KRAS 基因突變增加oxaliplatin化學治療藥物敏感性對轉移性結腸直腸癌的生物意義與臨床影響

The biological significance and clinical implication of KRAS mutant-mediated oxaliplatin chemotherapy sensitivity in metastatic colorectal cancer (mCRC)

林育麟(Yu-Lin Lin)

指導教授 ： 鄭安理 ； 葉坤輝

共同指導教授 ： 張逸良(Yih-Leong Chang)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.01770

摘要

結腸直腸癌是全世界導致癌症死亡的原因之一。雖然科技的發展不斷在進步，但在現今最好的標靶治療加上化學治療的情況下，末期結腸癌的平均存活仍只有20至30個月。

目前轉移性結腸直腸癌的治療，抗表皮生長因子接受器單株抗體（anti-epidermal growth factor receptor momoclonal antibody, anti-EGFR mAB）在結腸直腸癌治療上是一個有效的標靶治療。然而，轉移性結腸直腸癌病患若有KRAS基因突變，這樣的病人對此類抗表皮生長因子接受器單株抗體 (anti-EGFR mAB)是沒效的。因此，KRAS基因突變的結腸直腸癌病患比起KRAS基因野生型（wild-type）的病患在治療上有更多需要努力的地方，因為KRAS基因突變的結腸直腸癌病患他們無法使用抗表皮生長因子接受器單株抗體 (anti-EGFR mAB)，所以他們也少了一個有效的治療藥物。因此，替這群KRAS基因突變的結腸直腸癌病患在現存的化療藥物處方中，找出較有效的處方，或者針對KRAS基因突變的結腸直腸癌病患發明新的治療藥物，是有其需要的。

OPUS和PRIME這兩個臨床試驗，都是在研究轉移性結腸直腸癌病患第一線使用oxaliplatin為主的化學治療情況下，若再加上抗表皮生長因子接受器單株抗體，是否治療效果會更好的研究。 當我們只注意OPUS和PRIME這兩個臨床試驗的化學治療組病人時，很有趣地，我們會發現到，KRAS基因突變的結腸直腸癌病患第一線接受以oxaliplatin為主的化療時，其第一線疾病無惡化存活期（progression-free survival, PFS），會比第一線同樣接受oxaliplatin為主的化療的KRAS基因野生型的結腸直腸癌病患來的長。在OPUS研究中，KRAS基因突變的結腸直腸癌病患的疾病無惡化存活期是8.6個月，比上KRAS基因野生型的結腸直腸癌病患的疾病無惡化存活期是7.2個月。另外，在PRIME研究中，KRAS基因突變的結腸直腸癌病患的疾病無惡化存活期是8.8個月，比上KRAS基因野生型的結腸直腸癌病患的疾病無惡化存活期是8.0個月。相反地，CRYSTAL臨床試驗是研究轉移性結腸直腸癌病患第一線使用irinotecan為主的化學治療情況下，若再加上抗表皮生長因子接受器單株抗體，是否治療效果會更好的研究。在CRYSTAL臨床試驗中，若我們也只注意化學治療組，我們會發現到，KRAS基因突變的結腸直腸癌病患的疾病無惡化存活期，會比KRAS基因野生型的結腸直腸癌病患的疾病無惡化存活期來的短。 KRAS基因突變的結腸直腸癌病患在第一線使用以irinotecan為主的化療的疾病無惡化存活期是7.7個月，比上KRAS基因野生型的結腸直腸癌病患在第一線使用以irinotecan為主的化療的的疾病無惡化存活期是8.4個月。根據以上臨床試驗的觀察，我們的假說是以oxaliplatin為主的化學治療，對於KRAS基因突變的結腸直腸癌病患，其好處會比對於KRAS基因野生型的結腸直腸癌病患更多。

