mCRC Cases Research Articles

Efficacy of bevacizumab and chemotherapy in the first-line treatment of metastatic colorectal cancer: broadening KRAS-focused clinical view.

BackgroundThe aim of the present retrospective study was to analyze clinical outcome and risk factors associated with treatment outcomes according to KRAS status in patient with metastatic colorectal cancer (mCRC) treated with bevacizumab (bev) plus chemotherapy in the first-line setting.MethodsWe performed observational study on 1622 patients with mCRC treated with bev plus oxaliplatin- or irinotecan-based chemotherapy, and correlated treatment outcomes with KRAS mutation status. The primary endpoint was progression-free survival (PFS) and additionally overall survival (OS). Adverse events of bevacizumab and risk factors including location of metastases were evaluated.ResultsMutation in KRAS was present in 40.6% of mCRC cases. The median PFS in patients with wild-type KRAS (wtKRAS) vs mutant KRAS was 11.5 vs 11.4 months, respectively. The median OS was 30.7 vs 28.4 months (p = 0.312). Patients with KRAS mutation had lung metastases more frequently than wtKRAS individuals (32.0% vs 23.8%; p = 0.001). We observed no difference in clinical outcome between hepatic and extrahepatic metastatic disease.ConclusionKRAS mutation does not interfere with clinical benefit from first-line treatment with bevacizumab plus chemotherapy in mCRC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-015-0266-6) contains supplementary material, which is available to authorized users.

Analysis of correlation between oncogene mutation and response to chemotherapy in all RAS wild type metastatic colorectal cancer, using next-generation sequencing technology.

553 Background: Targeted therapies of monoclonal antibodies have changed the treatment of metastatic colorectal cancer (mCRC). A target therapy with chemotherapy regimen for mCRC was decided by KRAS mutation status (KRAS exon2 [codon12, codon13]). Currently, there are many reports suggesting that in addition to analysis of KRAS mutation status, the evaluation of EGFR gene copy number, levels of EGFR ligands, BRAF, NRAS, PIK3CA mutations could be helpful to have a more accurate selection of patients who may have a benefit from anti-EGFR targeted drugs. Methods: Mutation status of 50 oncogenes were analysed in 35 mCRC patients with all RAS wild type, using next-generation sequencing technology. The response for chemotherapy was classified response group (R group) and non-response group (N group) by RECIST. The relation between mutation status of 50 oncogenes and the response for chemotherapy was assessed. Results: There were 25 oncogene mutations in the 50 genes. Driver mutation associated with oncogenic mutation deeply were 5 oncogenes, which were PIK3CA, AKT1, BRAF, PDGFRA and TP53. Only BRAF mutation was significantly associated with poor chemo response in the 5 oncogenes. A case which had two driver mutations was only in the N group. One of the two driver mutations was tumor suppressor gene, TP53. Conclusions: BRAF mutation and the number of driver mutations are key predictors of chemosensitivity in the mCRC cases with all RAS wild type.

Controlling the quality of RAS mutation testing by a dichotomous approach: Ring trials and benchmarking mutation frequencies by a RAS monitor.

536 Background: The PRIME study (Douillard et al. New Engl. J. Med. 2014) demonstrated that testing all coding exons of both KRAS and NRAS proto-oncogenes (all-RAS) predicted response for anti-EGFR targeting antibody panitumumab better than testing only KRAS exon 2 (PFS: 8.9 vs 9.6 mo, OS: 20.2 vs. 26 mo). Thus, the EMA changed the label of this drug for Europe. Now all RAS testing has to be done on a high quality level. Therefore, an improved quality assurance (QA) system was developed. Methods: The German RAS Advisory Board as part of QuIP (Quality initiative Pathology) launched ring-trials to assure quality of RAS mutation detection. 12 test cases of metastatic colorectal cancers (mCRC) containing WT or mutated sequences in one of the three coding exons 2 – 4 of either the KRAS or the NRAS oncogenes. Additionally many QA passed participants supplied the results of all-RAS testing of mCRC for one year resulting in a RAS monitor. Results: In less than half a year an area-wide coverage of quality certified all-RAS testing laboratories was reached. In three ring-trials 89 of 93 (96%) institutions for pathology passed the QA using Sanger-Sequencing (60%), pyrosequencing (25%) or the COBAS system (15%). None of the methods was superior to another as comparable amounts of methods passed or failed the ring trials. As only 12 cases were tested the result is reliable with a lower limit of confidence of only 0.7. Thus, a RAS monitor was introduced as an additional instrument of QA. Here, the results of almost 4000 cases of mCRC tested for all-RAS were collected indicating RAS mutations in 45.7% of investigated cases (KRAS: 41.5%, NRAS: 4.2%). Conclusions: Ring-trials are a versatile tool to establish a network of quality tested diagnostic laboratories in a short time. In parallel data sets for benchmarking the frequencies of RAS mutations were generated region specifically. Thereby quality was brought to a higher level than without the RAS monitor.

