Characterization of rare transforming KRAS mutations in sporadic colorectal cancer

KRAS mutational status has been shown to be a predictive biomarker of resistance to anti-EGFR monoclonal antibody (mAb) therapy in patients with metastatic colorectal cancer. We report the spectrum of KRAS mutation in 1506 patients with colorectal cancer and the identification and characterization of rare insertion mutations within the functional domain of KRAS. KRAS mutations are found in 44.5% (670/1506) of the patients. Two cases are found to harbor double mutations involving both codons 12 and 13. The frequencies of KRAS mutations at its codons 12, 13, 61, and 146 are 75.1%, 19.3%, 2.5%, and 2.7%, respectively. The most abundant mutation of codon 12 is G12D, followed by G12V and G12C while G13D is the predominant mutation in codon 13. Mutations in other codons are rare. The KRAS mutation rate is significantly higher in women (48%, 296/617) than in men (42.1%, 374/889, P = 0.023). Tumors on the right colon have a higher frequency of KRAS mutations than those on the left (57.3% vs. 40.4%, P < 0.0001). Two in-frame insertion mutations affect the phosphate-binding loop (codon 10–16) of KRAS are identified. One of them has never been reported before. Compared with wild-type protein, the insertion variants enhance the cellular accumulation of active RAS (RAS-GTP) and constitutively activate the downstream signaling pathway. NIH3T3 cells transfected with the insertion variants show enhanced anchorage-independent growth and in vivo tumorigenicity. Potentially these mutations contribute to primary resistance to anti-EGFR mAb therapy but the clinical implication requires further validation.     

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy

The predictive value of KRAS mutation in metastatic colorectal cancer (MCRC) patients treated with cetuximab plus chemotherapy has recently been suggested. In our study, 59 patients with a chemotherapy-refractory MCRC treated with cetuximab plus chemotherapy were included and clinical response was evaluated according to response evaluation criteria in solid tumours (RECIST). Tumours were screened for KRAS mutations using first direct sequencing, then two sensitive methods based on SNaPshot and PCR-ligase chain reaction (LCR) assays. Clinical response was evaluated according to gene mutations using the Fisher exact test. Times to progression (TTP) were calculated using the Kaplan-Meier method and compared with log-rank test. A KRAS mutation was detected in 22 out of 59 tumours and, in six cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. Remarkably, no KRAS mutation was found in the 12 patients with clinical response. KRAS mutation was associated with disease progression (P=0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, P=0.015). Our study confirms that KRAS mutation is highly predictive of a non-response to cetuximab plus chemotherapy in MCRC and highlights the need to use sensitive molecular methods, such as SNaPshot or PCR-LCR assays, to ensure an efficient mutation detection.     

KRAS mutation analysis: a comparison between primary tumours and matched liver metastases in 305 colorectal cancer patients

Background:

KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor antibody in metastatic colorectal cancer (CRC). KRAS mutation analysis is usually performed on primary tumour tissue because metastatic tissue is often not available. However, controversial data are available on the concordance of test results between primary tumours and corresponding metastases. We assessed the concordance of KRAS mutation status in a study of 305 primary colorectal tumours and their corresponding liver metastases.

Methods:

Patients with histologically confirmed CRC who underwent surgical resection of the primary tumour and biopsy or surgical resection of the corresponding liver metastasis were included. KRAS mutation analysis was performed for codons 12 and 13.

Results:

KRAS mutation was detected in 108 out of 305 primary tumours (35.4%). In 11 cases (3.6%), we found a discordance between primary tumour and metastasis: 5 primary tumours had a KRAS mutation with a wild-type metastasis, 1 primary tumour was wild type with a KRAS mutation in the metastasis, and in 5 cases the primary tumour and the metastasis had a different KRAS mutation.

