Jon-Helge Angelsen

Impact of KRAS, BRAF, PIK3CA, TP53 status and intraindividual mutation heterogeneity on outcome after liver resection for colorectal cancer metastases.

We determined prognostic impact of KRAS, BRAF, PIK3CA and TP53 mutation status and mutation heterogeneity among 164 colorectal cancer (CRC) patients undergoing liver resections for metastatic disease. Mutation status was determined by Sanger sequencing of a total of 422 metastatic deposits. In univariate analysis, KRAS (33.5%), BRAF (6.1%) and PIK3CA (13.4%) mutations each predicted reduced median time to relapse (TTR) (7 vs. 22, 3 vs. 16 and 4 vs. 17 months; p < 0.001, 0.002 and 0.023, respectively). KRAS and BRAF mutations also predicted a reduced median disease‐specific survival (DSS) (29 vs. 51 and 16 vs. 49 months; p <0.001 and 0.008, respectively). No effect of TP53 (60.4%) mutation status was observed. Postoperative, but not preoperative chemotherapy improved both TTR and DSS (p < 0.001 for both) with no interaction with gene mutation status. Among 94 patients harboring two or more metastatic deposits, 13 revealed mutation heterogeneity across metastatic deposits for at least one gene. Mutation heterogeneity predicted reduced median DSS compared to homogeneous mutations (18 vs. 37 months; p = 0.011 for all genes; 16 vs. 26 months; p < 0.001 analyzing BRAF or KRAS mutations separately). In multivariate analyses, KRAS or BRAF mutations consistently predicted poor TRR and DSS. Mutation heterogeneity robustly predicted DSS but not TTR, while postoperative chemotherapy improved both TTR and DSS. Our findings indicate that BRAF and KRAS mutations as well as mutation heterogeneity predict poor outcome in CRC patients subsequent to liver resections and might help guide treatment decisions.

Intra-patient Inter-metastatic Genetic Heterogeneity in Colorectal Cancer as a Key Determinant of Survival after Curative Liver Resection.

Chromosomal instability is a well-defined hallmark of tumor aggressiveness and metastatic progression in colorectal cancer. The magnitude of genetic heterogeneity among distinct liver metastases from the same patient at the copy number level, as well as its relationship with chemotherapy exposure and patient outcome, remains unknown. We performed high-resolution DNA copy number analyses of 134 liver metastatic deposits from 45 colorectal cancer patients to assess: (i) intra-patient inter-metastatic genetic heterogeneity using a heterogeneity score based on pair-wise genetic distances among tumor deposits; and (ii) genomic complexity, defined as the proportion of the genome harboring aberrant DNA copy numbers. Results were analyzed in relation to the patients’ clinical course; previous chemotherapy exposure and outcome after surgical resection of liver metastases. We observed substantial variation in the level of intra-patient inter-metastatic heterogeneity. Heterogeneity was not associated with the number of metastatic lesions or their genomic complexity. In metachronous disease, heterogeneity was higher in patients previously exposed to chemotherapy. Importantly, intra-patient inter-metastatic heterogeneity was a strong prognostic determinant, stronger than known clinicopathological prognostic parameters. Patients with a low level of heterogeneity (below the median level) had a three-year progression-free and overall survival rate of 23% and 66% respectively, versus 5% and 18% for patients with a high level (hazard ratio0.4, 95% confidence interval 0.2–0.8, P = 0.01; and hazard ratio0.3,95% confidence interval 0.1–0.7, P = 0.007). A low patient-wise level of genomic complexity (below 25%) was also a favorable prognostic factor; however, the prognostic association of intra-patient heterogeneity was independent of genomic complexity in multivariable analyses. In conclusion, intra-patient inter-metastatic genetic heterogeneity is a pronounced feature of metastatic colorectal cancer, and the strong prognostic association reinforces its clinical relevance and places it as a key feature to be explored in future patient cohorts.

