Prognostic relevance of KRAS and BRAF mutations in Japanese patients with colorectal cancer

Background

Mutations of the KRAS or BRAF genes are now recognized as prognostic markers for colorectal cancer (CRC). They are also important predictive markers for resistance to the monoclonal antibodies that target the epidermal growth factor receptor.

Methods

In this retrospective study, KRAS and BRAF mutations were analyzed using a direct sequence method in 254 Japanese CRC patients, and the associations between KRAS or BRAF mutations and clinicopathological characteristics or outcome were evaluated.

Results

KRAS and BRAF mutations were detected in 33.5 and 6.7 % of all patients, respectively. Consistent with previous reports, BRAF mutations were significantly correlated with the anatomical site of the tumor (P < 0.001), tumor grade (P = 0.001) and high frequency of microsatellite instability (P < 0.001). BRAF mutations were correlated with poor overall survival in the full patient cohort (P = 0.009). KRAS mutations were significantly correlated with poor recurrence-free survival (P = 0.03), particularly in patients with stage II CRC (P = 0.007). Cox regression analysis showed that KRAS mutations were a negative predictor of recurrence-free survival in patients with stage II CRC.

Conclusion

KRAS mutation status could be a novel biomarker for predicting disease recurrence in Japanese patients with stage II CRC.     

EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients

To determine whether EGFR tyrosine kinase domain mutations are early events in the pathogenesis of lung adenocarcinomas, we tested for the presence of EGFR mutations in histologically normal bronchial and bronchiolar epithelia from lung adenocarcinomas bearing the common EGFR mutations. DNA was extracted from microdissected tissue obtained from 21 tumors with known EGFR mutations, 16 tumors without mutation, and 90 sites of normal bronchial and bronchiolar epithelium from the same surgical specimens. With the use of PCR and direct DNA sequencing, EGFR mutations identical to the tumors were detected in the normal respiratory epithelium in 9 of 21 (43%) patients with EGFR mutant adenocarcinomas but none in patients without mutation in the tumors. The finding of mutations being more frequent in normal epithelium within tumor (43%) than in adjacent sites (24%) suggests a localized field effect phenomenon. Our findings indicate that mutation of the tyrosine kinase domain of EGFR is an early event in the pathogenesis of lung adenocarcinomas, and suggest EGFR mutations as an early detection marker and chemoprevention target.     

Role of tyrosine kinase inhibitors in the treatment of advanced colorectal cancer

Colorectal cancer (CRC) is a common health problem in Western countries. In advanced disease, either FOLFOX (oxaliplatin/5-fluorouracil [5-FU]/leucovorin [LV]) or FOLFIRI (irinotecan/LV/5-FU) are accepted first-line chemotherapy regimens, but median survival appears to plateau with a chemotherapy-only approach. The use of epidermal growth factor receptor (EGFR)- and vascular endothelial growth factor (VEGF)-targeting monoclonal antibodies has increased the median survival of patients with advanced CRC beyond 20 months. However, the precise role of cetuximab, panitumumab and bevacizumab in combination with different chemotherapeutic regimens is still being determined in first- and second-line settings. The activity and tolerance of the EGFR tyrosine kinase inhibitors (TKIs), gefitinib erlotinib, and EKB-569, alone or in combination with chemotherapy, have been explored in patients with metastatic CRC. Regarding VEGF receptor TKIs, 2 phase III clinical trials determined the role of vatalanib in combination with FOLFOX. Efficacy of the oral multitargeted TKIs sorafenib and sunitinib is under investigation. This article aims to review the role of TKIs in advanced CRC.     

Absence of PIK3CA hotspot mutations in hepatocellular carcinoma in Japanese patients

A recent study revealed that the p110alpha (PIK3CA), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110alpha sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA. These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported.     