Conclusion:

We observed a high concordance of KRAS mutation status of 96.4% (95% CI 93.6–98.2%) between primary colorectal tumours and their corresponding liver metastases. In only six patients (2.0% 95% CI 0.7–4.2%), the discordance was clinically relevant. In this largest and most homogenous study to date, we conclude that both primary tumours and liver metastases can be used for KRAS mutation analysis.     

KRAS mutations in primary tumours and post-FOLFOX metastatic lesions in cases of colorectal cancer

Background:

KRAS mutations are predictive markers for the efficacy of anti-EGFR antibody therapies in patients with metastatic colorectal cancer. Although the mutational status of KRAS is reportedly highly concordant between primary and metastatic lesions, it is not yet clear whether genotoxic chemotherapies might induce additional mutations.

Methods:

A total of 63 lesions (23 baseline primary, 18 metastatic and 24 post-treatment metastatic) from 21 patients who were treated with FOLFOX as adjuvant therapy for stage III/IV colorectal cancer following curative resection were examined. The DNA samples were obtained from formalin-fixed paraffin-embedded specimens, and KRAS, NRAS, BRAF and PIK3CA mutations were evaluated.

Results:

The numbers of primary lesions with wild-type and mutant KRAS codons 12 and 13 were 8 and 13, respectively. The mutational status of KRAS remained concordant between the primary tumours and the post-FOLFOX metastatic lesions, irrespective of patient background, treatment duration and disease-free survival. Furthermore, the mutational statuses of the other genes evaluated were also concordant between the primary and metastatic lesions.

Conclusion:

Because the mutational statuses of predictive biomarker genes were not altered by FOLFOX therapy, specimens from both primary tumours and post-FOLFOX tumour metastases might serve as valid sources of DNA for known genomic biomarker testing.     

KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer

Background:

KRAS codons 12 and 13 mutations predict resistance to anti-EGFR monoclonal antibodies (moAbs) in metastatic colorectal cancer. Also, BRAF V600E mutation has been associated with resistance. Additional KRAS mutations are described in CRC.

Methods:

We investigated the role of KRAS codons 61 and 146 and BRAF V600E mutations in predicting resistance to cetuximab plus irinotecan in a cohort of KRAS codons 12 and 13 wild-type patients.

Results:

Among 87 KRAS codons 12 and 13 wild-type patients, KRAS codons 61 and 146 were mutated in 7 and 1 case, respectively. None of mutated patients responded vs 22 of 68 wild type (P=0.096). Eleven patients were not evaluable. KRAS mutations were associated with shorter progression-free survival (PFS, HR: 0.46, P=0.028). None of 13 BRAF-mutated patients responded vs 24 of 74 BRAF wild type (P=0.016). BRAF mutation was associated with a trend towards shorter PFS (HR: 0.59, P=0.073). In the subgroup of BRAF wild-type patients, KRAS codons 61/146 mutations determined a lower response rate (0 vs 37%, P=0.047) and worse PFS (HR: 0.45, P=0.023). Patients bearing KRAS or BRAF mutations had poorer response rate (0 vs 37%, P=0.0005) and PFS (HR: 0.51, P=0.006) compared with KRAS and BRAF wild-type patients.

Conclusion:

Assessing KRAS codons 61/146 and BRAF V600E mutations might help optimising the selection of the candidate patients to receive anti-EGFR moAbs.     

Association between KRAS mutation and lung metastasis in advanced colorectal cancer

Background:

KRAS mutations have been associated with lung metastases at diagnosis of metastatic colorectal cancer (mCRC), but the impact of this mutation on subsequent development of lung metastasis is unknown. We investigated KRAS mutation as a predictor of lung metastasis development.

Methods:

We retrospectively evaluated data from patients with mCRC whose tumour was tested for KRAS mutation from 2008 to 2010. The relationships of KRAS mutational status with time-to-lung metastasis (TTLM) and overall survival (OS) were analysed.