Bile duct injuries following laparoscopic cholecystectomy

Introduction: Bile duct injuries occur rarely but are among the most dreadful complications following cholecystectomies. Methods: Prospective registration of bile duct injuries occurring in the period 1992–2013 at a tertiary referral hospital. Results: In total, 67 patients (47 women and 20 men) with a median age of 55 (range 14–86) years had a leak or a lesion of the bile ducts during the study period. Total incidence of postoperative bile leaks or bile duct injuries was 0.9% and for bile duct injuries separately, 0.4%. Median delay from injury to repair was 5 days (range 0–68 days). In 12 patients (18%), the injury was discovered intraoperatively. Bile leak was the major symptom in 59%, and 52% had a leak from the cystic duct or from assumed aberrant ducts in the liver bed of the gall bladder. Following the Clavien–Dindo classification, 39% and 45% were classified as IIIa and IIIb, respectively, 10% as IV, and 6% as V. In all, 31 patients had injuries to the common bile duct or hepatic ducts, and in these patients, 71% were treated with a hepaticojejunostomy. Of patients treated with a hepaticojejunostomy, 56% had an uncomplicated event, whereas 14% later on developed a stricture. Out of 36 patients with injuries to the cystic duct/aberrant ducts, 30 could be treated with stents or sphincterotomies and percutaneous drainage. Conclusion: Half of injuries following cholecystectomies are related to the cystic duct, and most of these can be treated with endoscopic or percutaneous procedures. A considerable number of patients following hepaticojejunostomy will later on develop a stricture.

Population-based study on resection rates and survival in patients with colorectal liver metastasis in Norway

Detailed knowledge about the proportion of patients with colorectal liver metastases (CLM) undergoing resection is sparse. The aim of this study was to analyse cumulative resection rates and survival in patients with CLM.

Abstract 1878: Performance comparison of BRAF V600E detection assays in malignant melanoma and colorectal cancer specimens

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Personalized cancer care requires reliable biomarkers. One already implemented in the clinic is the BRAF V600E mutation. We present a comprehensive comparison of three detection methods including immunohistochemistry (IHC) using a mutation specific monoclonal antibody (VE1), Sanger sequencing and a probe-based high resolution melting assay, in melanoma and colorectal cancer specimens. Genomic DNA (gDNA) from fresh frozen tissue from 389 liver metastases and 6 primary tumors from 141 metastatic colorectal cancer (mCRC) patients as well as gDNA from 125 tumors from 75 malignant melanoma (MM) patients was available. Tissue microarrays (TMAs) containing formalin fixed paraffin embedded (FFPE) tissue from 285 of the CRC metastases from 123 patients were made. BRAF, exon 15 was amplified using the DyNazyme EXT polymerase system and sequenced by Sanger sequencing. IHC staining with the BRAF-VE1 antibody was performed on a Ventana BenchMark XT immunostainer. The intensity of staining was graded as 0 (no visible staining), grade 1 (weak diffuse background staining), 2 (moderate diffuse and granular) and 3 (strong granular staining). Grade 2 or 3 was regarded as positive. For high resolution melting analysis of V600-status, we used the LigthMix Kit BRAF V600E/K on a LightCycler 480 instrument.To establish the detection limits for the DNA-based methods used in this comparison, we analyzed a dilution series containing a ratio of 1:1- 1:107 BRAF V600E mutated DNA in BRAF wild-type DNA. We found the detection limit for the LightMix assay to be 1:1000 mutated alleles while it was 1:10 for Sanger sequencing. Among 125 samples from 75 MM patients, we found 60 samples from 31 patients to be positive for BRAF mutations by Sanger sequencing. By the LigthMix assay, we found 75 mutated samples from 37 patients. Among 395 samples form 141 mCRC patients, we found 22 samples from 7 patients to be BRAF V600E mutated by Sanger sequencing. 127 tumors from 42 of these patients were analyzed by the LigthMix assay and the BRAF V600E mutation was detected in 24 samples from the same 7 patients. IHC was performed on TMAs including tissue from 285 metastases from 123 of the mCRC patients. By this method 25 samples from 15 patients were found to be BRAF V600E mutated. These 15 patients included 5 of the 7 patients identified by DNA-based mutation analyses. Thus 8 patients revealing no mutation by either of the DNA-based methods showed positive immunostaining. Two patients found to carry the mutation by sequencing and melting point analysis were not identified by IHC. Our data show differences in sensitivity and specificity between the three methods. Compared to the LigthMix, assay Sanger sequencing showed inferior sensitivity. Further, we observed a large discrepancy between IHC and the two DNA-based methods, indicating IHC with the current antibody not to be recommendable for clinical tests alone. Citation Format: Inger Marie Loes, Heike Immervoll, Halfdan Sorbye, Jon-Helge Angelsen, Arild Horn, Per Eystein Lonning, Stian Knappskog. Performance comparison of BRAF V600E detection assays in malignant melanoma and colorectal cancer specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1878. doi:10.1158/1538-7445.AM2014-1878