RAS mutations affect pattern of metastatic spread and increase propensity for brain metastasis in colorectal cancer.

RAS and PIK3CA mutations in metastatic colorectal cancer (mCRC) have been associated with worse survival. We sought to evaluate the impact of RAS and PIK3CA mutations on cumulative incidence of metastasis to potentially curable sites of liver and lung and other sites such as bone and brain. We performed a computerized search of the electronic medical record of our institution for mCRC cases genotyped for RAS or PIK3CA mutations from 2008 to 2012. Cases were reviewed for patient characteristics, survival, and site-specific metastasis. Among the 918 patients identified, 477 cases were RAS wild type, and 441 cases had a RAS mutation (394 at KRAS exon 2, 29 at KRAS exon 3 or 4, and 18 in NRAS). RAS mutation was significantly associated with shorter median overall survival (OS) and on multivariate analysis independently predicted worse OS (HR, 1.6; P < .01). RAS mutant mCRC exhibited a significantly higher cumulative incidence of lung, bone, and brain metastasis and on multivariate analysis was an independent predictor of involvement of these sites (HR, 1.5, 1.6, and 3.7, respectively). PIK3CA mutations occurred in 10% of the 786 cases genotyped, did not predict for worse survival, and did not exhibit a site-specific pattern of metastatic spread. The metastatic potential of CRC varies with the presence of RAS mutation. RAS mutation is associated with worse OS and increased incidence of lung, bone, and brain metastasis. An understanding of this site-specific pattern of spread may help to inform physicians' assessment of symptoms in patients with mCRC.

BRAF mutation predicts for poor outcomes after metastasectomy in patients with metastatic colorectal cancer.

BRAF mutations occur in 5% to 11% of patients with metastatic colorectal cancer (mCRC) and have been associated with poor prognosis. The current study was undertaken to determine the clinicopathologic characteristics, PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) mutation frequency, and outcomes after metastasectomy in patients with BRAF-mutant mCRC. Data from 1941 consecutive patients with mCRC who underwent KRAS/BRAF mutation testing between 2009 and 2012 at a single institution were identified to identify BRAF-mutant mCRC cases (92 cases). BRAF wild-type mCRC cases from 2011 (423 cases) served as a control group. BRAF-mutated mCRC was found to be significantly associated with older age at diagnosis, female sex, right-sided location, poorly differentiated morphology, and mucinous histology compared with wild-type cases. BRAF-mutant cases more frequently progressed from stage III disease (32% vs 17%; P = .003) and among those patients with stage III disease, T4 disease was more common (48% vs 27%; P = .05). PIK3CA was found to be co-mutated in 5% of BRAF-mutant tumors versus 17% of KRAS-mutant tumors (P < .01) and 4% of BRAF/KRAS wild-type cases. Patients with BRAF-mutated mCRC presented more frequently with peritoneal involvement (26% vs 14%; P < 0.01) and less frequently with liver-limited metastases (41% vs 63%; P < .01). Patients with BRAF-mutated mCRC were less likely to undergo metastasectomy (41% vs 26% at 2 years from diagnosis of metastatic disease; P < .01) and were found to have lower overall survival (P < .01) after metastasectomy. BRAF-mutant mCRC is associated with worse clinical outcome. Patients with BRAF-mutant tumors more commonly develop peritoneal metastases, less frequently present with disease limited to the liver, and have shorter survival after metastasectomy compared with patients with BRAF wild-type tumors.