Results:

Of the 494 patients identified, 202 (41%) had tumours with KRAS mutation. KRAS mutations were associated with a shorter TTLM (median 15.2 vs 22.4 months; hazard ratio=1.40; P=0.002) and a two-fold greater odds of developing lung metastases during the disease course in patients with liver-limited mCRC at diagnosis (72 vs 56%, P=0.007). Overall survival did not differ by KRAS status.

Conclusions:

Lung metastasis was more likely to develop during the disease course in patients whose tumour had a KRAS mutation than in those whose tumour did not have a KRAS mutation. This finding may have an impact on decision making for surgical resection of metastatic disease.     

The prognostic role of KRAS,BRAF, PIK3CA and PTEN in colorectal cancer

Background

Mutations in KRAS, BRAF, PIK3CA and PTEN expression have been in focus to predict the effect of epidermal growth factor receptor-blocking therapy in colorectal cancer (CRC). Here, information on these four aberrations was collected and combined to a Quadruple index and used to evaluate the prognostic role of these factors in CRC.

Patients

We analysed the mutation status in KRAS, BRAF and PIK3CA and PTEN expression in two separate CRC cohorts, Northern Sweden Health Disease Study (NSHDS; n=197) and Colorectal Cancer in Umeå Study (CRUMS; n=414). A Quadruple index was created, where Quadruple index positivity specifies cases with any aberration in KRAS, BRAF, PIK3CA or PTEN expression.

Results

Quadruple index positive tumours had a worse prognosis, significant in the NSHDS but not in the CRUMS cohort (NSHDS; P=0.003 and CRUMS; P=0.230) in univariate analyses but significance was lost in multivariate analyses. When analysing each gene separately, only BRAF was of prognostic significance in the NSHDS cohort (multivariate HR 2.00, 95% CI: 1.16–3.43) and KRAS was of prognostic significance in the CRUMS cohort (multivariate HR 1.48, 95% CI: 1.02–2.16). Aberrations in PIK3CA and PTEN did not add significant prognostic information.

Conclusions

Our results suggest that establishment of molecular subgroups based on KRAS and BRAF mutation status is important and should be considered in future prognostic studies in CRC.     

Cost-effectiveness of first-line treatments for patients with KRAS wild-type metastatic colorectal cancer

Background

Combinations of chemotherapy regimens and monoclonal antibodies have been demonstrated to improve clinical outcomes in patients with metastatic colorectal cancer (mcrc). Although these combination treatment strategies are safe and effective in first-line treatment for mcrc, little is known about their economic consequences and resource allocation implications. In the present study, we evaluated the cost-effectiveness of bevacizumab plus folfiri, cetuximab plus folfiri, and panitumumab plus folfiri for patients with KRAS wild-type mcrc.

Methods

A Markov model simulated the lifetime patient outcomes and costs of each first-line treatment strategy and subsequent lines of treatment from the perspective of the health care payer in Ontario. The model was parameterized using data from the Ontario Cancer Registry, Ontario health administrative databases, and published randomized control trials. Patient outcomes were measured in quality-adjusted life years (qalys), and costs were measured in monetary terms. Costs and outcomes were both discounted at 5% and expressed in 2012 Canadian dollars.

Results

For mcrc patients with KRAS wild-type disease, the treatment strategy of bevacizumab plus folfiri was found to dominate the other two first-line treatment strategies. Sensitivity analyses revealed that the incremental cost-effectiveness ratio values were sensitive to the effectiveness of treatment, the costs of bevacizumab and cetuximab, and health utility values.

Conclusions

Evidence from Ontario showed that bevacizumab plus folfiri is the cost-effective first-line treatment strategy for patients with KRAS wild-type mcrc. The panitumumab plus folfiri and cetuximab plus folfiri options were both dominated, but the cetuximab plus folfiri strategy must be further investigated given that, in the sensitivity analyses, the cost-effectiveness of that strategy was found to be superior to that of bevacizumab plus folfiri under certain ranges of parameter values.     