我們整個的研究規劃是希望以細胞株研究與臨床研究來證實我們的假說。在細胞株的研究，我們將KRAS基因突變的結腸癌細胞株(DLD-1G13D and SW480G12V)內的KRAS表現用KRAS-si-RNA抑制，另一方面，將KRAS基因野生型的結腸癌細胞株(COLO320DM)內的KRAS表現用持續表現KRAS活性的載體（KRAS activating vector）將KRAS表現誘發。我們的研究發現到，在KRAS 野生型的結腸癌細胞株(COLO320DM)，KRAS表現上升時，會造成excision repair cross-complementation group 1 (ERCC1)的RNA與蛋白質合成減少，如此進一步造成結腸癌細胞對於oxaliplatin較敏感。相反地，在KRAS 突變的結腸癌細胞株(DLD-1G13D and SW480G12V)，KRAS表現被KRAS-si-RNA抑制時會造成ERCC1的RNA與蛋白質表現量上升，如此會造成結腸癌細胞對於oxaliplatin有抗藥性。此KRAS表現在結腸癌細胞株改變後，造成結腸癌細胞株對oxaliplatin敏感性改變的情況，在施加其他化療藥物，像irinotecan, 5FU，到KRAS表現改變的結腸癌細胞株時，都沒有觀察到。為了進一步證明ERCC1表現量的改變是造成KRAS表現改變結腸癌細胞株對於oxaliplatin敏感性改變的主要原因，我們也在KRAS 野生型的結腸癌細胞株(COLO320DM)中，用ERCC1-si-RNA抑制ERCC1的表現，然後再施加oxaliplatin，如此我們發現到此細胞對於oxaliplatin就變得較敏感。另一方面，將ERCC1表現增加，我們發現到該結腸癌細胞就會對oxaliplatin更具有抗藥性。整體來說，我們發現到，在結腸癌細胞中，KRAS基因突變與否是預測結腸癌細胞對於oxaliplatin是否具敏感性的一個指標，此敏感性產生的原因，與KRAS基因突變的結腸癌細胞中ERCC1表現量下降有關。

我們的第二篇臨床研究，是藉由收集結腸癌病人臨床治療資料與KRAS基因突變與否的結果，來分析是否KRAS基因突變的結腸癌病人其第一線疾病無惡化期與整體存活，比KRAS基因野生型的結腸癌病人更好。我們發現到，在第一線使用以oxaliplatin為主的結腸癌病人，KRAS基因突變的病人其第一線疾病無惡化期比KRAS基因野生型的病人有義意的延長。在KRAS基因突變的病人，其第一線疾病無惡化期是8.5個月，在KRAS基因野生型的病人，其第一線疾病無惡化期是5.8個月。相反地，第一線使用以irinotecan為主的結腸癌病人，KRAS基因突變的病人其第一線疾病無惡化期與KRAS基因野生型的病人沒有統計上顯著差別。在KRAS基因突變的病人，其第一線疾病無惡化期是3.9個月，在KRAS基因野生型的病人，其第一線疾病無惡化期是6.0個月。整體存活在兩組病人都沒差。最後，多變項分析中，仍可以發現，KRAS基因突變在第一線使用oxaliplatin為主的化療病人仍是一個獨立的預測因子，可以預測病人是否可達到較長的第一線無疾病惡化期。

我們的第三篇臨床研究進一步欲探討是否KRAS基因突變的結腸癌病人使用oxaliplatin為主的化療會真的比KRAS基因野生型的病人得到較長的整體存活期，我們將KRAS基因突變與沒有突變的病人，分成在第四期時曾經使用過oxaliplatin為主的化療或從來沒有使用過oxaliplatin為主化療的兩組病人，然後分析病人的存活。我們發現到，KRAS基因突變病人的整體存活，在曾經使用過以oxaliplatin為主化療的那組會比從來沒使用過oxaliplatin為主化療的病人有意義的延長。此現象在有使用與沒使用其他化療或標靶治療都不會發生。更重要的是，在KRAS基因野生型的病人，曾經使用過以oxaliplatin為主的化療的那組病人的整體存活並沒有有意義地比從來沒使用過以oxaliplatin為主的化療的那組病人延長。在多變項分析中，KRAS基因突變的結腸癌病人是否有使用過以oxaliplatin為主的化療處方仍是一個預測病人有較好預後的獨立預測因子。

總結我的研究，以oxaliplatin為主的化療處方對於KRAS基因突變的結腸癌病人的臨床獲益會比對KRAS基因野生型的病人來的大。

關鍵字

結腸癌 ； KRAS基因突變 ； oxaliplatin敏感性 ； ERCC1 ； oxaliplatin為主的化學治療 ； 疾病無惡化期 ； 整體存活期

並列摘要

Colorectal cancer (CRC) is one of the leading cause of cancer death worldwide. Although cutting-edge technology and intervention continuously advance, median overall survival (mOS) of metastatic colorectal cancer (mCRC), under currently state-of-art treatment with molecularly targeted therapy plus chemotherapy, remains only 20-30 months.