High-sensitivity PCR method for detecting BRAF V600Emutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles

The response to epidermal growth factor receptor (EGFR)-targeted therapy in metastatic colorectal cancer (mCRC) is variable because of intra-tumor heterogeneity at the genetic level, and consequently, it is important to develop sensitive and selective assays to predict patient responses to therapy. Low-abundance BRAF V600E mutations are associated with poor response to treatment with EGFR inhibitors. We developed a method for the detection of BRAF V600E mutations in mCRC using real-time wild-type blocking PCR (WTB-PCR), in which a chimera composed of locked nucleic acids and DNA is incorporated to amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. Mixing experiments showed that this method is exquisitely sensitive, with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000. To demonstrate the applicability of this approach for mCRC patients, we assessed the V600E mutations in 50 clinical cases of mCRC by real-time WTB-PCR. The percentage of patients with V600E mutation as determined by WTB-PCR (16%, 8/50) was higher than by traditional PCR (10%, 5/50), suggesting an increased sensitivity for WTB-PCR. By calculating the ΔC q for real-time traditional PCR, which amplifies all BRAF alleles, versus WTB-PCR, which selectively amplifies mutant BRAF, we demonstrated that among the V600E-positive mCRC patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 0.05 to 52.32%. In conclusion, WTB-PCR provides a rapid, simple, and low-cost method to detect trace amounts of mutated BRAF V600E gene.

MicroRNA Signature in Metastatic Colorectal Cancer Patients Treated With Anti-EGFR Monoclonal Antibodies

BackgroundTo investigate whether microRNAs are predictive of sensitivity to anti–epidermal growth factor receptor (EGFR) monoclonal antibodies in patients with metastatic colorectal cancer (mCRC). MethodsA total of 183 mCRC cases from 2 independent cohorts (cohort 1: 74 cases; validation cohort: 109 cases) treated with cetuximab or panitumumab were included in the study. MiRNA arrays were analyzed using Agilent's miRNA platform. ResultsThe study identified the cluster Let-7c/miR-99a/miR-125b as a signature associated with an outcome different from that of anti-EGFR therapies. In the first cohort, patients with high-intensity signatures had a significantly longer progression-free survival (PFS) (6.1 vs. 2.3 mo; P = .02) and longer overall survival (OS) ( 29.8 vs. 7.0 mo, P = .08) than patients with low-intensity signatures. In the validation cohort, patients with high signature had significantly longer PFS and OS than individuals with low-intensity signatures (PFS 7.8 vs. 4.3 mo, P = .02; OS 12.8 vs. 7.5 mo, P = .02). In the KRAS wild-type population (n = 120), high-intensity signature patients had a significantly longer PFS (7.8 vs. 4.6 mo, P = .016) and longer OS (16.1 vs. 10.9 mo, P = .09) than low-signature individuals, with no difference in KRAS mutated patients. ConclusionThe MiR-99a/Let-7c/miR-125b signature may improve the selection of patients with KRAS wild-type mCRC as good candidates for anti-EGFR therapy.

Cetuximab as salvage monotherapy in chemotherapy-refractory metastatic colorectal cancer: A single-center report

In July 2008, cetuximab, a monoclonal antibody against epidermal growth factor receptor (EGFR), was approved in Japan for clinical use against chemotherapy-refractory metastatic colorectal cancer (mCRC). At Shiga University of Medical Science, between December 2007 and April 2012, a total of 24 EGFR-positive mCRC cases were administered immunohistochemistry with cetuximab as salvage monotherapy. The safety, side-effects and clinical efficacy of the treatment, including response rate, time to treatment failure, progression-free and overall survival, K-ras mutation status and impact on outcome, were investigated. The patient tumor growth control rate (TCR) was 38%, the mean time to progression (TTP) was 9.8 weeks [95% confidence interval (CI), 7.2–12.4] and the mean overall survival (OS) was 49.4 weeks (95% CI, 30.1–68.8). The most common adverse reactions reported were skin reactions, including acne (67%), hand-foot syndrome (16.7%) and paronychia (16.7%), followed by hypocalcemia (50%), hypomagnesemia (16%), stomatitis (20%) and gastrointestinal disorders (12%). The results of the present single-center study demonstrated that cetuximab monotherapy is beneficial for the treatment of chemotherapy-refractory patients with mCRC and that it has an acceptable level of safety and manageable side-effects.

ALK and ROS1 gene rearrangements detected in colorectal cancer (CRC) by fluorescence in situ hybridization (FISH).