TP53 mutations predict disease control in metastatic colorectal cancer treated with cetuximab-based chemotherapy

Recent studies have suggested that activation of the EGFR pathway leads to malignant transformation only if the p53 protein is inactivated. Therefore, we evaluated the impact of TP53 mutations on cetuximab-based chemotherapy (CT) sensitivity in combination with KRAS mutations that have been associated with cetuximab resistance. KRAS and TP53 status were assessed in tumours from 64 metastatic colorectal cancer patients treated with cetuximab-based CT and correlated to clinical response using the Fisher''s exact test. Times to progression (TTPs) according to gene status were calculated using the Kaplan–Meier method and compared with log-rank test. TP53 mutations were found in 41 patients and were significantly associated with controlled disease (CD), as defined as complete response, partial response or stable disease (P=0.037) and higher TTP (20 vs 12 weeks, P=0.004). Remarkably, in the subgroup of 46 patients without KRAS mutation, but not in patients with KRAS mutation, TP53 mutations were also associated with CD (P=0.008) and higher TTP (24 vs 12 weeks, P=0.0007). This study suggests that TP53 mutations are predictive of cetuximab sensitivity, particularly in patients without KRAS mutation, and that TP53 genotyping could have a clinical interest to select patients who should benefit from cetuximab-based CT.     

Prognostic and predictive value of TOPK stratified by KRAS and BRAF gene alterations in sporadic, hereditary and metastatic colorectal cancer patients

Background:

Our aim was to investigate the prognostic and predictive value of the oncogenic MAPKK-like protein T-cell-originated protein kinase (TOPK) stratified by KRAS and BRAF mutations in patients with sporadic, hereditary and metastatic colorectal cancer (CRC) treated with anti-EGFR therapy.

Methods:

Immunohistochemistry (IHC) for TOPK was performed on four study groups. Group 1 included two subgroups of 543 and 501 sporadic CRC patients used to test the reliability of TOPK expression by IHC. In Group 2, representing an additional 222 sporadic CRCs, the prognostic effect of TOPK stratified by KRAS and BRAF was assessed. The prognostic effect of TOPK was further analysed in Group 3, representing 71 hereditary Lynch syndrome-associated CRC patients. In Group 4, the predictive and prognostic value of TOPK was analysed on 45 metastatic patients treated with cetuximab or panitumumab stratified by KRAS and BRAF gene status.

Results:

In both sporadic CRC subgroups (Group 1), associations of diffuse TOPK expression with clinicopathological features were reproducible. Molecular analysis of sporadic CRCs in Group 2 showed that diffuse TOPK expression was associated with KRAS and BRAF mutations (p<0.001) and with poor outcome in patients with either mutation in univariate and multivariate analysis (P=0.017). In hereditary patients (Group 3), diffuse TOPK was linked to advanced pT stage. In metastatic patients treated with anti-EGFR therapy (Group 4), diffuse TOPK expression was linked to dismal outcome despite objective response to treatment (P=0.01).

Conclusions:

TOPK expression is an unfavourable prognostic indicator in sporadic patients with KRAS or BRAF mutations and also in patients with metastatic disease experiencing a response to anti-EGFR therapies. The inhibition of TOPK, which could benefit 30–40% of CRC patients, may represent a new avenue of investigation for targeted therapy.     

Molecular determinants of anti-EGFR sensitivity and resistance in metastatic colorectal cancer