Mutation of the KRAS gene in mCRC has been identified as a negative predictor to anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (mAB), which is one of the currently available molecularly targeted therapy approved to be used in mCRC patients. Therefore, KRAS mutant mCRC patients have therapeutically unmet needs because they cannot use anti-EGFR mAB and have fewer treatment options than KRAS wild-type mCRC patients. To identify potentially better regimens from currently available systemic treatments or to explore newer agents for the treatment of KRAS mutant mCRC patients is thus warranted.

If we primarily focus on chemotherapy alone group in the OPUS and PRIME studies, in which first-line oxaliplatin-based chemotherapy (FOLFOX, oxaliplatin/5-fluorouracil (5-FU)/leucovorin) was given in both studies, it is intriguing to find that the progression-free survival (PFS) in KRAS mutant group was longer than the PFS in KRAS wild-type group, with 8.6 versus 7.2 months in the OPUS study and 8.8 versus 8.0 months in the PRIME study. In contrast, in the CRYSTAL study, if we focused on chemotherapy alone group, in which first-line irinotecan-based chemotherapy (FOLFIRI, irinotecan/5-FU/leucovorin) was given, the median PFS was 7.7 months in KRAS mutant group and 8.4 months in the KRAS wild-type group. According to these observations, we hypothesized that oxaliplatin-based regimens may be more beneficial to KRAS mutant than wild-type mCRC patients.

We conducted in vitro and clinical studies trying to prove our hypothesis. In the in vitro study, KRAS gene was knocked-down in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V) and overexpressed in KRAS-wild-type CRC cells (COLO320DM) to generate paired CRC cells. In KRAS wild-type CRC cells (COLO320DM), we found KRAS overexpression caused excision repair cross-complementation group 1 (ERCC1) down regulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS mutant CRC cells (DLD-1G13D and SW480G12V), KRAS knocked-down by KRAS-siRNA led to ERCC1 up-regulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU did not change in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our first study suggests that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation.

Our second clinical study, evaluating clinicopathologic features and treatment outcome of mCRC patients including first-line PFS and overall survival (OS) in association with KRAS mutation status by using the Cox proportional hazard model, demonstrated that in patients who received first-line oxaliplatin-based regimens, the first-line PFS was significantly longer in KRAS mutant patients than that in KRAS wild-type patients. The median PFS was 8.5 months in KRAS mutant versus 5.8 months in KRAS wild-type patients (P = .008). In contrast, in patients who received first-line irinotecan-based regimens, the PFS was shorter in KRAS mutant patients (N = 15) than that in KRAS wild-type patients (N = 20). Median PFS was 3.9 months in KRAS mutant versus 6.0 months in KRAS wild-type patients (P = .23). Median OS between KRAS mutant and wild-type patients was not significantly different in both oxaliplatin-based and irinotecan-based regimens. In multivariate analyses, KRAS mutation remains an independent predictive factor for longer PFS in first-line oxaliplatin-based regimens.

In our third study, clinicopathologic features and OS of KRAS mutant and wild-type mCRC patients who had used oxaliplatin-based, irinotecan-based, bevacizumab-based, as well as cetuximab-based regimens were compared to those who had never-used oxaliplatin-based, irinotecan-based, bevacizumab-based, as well as cetuximab-based regimens, respectively. We demonstrated that in KRAS mutant patients who had used oxaliplatin-based regimens (N = 131), the OS was significantly longer than that in KRAS mutant patients who had never-used oxaliplatin-based regimens (N = 38). The OS was 28.8 months in KRAS mutant patients who had used oxaliplatin-based regimens versus 17.8 months in KRAS mutant patients who had never-used oxaliplatin-based regimens (P = 0.026). Notably, OS in KRAS wild-type mCRC patients who had used oxaliplatin-based regimens (N = 185) was not significantly longer than that in KRAS wild-type mCRC patients who had never used oxaliplatin-based regimens (N = 40) (P = 0.25). Furthermore, the OS in KRAS mutant patients who had used either irinotecan-based, bevacizumab-based or cetuximab-based regimens was not significantly different than that in KRAS mutant patients who had never-used either irinotecan-based, bevacizumab-based or cetuximab-based regimens, respectively. In multivariate analyses, patients who had used oxaliplatin-based regimens remains an independent prognostic factor for longer OS in KRAS mutant mCRC patients. In conclusion, oxaliplatin-based regimens may be more beneficial in KRAS mutant than in KRAS wild-type mCRC patients.

並列關鍵字

colon cancers ； KRAS mutation ； oxaliplatin sensitivity ； ERCC1 ； oxaliplatin-based chemotherapy ； progression-free survival ； overall survival

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