3545 Background: Activation of ROS1 and ALK tyrosine kinases through gene fusions lead to unchecked cell proliferation and transformation. ROS1 and ALK gene fusions were found in about 5% and 2% of lung adenocarcinomas and are highly sensitive to specific tyrosine kinase inhibitors. This study aimed at identifying the presence of ROS1 and ALKrearrangements in CRC using FISH technology. Methods: Arrayed specimens of metastatic CRC (mCRC) patients were tested with a 4-target, 4-color break-apart FISH probe set (Abbott Molecular) designed to simultaneously evaluate the genomic status of ROS1 and ALK. Fused 3’/5’ signals of each gene were considered negative for rearrangement; single 3’/single 5’ (for ROS1) and split 3’-5’ or single 3’ (for ALK) were considered positive for rearrangement. Results: Among 236 mCRC patients tested, two were positive for ROS1 (single 3’ROS1 signals in 39% and 61% of tumor cells) and one was positive for ALK rearrangement (single 3’ALK in 41% of tumor cells). The upper cut-off for positive FISH patterns in the negative specimens was identified as &lt;15% both for ROS1 and ALK. Interestingly, the ALK+ patient displayed intra-tumoral heterogeneity, detected in the tissue cores and confirmed in two resection blocks. The fusion partner for ALK was identified as EML4 by PCR-based tests and sequencing. The fusion partner(s) for ROS1 remains to be identified by other technologies. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. Conclusions: The novel FISH probe set was effective to identify the first cases of ROS1 rearrangements in CRC and re-confirm the occurrence of ALK rearrangements. This supports further evaluation of mCRC cases for ROS1 and ALK gene fusions as these may represent new targets for evaluation in clinical trials. Tumor heterogeneity in the ALK rearrangement must be addressed for screening tests. (Partially supported by research grant from Abbott Molecular and the Colorado CCSG P30CA046934).

High resolution melting analysis of KRAS, BRAF and PIK3CA in KRAS exon 2 wild-type metastatic colorectal cancer.

BackgroundKRAS is an EGFR effector in the RAS/RAF/ERK cascade that is mutated in about 40% of metastatic colorectal cancer (mCRC). Activating mutations in codons 12 and 13 of the KRAS gene are the only established negative predictors of response to anti-EGFR therapy and patients whose tumors harbor such mutations are not candidates for therapy. However, 40 to 60% of wild-type cases do not respond to anti-EGFR therapy, suggesting the involvement of other genes that act downstream of EGFR in the RAS-RAF-MAPK and PI3K-AKT pathways or activating KRAS mutations at other locations of the gene.MethodsDNA was obtained from a consecutive series of 201 mCRC cases (FFPE tissue), wild-type for KRAS exon 2 (codons 12 and 13). Mutational analysis of KRAS (exons 3 and 4), BRAF (exons 11 and 15), and PIK3CA (exons 9 and 20) was performed by high resolution melting (HRM) and positive cases were then sequenced.ResultsOne mutation was present in 23.4% (47/201) of the cases and 3.0% additional cases (6/201) had two concomitant mutations. A total of 53 cases showed 59 mutations, with the following distribution: 44.1% (26/59) in KRAS (13 in exon 3 and 13 in exon 4), 18.6% (11/59) in BRAF (two in exon 11 and nine in exon 15) and 37.3% (22/59) in PIK3CA (16 in exon 9 and six in exon 20). In total, 26.4% (53/201) of the cases had at least one mutation and the remaining 73.6% (148/201) were wild-type for all regions studied. Five of the mutations we report, four in KRAS and one in BRAF, have not previously been described in CRC. BRAF and PIK3CA mutations were more frequent in the colon than in the sigmoid or rectum: 20.8% vs. 1.6% vs. 0.0% (P=0.000) for BRAF and 23.4% vs. 12.1% vs. 5.4% (P=0.011) for PIK3CA mutations.ConclusionsAbout one fourth of mCRC cases wild-type for KRAS codons 12 and 13 present other mutations either in KRAS, BRAF, or PIK3CA, many of which may explain the lack of response to anti-EGFR therapy observed in a significant proportion of these patients.

Bevacizumab-based treatment in colorectal cancer with a NRAS Q61K mutation

Despite development of new therapies, metastatic colorectal cancer (mCRC) largely remains an incurable disease. Approximately 2-6% of colorectal cancers harbor NRAS mutations. The anti-VEGF antibody bevacizumab is a backbone of most therapeutic regimens; however, biomarkers predicting its activity are not known. We report two cases of mCRC with a Q61K NRAS mutation that had a favorable response to bevacizumab and the histone deacetylase inhibitor valproic acid. In contrast, none of ten patients with wild-type NRAS or unknown NRAS status and mutated KRAS (NRAS and KRAS mutations are mutually exclusive) responded to the same regimen. These results suggest that NRAS mutation merits further investigation as a potential biomarker predicting the efficacy of bevacizumab-based treatment.

Hepatic imaging response to 90y-microsphere therapy administered for tumor progression during systemic chemotherapy in patients with colorectal liver metastases.