Since 2004, the clinical impact of monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) on patients with metastatic colorectal cancer (MCRC) has been clearly established. The combination of these biological agents with conventional chemotherapy has led to a significant improvement in response rate, progression-free survival and overall survival in first-line as well as in second- or third-line treatment of MCRC. However, the high variability of response and outcome in MCRC patients treated with these anti-EGFR mAbs has highlighted the need of identifying clinical and/or molecular predictive markers to ensure appropriate use of targeted therapies. The presence of somatic KRAS mutations has been clearly identified as a predictive marker of resistance to anti-EGFR in MCRC, and the use of anti-EGFR mAbs is now restricted to patients with no detectable KRAS mutation. Several studies have indicated that amplification of EGFR, overexpression of the EGFR ligands and inactivation of the anti-oncogene TP53 are associated with sensitivity to anti-EGFR mAbs, whereas mutations of BRAF and PIK3CA and loss of PTEN expression are associated with resistance. Besides these somatic variations, germline polymorphisms such as those affecting genes involved in the EGFR pathway or within the immunoglobulin receptors may also modulate response to anti-EGFR mAbs. Until now, all these markers are not completely validated and only KRAS genotyping is mandatory in routine practice for use of the anti-EGFR mAbs in MCRC.     

KRAS mutations detected by the amplification refractory mutation system-Scorpion assays strongly correlate with therapeutic effect of cetuximab

Background:

We aimed to compare the sensitive and quality-controlled KRAS testing with direct sequencing and to assess the impact on decision making of treatment.

Patients and methods:

We analysed genomic DNA isolated from macrodissected formalin-fixed paraffin-embedded specimens by direct sequencing and an amplification refractory mutation system–Scorpion assay (ARMS/S) method. Cetuximab was administered to patients identified as having wild-type (WT) KRAS using direct sequencing. Therapeutic effects were evaluated according to their KRAS status as determined by ARMS/S.

Results:

Among the 159 patients, the overall mutation rate was determined to be 37.0% by direct sequencing and 44.0% by ARMS/S. For the patients diagnosed as WT by direct sequencing and treated with cetuximab (n=47), a response rate of 16.0% was observed for 38 ARMS/S WT patients, whereas 9 ARMS/S mutant (MUT) patients failed to respond. The ARMS/S WT patients showed significant improvement in progression-free survival (PFS) and overall survival (OS) compared with ARMS/S MUT patients (PFS median 5.0 vs 1.7 months, hazards ratio (HR)=0.29, P=0.001; OS median 12.1 vs 3.8 months, HR=0.26, P=0.001).

Conclusion:

Sensitive and quality-controlled KRAS testing may provide improved predictive power to determine the efficacy of anti-epidermal growth factor antibodies.     

KRAS as a predictor of poor prognosis and benefit from postoperative FOLFOX chemotherapy in patients with stage II and III colorectal cancer

Purpose

The KRAS gene frequently mutates in colorectal cancer (CRC). Here we investigated the prognostic and predictive role of KRAS mutation in patients with stage II or III CRC.

Experimental design

A consecutive cohort of patients with stage II or III CRC from a single center database was studied. The association between KRAS status, adjuvant FOLFOX therapy, and 3‐year disease‐free survival (3‐y DFS) was analyzed.

Results

Of our 433 patients, 166 (38.3%) exhibited the KRAS mutation. Among the 190 patients who did not receive adjuvant therapy, those with KRAS mutation tumors had a worse 3‐y DFS (hazard ratio [HR], 1.924; 95% confidence interval [CI], 1.078–3.435; P = 0.027). Among patients who received adjuvant chemotherapy, KRAS mutation was not correlated with worse 3‐y DFS (HR, 1.083; 95% CI, 0.618–1.899; P = 0.781). Adjuvant chemotherapy improved 3‐y DFS only among patients with KRAS mutant tumors (78.0% vs 69.2%) on multivariate analysis adjusted for age, stage, grade, site, vessel invasion, and carcinoembryonic antigen level (HR, 0.454; 95% CI, 0.229–0.901; P = 0.024). In contrast, there was no benefit of adjuvant chemotherapy in the KRAS wild‐type group (84.3% vs 82.0%).

Conclusions

KRAS mutation indicates poor prognosis. FOLFOX adjuvant chemotherapy benefits patients with stage II or III colorectal cancer with KRAS mutant tumors and is worth further investigation.     