The c-kit tyrosine kinase inhibitor STI571 for colorectal cancer therapy

The c-kit tyrosine kinase inhibitor STI571 exhibits a substantial therapeutic activity in patients with chronic myeloid leukemia and gastrointestinal stromal tumors respectively associated with constitutive activation of the BCR-ABL and c-kit tyrosine kinases. Human colorectal tumors also express the c-kit proto-oncogene. The present study focuses on the anticancer activity of STI571 in human colorectal tumor cells in vitro and in vivo. The c-kit receptor was identified as a M(r) 145,000 immunoreactive band in human colon cancer cells HT29, HCT8/S11, and HCT116. Cellular invasion induced by 10 ng/ml stem cell factor (EC(50) = 3 ng/ml) in HT29 cells was blocked by 1 micro M STI571 (IC(50) = 56 nM) and pharmacological inhibitors of several oncogenic signaling pathways, namely, phosphatidylinositol 3-kinase (LY294002), Rho GTPases (Clostridium botulinum exoenzyme C3 transferase), and Rho-kinase (Y27632). STI571 inhibited HT29 cell proliferation (IC(50) = 6 micro M) and induced apoptosis in vitro. These cellular effects were associated with a decrease in tumor growth. We also demonstrated that stem cell factor is a proangiogenic factor in vivo and in vitro. These encouraging results warrant further preclinical investigations and clinical trials on the use of the c-kit inhibitor STI571 as a chemotherapeutic agent in colon cancer prevention and in treatment of advanced colorectal cancers associated with liver metastases.     

酪氨酸激酶抑制剂在非小细胞肺癌中的耐药机制

EGFR-TKIs在EGFR突变型非小细胞肺癌治疗中是非常有效的。然而在非小细胞肺癌EGFR突变患者中约有20%天然耐药,即使治疗有效,在10~16月左右会产生继发耐药。迄今为止,研究发现可能的继发性耐药机制包括EGFR二次突变、旁路激活、下游信号激活、小细胞转化及上皮-间质转化（EMT）等。近期,有研究发现BIM基因与TKI耐药相关。本文就有关耐药机制的近期研究进展作一综述。     

FMS‐like tyrosine kinase 3 (FLT3) amplification in patients with metastatic colorectal cancer

FMS‐like tyrosine kinase 3 (FLT3) plays a key role in hematopoiesis. However, the oncogenic role of FLT3 amplification in patients with metastatic colorectal cancer (mCRC) remains unclear. Here, we aimed to evaluate the characteristics, prognosis, and treatment efficacy of an FLT3 inhibitor (regorafenib) in patients with mCRC with FLT3 amplifications. Tumor tissue samples from 2329 patients were sequenced using NGS in the Nationwide Cancer Genome Screening Project in Japan. The effects of clinicopathological features, co‐altered genes, prognosis, and efficacy of regorafenib were investigated. Between April 2015 and June 2018, 85 patients with mCRC with FLT3 amplification were observed. There were no differences in baseline characteristics between patients with or without FLT3 amplification. The frequency of RAS or other gene co‐alterations was inversely correlated with the copy number status. Median survival time in patients with FLT3 amplification was significantly shorter compared with those with non‐FLT3 amplification. Further investigations of FLT3 amplification as a potential treatment target in mCRC are warranted.     

受体型酪氨酸激酶RON在结直肠癌侵袭及转移中的作用

RON (recepteur d'origine nantais)属于对肿瘤发生、发展有显著作用的受体酪氨酸激酶的亚家族.RON在胚胎发育、调节机体某些生理过程、癌症演进及其恶性程度方面具有重要意义.在大量结直肠癌患者中RON过表达,并且常常伴随不同拼接变异体的生成.本文对RON在结直肠癌侵袭及转移中的作用(包括RON的致瘤活性、RON介导上皮基质转化、RON介导信号转导和RON突变)以及RON作为肿瘤治疗靶点的可能性进行简要综述.     

Current status of small-molecule tyrosine kinase inhibitors targeting epidermal growth factor receptor in colorectal cancer

The epidermal growth factor receptor (EGFR) is expressed in the majority of colorectal cancers (CRCs) and is associated with poor clinical outcome. Ample evidence suggests that inhibition of this pathway by monoclonal antibodies directed against EGFR leads to antitumor activity in CRC. Small-molecule tyrosine kinase inhibitors (TKIs) provide distinct advantages over monoclonal antibodies by virtue of lower production costs, ease of oral administration, and ability to target multiple cellular survival pathways. Despite theoretical advantages, multiple early-phase trials of EGFR TKIs fail to demonstrate single-agent activity in CRC. However, the unusually high response rates observed when gefitinib, an EGFR TKI, is combined with chemotherapy for patients with metastatic CRC suggest a possible synergistic effect. This effect is not seen in non-small-cell lung cancer (NSCLC), for which larger phase III trials have been conducted. The differences between NSCLC and CRC with respect to EGFR expression and mutation status do not completely explain this dichotomy, and further investigation into the pharmacogenomics of EGFR tyrosine kinase inhibition in CRC is under way. Significant effort is directed toward newer strategies targeted at the EGFR in CRC. A new generation of small-molecule TKIs is emerging in which multiple receptor pathways, including ErbB2 and vascular endothelial growth factor receptor, can be simultaneously targeted with EGFR. These agents are still in early-phase clinical trials, and specific data for patients with CRC are forthcoming.     