270 Background: Hepatic tumor progression after one or more lines of 5FU-based systemic chemotherapy, with or without biologic agents, is an indication for radioembolization using 90Y microspheres. Methods: An independent imaging review following resin-only 90Y microsphere treated cases of mCRC from July 2002 to December 2011 at 9 US institutions was conducted. A board certified radiologist systematically reviewed hepatic Computed Tomography (CT) images (portal-venous phase) at baseline and 3 months after 90Y treatment. Tumor response was assessed using RECIST 1.0 criteria, based on a maximum of 5 target lesions. Peri-tumoral edema and necrosis; known artifacts which can affect the interpretation of RECIST response, were documented for each lesion. Kaplan Meier analysis compared survival for responders [Partial Response (PR)] vs. non-responders [Stable Disease (SD) or Progressive Disease (PD)]. Results: A total of 184 patients were studied; male (61%) and Caucasians (61%) most common, mean age 62.5 years received a median of 2 (range 1–5) lines of chemotherapy prior to 90Y therapy. Median tumor/liver ratio at 90Y therapy was 15% (IQR 20%). Median 90Y activity administered was 1.18 GBq (IQR 0.55). RECIST response at 3 months was 9.8% PR (n=18), 72.3% SD (133) and 17.9% PD (33); Disease Control Rate = 82.1%. Peri-tumoral edema was documented in 33% (n=60); necrosis in 42% (79); both in 22% (40) of cases, respectively. No significant differences in background characteristics between responders and non-responders were evident (p&gt;0.05). RECIST response at 3 months predicted survival: PR median 13.9 months (95% CI 9.2-30.3) vs. SD 11.0 (8.9-13.5) vs. PD 6.7 (5.5-8.1) [p=0.002]. Conclusions: Radiological lesion response to 90Y therapy at 3 months must be interpreted with caution due to the significant proportions of peri-tumoral edema and necrosis encountered. Both of these artifacts may lead to either the under estimation of PR/SD or the overestimation of PD, respectively. Given these caveats, early (3 month) hepatic radiological response to 90Y therapy appears to predict longer term prognosis.

A retrospective cohort study to determine the prognostic factors in metastatic colorectal cancer (mCRC) patients (pts) undergoing lung metastatectomy.

565 Background: Resection of liver metastases is now accepted as a potentially curative strategy in some pts with mCRC. Resection of lung metastases (LMs) may also be beneficial but this is not well established. We hypothesized that resection of LMs may lead to prolonged survival in highly selected pts with mCRC. Methods: In this IRB-approved retrospective cohort study, cases were defined as mCRC pts with LMs as their only site of disease who underwent metastatectomy. Controls consisted of mCRC pts who had LMs but did not undergo resection. Charts were reviewed for clinical characteristics; survival data was obtained from the institutional tumor registry. The primary endpoint was overall survival (OS) in the cases versus controls. The secondary endpoint was to determine prognostic factors associated with better survival. Results: We included 26 consecutive cases of mCRC undergoing resection of LMs and randomly selected 26 controls. All pts in the both groups were treated with contemporary chemotherapy, as well as bevacizumab and anti-EGFR monoclonals when appropriate. The median OS from the time of diagnosis with LMs was 29.8 months (95% CI 22.9-36.6) in the control group versus 56.6 months (95% CI 47.3-65.8) for the cases who underwent resection (log rank p=0.04). A number of baseline factors were significantly different between the cases and controls. The control group contained more pts with poorly differentiated tumors (p=0.016); had more pts with synchronous versus metachronous presentation of LM's (p=0.001); had more pts with multiple vs solitary LM's (p&lt;0.001); and had more pts with bilateral vs unilateral LMs (p&lt;0.001). The interval from initial diagnosis to the onset of LMs was significantly longer in the resection group vs controls (22.9 vs 2.9 months, p&lt;0.001). Conclusions: In this retrospective study, pts with mCRC to the lung who were referred for resection were more likely to have well differentiated, solitary, unilateral tumors, with a long interval between the original diagnosis and the onset of LMs. Pts selected using these criteria may have a significant survival benefit from surgical resection of their LMs.