BRAF mutation is a powerful prognostic factor in advanced and recurrent colorectal cancer

Background:

Activating mutation of KRAS and BRAF are focused on as potential prognostic and predictive biomarkers in patients with colorectal cancer (CRC) treated with anti-EGFR therapies. This study investigated the clinicopathological features and prognostic impact of KRAS/BRAF mutation in advanced and recurrent CRC patients.

Method:

Patients with advanced and recurrent CRC treated with systemic chemotherapy (n=229) were analysed for KRAS/BRAF genotypes by cycleave PCR. Prognostic factors associated with survival were identified by univariate and multivariate analyses using the Cox proportional hazards model.

Results:

KRAS and BRAF mutations were present in 34.5% and 6.5% of patients, respectively. BRAF mutated tumours were more likely to develop on the right of the colon, and to be of the poorly differentiated adenocarcinoma or mucinous carcinoma, and peritoneal metastasis. The median overall survival (OS) for BRAF mutation-positive and KRAS 13 mutation-positive patients was 11.0 and 27.7 months, respectively, which was significantly worse than that for patients with wild-type (wt) KRAS and BRAF (40.6 months) (BRAF; HR=4.25, P<0.001, KRAS13; HR=2.03, P=0.024). After adjustment for significant features by multivariate Cox regression analysis, BRAF mutation was associated with poor OS (HR=4.23, P=0.019).

Conclusion:

Presence of mutated BRAF is one of the most powerful prognostic factors for advanced and recurrent CRC. The KRAS13 mutation showed a trend towards poor OS in patients with advanced and recurrent CRC.     

Down-regulation of KRAS-interacting miRNA-143 predicts poor prognosis but not response to EGFR-targeted agents in colorectal cancer

Background:

MicroRNA-143 (miRNA-143) is frequently down-regulated in colorectal cancer (CRC) and may influence CRC cell proliferation, apoptosis and sensitivity to 5-fluorouracil. mRNA encoded by the KRAS oncogene has been identified as a target of miRNA-143. However, the prognostic significance of miRNA-143 expression and the ability to predict patient response to epidermal growth factor receptor (EGFR)-targeted agents have not yet been explored.

Methods:

We examined 77 CRC patients who were identified by pyrosequencing to have wild-type KRAS and were subsequently treated with EGFR-targeted therapy with the monoclonal antibodies cetuximab or panitumumab. MicroRNA-143 expression was measured in CRC tissue and corresponding non-neoplastic colon tissue by RT–PCR and its expression level was correlated with clinico-pathological characteristics. Univariate and multivariate analyses were used to calculate cancer-specific survival (CSS). The progression-free survival (PFS) and objective response rates on EGFR-targeted therapy were also evaluated.

Results:

Down-regulation of miRNA-143 was observed in 47 out of 77 (61%) tumours. Multivariate Cox regression analysis identified low levels of miRNA-143 expression as an independent prognostic factor with respect to CSS (hazard ratio=1.92, confidence interval=1.1–3.4, P=0.024). A significant difference was also observed with regard to PFS on EGFR-targeted therapy (P=0.031), but there were no significant differences with regard to the objective response rates.

Conclusion:

Our data indicate that miRNA-143 expression levels serve as an independent prognostic biomarker for CRC in KRAS wild-type patients. No role for miRNA-143 expression as a predictive biomarker for EGFR-targeted agents could be identified. Given its negative impact on CSS and PFS, miRNA-143 represents a novel prognosticator and a promising drug target for patients with CRC.     

Sorafenib and irinotecan (NEXIRI) as second- or later-line treatment for patients with metastatic colorectal cancer and KRAS-mutated tumours: a multicentre Phase I/II trial

Background:

This trial evaluated the feasibility and efficacy of combined sorafenib and irinotecan (NEXIRI) as second- or later-line treatment of patients with KRAS-mutated metastatic colorectal cancer (mCRC), who had progressed after irinotecan-based chemotherapy.