The prognostic value of KRAS mutations in patients with colorectal cancer

Our aim was to evaluate the KRAS genotypes of Japanese colorectal cancer (CRC) patients and to assess the effect of these genotypes on clinical outcome. A total of 99 patients with stage I-IV CRC who underwent resection were prospectively studied for KRAS mutations by direct sequencing. KRAS mutations were found in 37 (37.4%) of 99?patients. Of these, 11.1% were the KRAS p.G13D mutation and the remaining 26.2% were other KRAS mutations. The cumulative 5-year survival rates for patients with wild-type KRAS, KRAS 12 and KRAS p.G13D mutations were 81.4, 61.4 and 42.0%, respectively (P=0.0397). The KRAS genotype had no effect on stage IV patient prognosis without anti-epithelial growth factor receptor (EGFR) antibody therapy. However, in stage I-III patients significant or trends in prognostic factors for disease-free survival (DFS) were pathological T stage, lymphatic vessel involvement and KRAS p.G13D. Multivariate analysis identified T4 pathological stage (P=0.0076) and the KRAS p.G13D mutation (P=0.0499) as the most significant independent prognostic factors associated with DFS. In Japanese CRC patients KRAS p.G13D had prognostic impact on DFS in stage I-III disease, while the prognosis of stage IV patients without anti-EGFR antibody therapy was unaffected by KRAS status.     

Gefitinib-sensitive mutations of the epidermal growth factor receptor tyrosine kinase domain in chinese patients with non-small cell lung cancer.

PURPOSE: Studies have shown that mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain are associated with response of lung cancer to gefitinib (Iressa, AstraZeneca Corp., Shanghai, China). A higher incidence of EGFR mutation was observed in non-small cell lung cancer (NSCLC) patients of Japanese origin compared with those of American origin. However, no data about such mutations in Chinese patients with NSCLC could be obtained. METHODS: Primary NSCLC tissues were obtained for analysis of mutations in exons 18 to 21 of EGFR from a total of 76 patients, of whom 54 did not receive gefitinib therapy and 22 did. PCR products were sequenced directly and mutations were confirmed by an independent PCR and sequence analysis. All types of mutation were cloned and sequenced. RESULTS: A total of 10 types of mutation were found in the series of patients, including two different silent mutations in exon 20 from 11 patients. More than half of the silent mutations (6 of 11) in exon 20 coexisted with other mutations. Mutations were more frequent in adenocarcinoma (17 of 35; 48.6%) compared with squamous carcinoma (1 of 19; 5.3%) among untreated patients. Similar mutations were observed in all seven gefitinib-treated patients with partial response, and no mutations were detected in all eight patients with progressive disease (P < 0.001), except two silent mutations. Three mutations were observed in seven patients with stable disease. CONCLUSIONS: Mutations in the epidermal growth factor receptor tyrosine kinase domain in lung adenocarcinomas from Chinese patients were more frequent than reported previously in lung adenocarcinomas from American patients. Such mutations were well correlated with tumor response to gefitinib.     

A pharmacodynamic study of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in metastatic colorectal cancer patients.

PURPOSE: Epidermal growth factor receptor (EGFR) appears to play an important role in the pathogenesis of colorectal cancer. We have performed a Phase I/II study of the EGFR tyrosine kinase inhibitor ZD1839 in metastatic colorectal cancer patients in which serial biopsies were taken pre- and posttreatment to assess biological activity. Experimental Design: Paired biopsies were obtained from colorectal cancer patients before and after treatment. Proliferation and apoptosis were assessed using Ki67 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated nick end labeling assays, respectively. Immunohistochemistry for EGFR, activated EGFR, phosphorylated Akt, phosphorylated ERK, p27(Kip1), and beta-catenin was also performed. RESULTS: Posttreatment samples showed a statistically significant reduction in the cancer cell proliferation index (mean proliferation index pretreatment 31%; posttreatment 21%; P = 0.047). The mean cancer cell apoptosis index also increased from 6 to 12% in posttreatment samples, although this difference did not achieve statistical significance. All pretreatment samples showed strong staining for EGFR. Loss of immunohistochemical staining for activated EGFR, phosphorylated Akt, and phosphorylated ERK in cancer cells was observed in some patients after treatment. p27(Kip1) was absent in the cancer cells of most pretreatment biopsies; two patients showed a marked increase in staining for nuclear p27(Kip1) after treatment with ZD1839. These two patients also showed large increases in apoptotic index. CONCLUSIONS: ZD1839 inhibits EGFR signaling and proliferation in the cancer cells of patients with metastatic colorectal cancer. ZD1839 may also induce cancer cell apoptosis in a subset of colorectal cancer patients via up-regulation of p27(Kip1).     