Low Percentage of KRAS Mutations Revealed by Locked Nucleic Acid Polymerase Chain Reaction: Implications for Treatment of Metastatic Colorectal Cancer

Metastatic colorectal cancer (mCRC) is frequently characterized by the presence of mutations of the KRAS oncogene, which are generally associated with a poor response to treatment with anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies. With the methods currently used, a case is classified as KRAS-mutated when approximately 20% of the cells bear an activating KRAS mutation. These considerations raise the question of whether cells with a mutated KRAS can be found in mCRC cases classified as KRAS wild-type when more sensitive methods are used. In addition, the issue arises of whether these mCRC cases with low proportion of KRAS-mutated cells could account at least in part for the therapeutic failure of anti-EGFR therapies that occur in 40-60% of cases classified as KRAS wild type. In this study, we compared the classical assays with a very sensitive test, a locked nucleic acid (LNA) polymerase chain reaction (PCR), capable of detecting KRAS-mutated alleles at extremely low frequency (detection sensitivity limit 0.25% mutated DNA/wild-type DNA). By analyzing a cohort of 213 mCRC patients for KRAS mutations, we found a 20.6% discordance between the sequencing/TheraScreen methods and the LNA-PCR. Indeed, 44 mCRC patients initially considered KRAS wild type were reclassified as KRAS mutated by using the LNA-PCR test. These patients were more numerous among individuals displaying a clinical failure to anti-EGFR therapies. Failure to respond to these biological treatments occurred even in the absence of mutations in other EGFR pathway components such as BRAF.

Comparison of COBAS 4800 KRAS, PCR TaqMan, and PCR high-resolution melting assays for the detection of KRAS somatic mutations in formalin-fixed paraffin-embedded colorectal tumors.

103 Background: Colorectal cancer (CRC) is the 2nd most common cancer with more than one million new cases diagnosed every year. From 2006 to 2008, several studies showed the importance of the KRAS oncogene in the treatment of metastatic CRC (mCRC) and response to anti-EGFR therapies like cetuximab or panitumumab. KRAS is a small G protein which can bear activating mutations in 40% cases of mCRC. Numerous methods have been described for the detection of KRAS mutations in FFPE tissues. Direct sequencing is the gold standard but the most sensitive methods are real time PCR assays based. The new COBAS4800 KRAS developed by Roche Diagnostics is a CE IVD validated assay using real time PCR and TaqMelt technology. This assay is validated for the detection of 19 KRAS somatic mutations on exon 2 (codons 12/13) and exon 3 (codon 61) in specimen containing at least 5% of mutated tumoral cells. Methods: We compared COBAS with previously validated PCR TaqMan and PCR High Resolution Melting (HRM) assays. The PCR TaqMan and HRM are a real time PCR based assays. TaqMan can detect accurately 0.5 to 1% of mutated tumoral DNA on codon 12 (G12A, G12C, G12D, G12R, G12S and G12V) and on codon 13 (G13D). HRM can detect 5 to 10% of tumoral DNA on codon 12 and 13. 80 FFPE samples of colorectal tumors resections or biopsies have been collected and processed with the 3 assays. Results have been compared using kappa statistics. Results: On 80 samples, 69 were interpretable with COBAS and TaqMan and 62 with COBAS and HRM. No statistically significant difference between COBAS and TaqMan was found (k=0.942 ; p&lt;0.001). Only 2 discordant cases were found mainly regarding codon 61 mutations not detected with TaqMan. No statistically significant difference between COBAS and HRM was found (k=0.903 ; p&lt;0.001). Three discordant cases were found two regarding codon 61, not detected with HRM and one G12S mutation (detected by COBAS and TaqMan). Conclusions: The 3 assays can detect accurately KRAS mutations. Less ininterpretable results were found with COBAS and TaqMan assays. A pre screening with COBAS and mutation characterization with TaqMan seem to be a good scheme for routine diagnostic of KRAS mutations.