Methods:

In Phase I, in a 3+3 dose escalation schedule, patients received irinotecan (125, 150 or 180 mg m⁻² every 2 weeks), in combination with 400 mg sorafenib b.d. The primary end point was the maximum-tolerated dose of irinotecan. In Phase II, the primary end point was disease control rate (DCR). Secondary end points were progression-free survival (PFS), overall survival (OS) and toxicity.

Results:

Phase I included 10 patients (median age 63 (49–73)); no dose-limiting toxicity was seen. In Phase II, 54 patients (median age 60 (43–80) years) received irinotecan 180 mg m⁻² every 2 weeks with sorafenib 400 mg b.d. Nine patients (17%) remained on full-dose sorafenib. The DCR was 64.9% (95% CI, 51–77). Median PFS and OS were 3.7 (95% CI, 3.2–4.7) and 8.0 (95% CI, 4.8–9.7) months, respectively. Toxicities included Grade 3 diarrhoea (37%), neutropenia (18%), hand-foot syndrome (13%) and Grade 4 neutropenia (17%).

Conclusion:

The NEXIRI regimen showed promising activity as second- or later-line treatment in this heavily pretreated mCRC population (ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00989469","term_id":"NCT00989469"}}NCT00989469).     

Evaluation of efficacy and safety markers in a phase II study of metastatic colorectal cancer treated with aflibercept in the first-line setting

Background:

Aflibercept (ziv-aflibercept) is an anti-angiogenic agent recently approved in combination with FOLFIRI for the treatment of metastatic colorectal cancer (mCRC) patients previously treated with oxaliplatin. Despite heterogeneity in response to aflibercept, no biomarkers for efficacy or adverse effects have been identified. Here we present biomarker data from the randomised phase II AFFIRM trial assessing aflibercept in combination with mFOLFOX6 first line in mCRC.

Methods:

Ninety-six somatic mutations in key oncogenic drivers of mCRC and 133 common single-nucleotide polymorphisms (SNPs) in vascular endothelial growth factor (VEGF) pathway genes were analysed, and 27 plasma markers measured at baseline, during and after treatment. We assessed correlations of these three classes of biomarkers with progression-free survival (PFS) and adverse events (AEs).

Results:

Somatic mutations identified in KRAS, BRAF, NRAS, PIK3CA and PIK3R1 did not significantly correlate with PFS (multiple testing-adjusted false discovery rate (FDR) or multiple testing-adjusted FDR>0.3). None of the individual SNPs correlated with PFS (multiple testing-adjusted FDR>0.22), but at the gene level variability in VEGFB significantly correlated with PFS (multiple testing-adjusted FDR=0.0423). Although none of the plasma markers measured at baseline significantly correlated with PFS, high levels of circulating IL8 at baseline together with increased levels of IL8 during treatment were significantly associated with reduced PFS (multiple testing-adjusted FDR=0.0478). No association was found between biomarkers and AEs.

Conclusions:

This represents the first biomarker study in mCRC treated with aflibercept. High IL8 plasma levels at baseline and subsequent increases in IL8 were associated with worse PFS, suggesting that IL8 may act as a potentially predictive biomarker of aflibercept treatment outcome.     

c-Ki-ras mutations in colorectal adenocarcinomas from a country with a rapidly changing colorectal cancer incidence.

We have examined the incidence of mutation of the c-Ki-ras proto-oncogene in colorectal adenocarcinomas from two different time periods, namely 1962-1966 and 1994-1996. The first cohort of samples consisted of formalin-fixed, archival paraffin block and represent the oldest colorectal cancer samples for which ras mutation has been examined, while the second cohort of tumours were fresh, flash-frozen samples representative of genetic events occurring in contemporary times. Analysis of mutation status was undertaken by a mismatch-specific oligonucleotide hybridization analysis of exon 1 of the c-Ki-ras proto-oncogene after amplification by the polymerase chain reaction. Mutations in codon 12 or 13 of c-Ki-ras were detected in 28% (14/50) of contemporary samples, a figure consistent with locally established mutation rates. In contrast no mutation was detected in any of the 18 samples from the earlier period, a result that is statistically significant (P = 0.007). Age-standardized rates of colorectal cancer in Singapore have seen a marked increase over the last 30 years, and for the first time we have shown that such an increase in colorectal cancer is associated, at least in part with an increase in incidence of a specific mutagenic change.     

