cancer, the prognosis was significantly poor in the high LAT1-expressing group (p = 0.0270, FIG. 5b). Especially when limited to stage IB and II non-skilled gastric cancer, high LAT1 had a significantly worse prognosis (p = 0.0156, Fig. 5c).
Furthermore, in gastric cancer cases excluding type I (intestinal type) and undifferentiated cancer, the high LAT1 score group tended to have a poor prognosis (p = 0.0570, FIG. 6a; p = 0.0558, FIG. 6b, respectively).
Furthermore, in undifferentiated cancers (33 cases) excluding Skills-type gastric cancer, the high LAT1 score group had a significantly poor prognosis (p = 0.0177, Fig. 6c).
(8) Cox hazard テスト
87症例全体では、癌の進行度stage のみが予後規定因子であった(表３)。非スキルス型胃癌に限定すると、LAT1 スコアとstage が単変量解析で、予後規定因子として認められた。続いて多変量解析でも両者は個々の独立した予後因子であった(表４)。
In all 87 cases, only the stage of cancer progression was the prognostic factor (Table 3). When limited to non-skilled gastric cancer, LAT1 score and stage were recognized as prognostic factors in univariate analysis. Subsequently, both were independent prognostic factors in multivariate analysis (Table 4).
文献
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Literature
1. Kanai Y, Segawa H, Miyamoto K, Uchino H, Takeda E, Endou, H.
Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98) .J Biol Chem 1998; 273: 23629-32.
2. Rajan DP, Kekuda R, Huang W et al. Cloning and expression of ab (0, +)-like amino acid transporter functioning as a heterodimer with 4F2hc instead of rBAT.A new candidate gene for cystinuria.J Biol Chem 1999; 274: 29005-10.
3. Sakata T, Ferdous G, Tsuruta T et al. L-type amino-acid transporter 1 as a novel biomarker for high-grade malignancy in prostate cancer.Pathol Int 2009; 59: 7-18.
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