Abstract 4527: The HER3-EREG axis and its role in colorectal cancer aggression

Abstract Yearly, 50,000 Americans die of colorectal cancer metastases to liver and lung. EGFR-targeted agents improve survival in a subset of metastatic colorectal cancer (mCRC) cases, and overexpression of the HER-family ligands amphiregulin (AREG) and epiregulin (EREG) correlates with EGFR-targeted agent susceptibility. Here we present evidence of association between EREG overexpression and disease aggression by showing that knockdown of EREG or HER3 impede anchorage independent growth in the HCT 116 CRC cell line. We also find that a HER3-EREG axis gene signature groups mCRC tumors into prognostically distinct groups. Snap-frozen CRC hepatic metastases (65) with patient matched normal liver, as well as 71 primary CRC tumors with patient matched normal colonic mucosa were analyzed via qRT-PCR for receptors (HER1-4) and ligands (AREG, and EREG). Among the mCRC cases HER2, HER3, AREG, and EREG were expressed at higher levels than unmatched normal mucosa (p&amp;lt;0.005). HER2, AREG, and EREG were also overexpressed in mCRC compared to unmatched primary CRC tissue (p&amp;lt;0.005). Expression of AREG and EREG were highly correlated (Rs=0.90), and higher EREG mRNA expression was associated with markedly decreased post-hepatectomy overall survival (OS) (p=0.018). In a panel of 9 CRC cell lines HCT 116 had the highest EREG expression. Using the pLKO.1 viral vector, shRNAs targeting EREG, and HER3 were introduced to HCT 116 cells. Empty vector was used as a control. mRNA levels for HER3 and EREG were knocked down by 87% and 73% respectively and protein downregulation was confirmed by western blot. Knockdown and control cells were grown in 0.35% soft agar at a density of 5000 cells per well in 6 well plates. Knockdowns impeded anchorage independent growth with a mean colony count (&amp;gt;50 um diameter) of 170 for HER3, and 285 for EREG as compared to 530 for the empty vector (p&amp;lt;0.005). RNA from 3 distinct sets of HER3, EREG, and empty vector infections was assayed on Agilent Human GE 4x44K v2 microarrays. Unsupervised Principle Component Analysis (PCA) suggested broad concordance in the impact of EREG or HER3 knockdown upon the expression profile. Statistical Analysis of Microarrays (SAM) comparing HER3, and EREG knockdowns to empty vector control revealed 205, and 432 differentially expressed genes with at least a 1.5 fold-change and a false discovery rate of 0%. When HER3 and EREG differential gene lists were compared, 95 overlapping genes with 100% directional agreement were observed. For 51 mCRC cases RNA was available for microarray analysis. A predictor of HER3-EREG activity was constructed from 250 genes that discriminate HER3/EREG knockdown and empty vector control cells and are generally variable in tumors. The predictor was used to assign a HER3-EREG activity score to each mCRC case. This score is significantly associated with OS (p=0.005) with the top 50% of patients by HER3-EREG score demonstrating 5 year OS of 23% as compared to 49% for cases in the lower half of HER3-EREG activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4527. doi:1538-7445.AM2012-4527

PS1-14: Utilization and Effectiveness of KRAS Testing for Colorectal Cancer

Background: New genomic and personalized medicine (GPM) applications have the potential to enhance our ability to screen for disease predisposition and treatment responsiveness. Surveying colorectal cancer (CRC) tissue for mutations in the KRAS gene is one such test that can help decide how to treat patients with metastatic colorectal cancer (mCRC). KRAS testing provides an opportunity for oncologists to tailor the use of anti-EGFR therapy to increase treatment effectiveness and potentially minimize adverse events. The FDA has recently changed the labeling of anti-EGFR therapies to indicate that it is not effective for the treatment of patients with KRAS mutations. KRAS testing has already become standard clinical practice in many settings. In this study, we will assess the patterns of diffusion and utilization of KRAS testing and evaluate the characteristics of patients, providers, and health systems as they relate to the uptake of KRAS testing. We will also conduct a comparative analysis of KRAS testing for mCRC patients treated in community practices to evaluate its effectiveness in terms of both progression-free survival and overall survival. Methods: This study will include a total of 800 mCRC cases diagnosed between January 1, 2005 and July 31, 2010 from 7 HMORN sites. Using electronic records at each site, we will capture information on relevant patient characteristics including disease characteristics, comorbidities, history of all cancer-directed therapies, acute complications, receipt of palliative care and contextual socioeconomic variables. We will use chart review to supplement, or in some cases verify, the electronic data. Conclusion: The study will serve as a model for evaluating the potential impact of GPM applications to improve health outcomes, reduce costs, and influence patient and physician decision-making in the area of personalized therapies.

Success and consequences of the German quality assurance system for the molecular-pathological detection of KRAS mutations in the treatment of metastatic colorectal cancer.