Relationship of increased aurora kinase A gene copy number, prognosis and response to chemotherapy in patients with metastatic colorectal cancer

Background:

Increased Aurora kinase A gene copy number (AURKA-CN) has been reported in metastatic colorectal cancer (mCRC), with unknown relationship to clinical outcome. We correlated increased AURKA-CN in mCRC tumours with KRAS mutation status, overall and progression-free survival (OS, PFS).

Methods:

Sixty-one mCRC tumours were analysed for AURKA-CN using q-PCR, and KRAS mutation status by direct sequencing. Expression of AURKA protein was analysed by immunohistochemistry. Cox-proportional hazard method, Kaplan–Meier curves and log-rank statistics were used to estimate and compare the hazard ratios and median survival between the groups.

Results:

In all, 68% of tumour exhibited high AURKA-CN, and 29% had a KRAS mutation, without correlation between the two. Patients with high AURKA-CN tumours had longer median OS (48.6 vs 18.8 months, P=0.01), with stronger trend among KRAS wild-type tumours (median OS not reached vs 18.8 months, P=0.003). Progression-free survival was longer on first-line or second-line chemotherapy among patients with KRAS wild-type and high vs low AURKA-CN (first: 17.6 vs 5.13 months, P=0.04; second: 10.4 vs 5.1 months, P=0.01). AURKA-CN level did not affect outcomes among patients with KRAS mutant tumours.

Conclusion:

Increased AURKA-CN is common in mCRC tumours and is associated with longer OS and longer PFS during chemotherapy, particularly in KRAS wild-type tumours.     

Detection of KRAS mutations in circulating tumor cells from patients with metastatic colorectal cancer

Background: Quantification of Circulating Tumor Cells (CTCs) as a prognostic marker in metastatic colorectal cancer (mCRC) has already been validated and approved for routine use. However, more than quantification, qualification or characterization of CTCs is gaining importance, since the genetic characterization of CTCs may reflect, in a real time fashion, genetic profile of the disease. Objective: To characterize KRAS mutations (codon 12 and 13) in CTCs from patients with mCRC and to compare with matched primary tumor. Additionally, correlate these mutations with clinical and pathological features of patients. Methods: Blood samples were collected from 26 patients with mCRC from the AC Camargo Cancer Center (São Paulo-Brazil). CTCs were isolated by ISET technology (Isolation by Size of Epithelial Tumors; Rarecells Diagnostics, France) and mutations analyzes were performed by pyrosequencing (QIAGEN). Results:KRAS mutation was detected in 7 of the 21 cases (33%) of samples from CTCs. In matched primary tumors, 9 of the 24 cases (37.5%) were found KRAS mutated. We observed that 5 of the 9 samples with KRAS mutation in their primary tumor had also KRAS mutation in CTCs, meaning a concordance of 71% of matched cases (P = 0.017). KRAS mutation neither on primary tumor nor in CTCs was associated with clinical-pathological parameters analyzed. Conclusion: Faced with a polyclonal disease like colorectal cancer, which is often treated with alternating and successive lines of chemotherapy, real time genetic characterization of CTCs, in a fast and feasible fashion, can provide important information to clinical management of metastatic patients. Although our cohort was limited, it was possible to show a high grade of concordance between primary tumor and CTCs, which suggests that CTCs can be used as surrogate of primary tumors in clinical practice, when the knowledge of mutation profile is necessary and the primary tumor is not available.