The relationship between tyrosine kinase inhibitor therapy and overall survival in patients with non-small cell lung cancer carrying EGFR mutations

For patients with epidermal growth factor receptor (EGFR) mutation-positive lung cancer, the relationship between the dose or duration of treatment with tyrosine kinase inhibitor (TKI) and overall survival remains unclear. Here, we analyzed clinical data of 39 patients who were diagnosed with EGFR mutation-positive non-small cell lung cancer and treated with TKI, but subsequently died. Several parameters were measured in this study: overall survival; first, second, and overall TKI therapy durations; first TKI intensity (actual dose/normal dose); and TKI rate (overall TKI therapy duration/overall survival). The response rate to TKI therapy was 50% , and the median survival was 553 days. After TKI therapy failed, 38.5% patients were re-challenged with TKI. We observed a moderate relationship [r = 0.534, 95% confidential interval (CI) = 0.263 to 0.727, P 0.001] between overall TKI therapy duration and overall survival. However, we found no relationship between overall survival and first TKI intensity (r = 0.073, 95% CI = -0.380 to 0.247, P = 0.657) or TKI rate (r = 0.0345, 95% CI = -0.284 to 0.346, P = 0.835). Nonsmall cell lung cancer patients with mutation-positive tumors remained on TKI therapy for, on average, 33% of the overall survival time. These findings suggest that patients with EGFR mutation-positive tumors should not stick to using TKIs.     

Caspase recruitment domain-containing protein 15 mutations in patients with colorectal cancer

The caspase recruitment domain-containing protein 15 (CARD15) plays a crucial role in mediating the innate immune response. Mutations within this protein have been shown to be independent risk factors for the development of Crohn's disease in Caucasians. As Crohn's disease patients are at increased risk of developing sporadic colorectal cancer, it is conceivable that genetic variability within CARD15 may also play a role in determining susceptibility to this gastrointestinal malignancy in individuals without Crohn's disease. This hypothesis is supported by the findings of two case-control studies that found the frequencies of CARD15 mutations were significantly elevated in Polish and Greek colorectal cancer patients. Given the results of these previous studies, we examined whether the high incidence of sporadic colorectal cancer observed in New Zealand Caucasians was due to mutations within CARD15. To answer this question, we genotyped 133 colorectal cancer patients and 201 Caucasian controls for R702W, G908R, 1007fs, and P268S. Chi(2) Testing found that the combined frequency of R702W, G908R, and 1007fs was significantly elevated in colorectal cancer patients compared with controls (P = 0.001; odds ratio, 2.8; 95% confidence interval, 1.5-5.4), but no association was detected between tumor behavior or age of disease onset and CARD15 mutations in our colorectal cancer cohort. This study is the first to explore the link between CARD15 mutations and colorectal cancer in New Zealand Caucasians. Our results strongly suggest that CARD15 influences susceptibility to colorectal cancer, but we have found no evidence to indicate that CARD15 mutations predict the clinicopathologic characteristics of this disease.     

The use of first-generation tyrosine kinase inhibitors in patients with NSCLC and somatic EGFR mutations

Initial studies with the first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib in patients with non-small cell lung cancer (NSCLC) showed that, although most did not have an objective radiographic response, a minority of patients had dramatic and durable clinical and radiographic responses. The discovery of EGFR mutations in tumours from patients with NSCLC and the association of these mutations with clinical response to gefitinib and erlotinib provided an opportunity to tailor treatment to the mutation profile of the tumour. A number of retrospective reviews and prospective trials have established that gefitinib or erlotinib therapy leads to radiographic responses in approximately 75-80% of patients with NSCLC with EGFR mutations. Although a variety of mutations in EGFR have been identified, the two most common somatic activating EGFR mutations are the LREA deletions in exon 19 and the L858R substitution in exon 21. Together, these mutations make up 85-90% of EGFR mutations. At least two retrospective reviews have indicated a difference in the outcome of patients with different EGFR mutations: after treatment with gefitinib or erlotinib, patients with exon 19 deletions have an increased survival compared with those patients whose tumours have an L858R substitution. These findings remain to be confirmed in prospective studies. Improved understanding of the association of EGFR mutations with clinical outcome may improve the ability of physicians to match treatment to mutation status for patients with NSCLC.