e14016 Background: One option in the therapy of metastatic colorectal cancer (mCRC) is the targeting of the epidermal growth factor receptor (EGFR) using the antibodies panitumumab or cetuximab. Here, a biomarker, the KRAS gene, allows the prediction of the response on an individual basis as mutated forms of KRAS lead to a deregulation of the EGFR- signaling pathway by which the antibodies become useless. Therefore, the approval of the European Medicines Agency (EMEA) as well as a recommendation by the Food and Drug Administration (FDA) included that anti-EGFR targeted therapy is only valid in case of mCRCs with wildtypic (WT) KRAS sequences. Thus, the correct determination of the sequence of the KRAS oncogene became an important piece of work in the targeted therapy of mCRCs. Methods: Therefore, the German Society for Pathology (DGP) and the Federation of the German Pathologists (BDP) initiated under the umbrella of the Quality Initiative in Pathology (QuIP) a quality assurance (QA) system based on the testing of histological sections of 10 cases of mCRC. Two parameters were defined for passing the QA test: 1) Correctness or quality of the results independent of the applied testing method (table). 2) Time to completion (14 days). Results: 159 institutions for pathology participated in this QA system in the last two years resulting in a network of now 82 institutions in Germany that analyze the mutational status of the KRAS gene fast and with a high grade of quality. This was interestingly independent of the applied method (table-some institutes participated repeatedly). Conclusions: The results and consequences of the German QA system are presented here. Method No. of participants Sensitivity(true positives) Specificity(true negatives) ARMS-PCR 24 0.99 1.00 DHPLC 0 0.99 1.00 Didesoxy-sequencing 75 0.93 0.99 Hybridization 11 0.93 0.99 Melting point analysis 7 1.00 1.00 Other 7 0.97 1.00 Pyrosequencing 21 0.99 1.00 Restriction-fragment-length-polymorphism 3 0.93 0.93 Snapshot 1 1.00 1.00 Single strand conformational polymorphism 2 1.00 1.00 All 151 0.96 0.99 Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen, Merck Serono Amgen, Merck Serono

KRAS and BRAF Mutational Status in Primary Colorectal Tumors and Related Metastatic Sites: Biological and Clinical Implications

KRAS and BRAF mutations in primary colorectal tumors (PT) are predictive of nonresponse to anti-epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer (mCRC). The question of primary resistance to anti-EGFR treatment as a result of the presence of KRAS or BRAF mutations only in metastases has been raised but not resolved. We analyzed the mutational status of KRAS and BRAF in 64 new patients with mCRC and performed a systematic review of published data from 285 patients. A total of 285 and 95 matched PT/metastases were available for the analysis of the KRAS and the BRAF status, respectively. An identical mutational pattern of KRAS in PT and the matching metastases were reported in all the cases but 14 (5%). In six cases (2%), KRAS was mutated in the PT and wild type in the metastatic site, whereas in eight cases (3%), KRAS was wild type in the PT and mutated in the metastatic site. An identical mutational pattern of BRAF in PT and the matching metastases was reported in all but two cases (3%). In one case (1.5%), BRAF was mutated in the PT and wild type in the metastatic site, whereas in one case (1.5%), BRAF was wild type in the PT and mutated in the metastatic site. The acquisition by metastases of a KRAS or a BRAF mutation that was not present in the PT is a rare event, occurring in 5% of cases of mCRC. This is not a frequent mechanism of primary resistance to anti-EGFR treatments in mCRC.

Estimating costs of care for patients with newly diagnosed metastatic colorectal cancer (mCRC)

6082 Background: The cost of treatment for patients with metastatic colorectal cancer (mCRC) has become a significant component of overall cancer care due to its high incidence and the increasing duration of patient survival. This study examines the resource use patterns and costs of care for patients with incident metastatic colorectal cancer (mCRC) based on analyses of retrospective claims data from selected health plans in the United States. Methods: A case-control analysis was performed using claims data from over 70 US health plans representing 40 million lives from years 1998–2004. Incident mCRC cases were identified based on evidence of a colorectal cancer diagnosis and a metastatic disease diagnosis. Incident mCRC cases could have no other evidence of cancer in the one-year period prior to the date of their first mCRC diagnosis. Cases were matched to non-mCRC controls based on age, gender, geographic region and duration of plan enrollment. Costs were evaluated by phase of disease: diagnosis, treatment, or death phases. Ordinary least squares regressions were performed to evaluate impact of covariates in each phase. Results: Total costs in the follow-up period averaged $97,031 higher for mCRC cases (N=) than for controls. The main cost drivers for mCRC were hospitalizations ($37,369) and specialist visits ($34,582) which included chemotherapy administration. Approximately 40% of the 672 mCRC patients who qualified for the phase analysis were identified with a fatal event during follow-up. Monthly costs were similar in the diagnostic phase ($12,394) and death phase ($12,069), but were significantly lower in the treatment phase ($4,653). Both mean/median monthly costs increased over time during the study period, regardless of disease phase. Conclusion: The economic burden of mCRC is substantial for patients in commercial health plans in the U.S., and costs of care have increased substantially in recent years. Further research is needed to assess the cost impact of newer targeted therapies for the treatment of mCRC. No significant financial relationships to disclose.