BRIP1, a Gene Potentially Implicated in Familial Colorectal Cancer Type X.

Familial colorectal cancer Type X (FCCTX) comprises a heterogeneous group of families with an increased risk of developing colorectal cancer and other related tumors, but with mismatch repair-proficient, microsatellite-stable (MSS) tumors. Unfortunately, the genetic basis underlying their cancer predisposition remains unknown. Although pathogenic germline variants in BRIP1 increase the risk of developing hereditary ovarian cancer, the involvement of BRIP1 in hereditary colorectal cancer is still not well known. In order to identify new BRIP1 variants associated with inherited colorectal cancer, affected and nonaffected individuals from 18 FCCTX or high-risk MSS colorectal cancer families were evaluated by whole-exome sequencing, and another 62 colorectal cancer patients from FCCTX or high-risk MSS colorectal cancer families were screened by a next-generation sequencing (NGS) multigene panel. The families were recruited at the Genetic Counseling Unit of Hospital Clínico San Carlos of Madrid. A total of three different BRIP1 mutations in three unrelated families were identified. Among them, there were two frameshift variants [c.1702_1703del, p.(Asn568TrpfsTer9) and c.903del, p.(Leu301PhefsTer2)] that result in the truncation of the protein and are thus classified as pathogenic (class 5). The remaining was a missense variant [c.2220G>T, p.(Gln740His)] considered a variant of uncertain significance (class 3). The segregation and loss-of-heterozygosity studies provide evidence linking the two BRIP1 frameshift variants to colorectal cancer risk, with suggestive but not definitive evidence that the third variant may be benign. The results here presented suggest that germline BRIP1 pathogenic variants could be associated with hereditary colorectal cancer predisposition.Prevention Relevance: We suggest that BRIP1 pathogenic germline variants may have a causal role in CRC as moderate cancer susceptibility alleles and be associated with hereditary CRC predisposition. A better understanding of hereditary CRC may provide important clues to disease predisposition and could contribute to molecular diagnostics, improved risk stratification, and targeted therapeutic strategies.

Association Between Baseline Circulating Tumor Cells, Molecular Tumor Profiling, and Clinical Characteristics in a Large Cohort of Chemo-naïve Metastatic Colorectal Cancer Patients Prospectively Collected.

BACKGROUND: Clinicopathologic characteristics and prognostic and predictive factors offer valuable guidance when selecting optimal first-line treatment in patients with metastatic colorectal cancer (CRC). The association between baseline circulating tumor cell (bCTC) count, molecular tumor profile, and clinicopathologic features was analyzed in a chemo-naïve metastatic CRC population. PATIENTS AND METHODS: A total of 1202 patients from the Spanish VISNÚ-1 (FOLFIRINOX/bevacizumab vs. FOLFOX/bevacizumab) and VISNÚ-2 (FOLFIRI/bevacizumab vs. FOLFIRI/cetuximab; RAS-wildtype) studies were analyzed for mutational status and bCTC count. The association between clinicopathologic characteristics and bCTC count, mutational status, and microsatellite instability (MSI) was analyzed in 589 eligible patients. RESULTS: Interestingly, 41% of the population studied presented ≥3 bCTC count. bCTC count ≥3 was associated with worse performance status (according Eastern Cooperative Oncology Group scale), stage IV at diagnosis, at least 3 metastatic sites, and elevated carcinoembryonic antigen (CEA) levels; but not with RAS or BRAF mutations or high MSI. BRAFmut (BRAF mutated) tumors were associated with right-sided primary tumors, peritoneum, distant lymph node metastasis, and less frequent liver involvement. RASmut (RAS mutated) was associated with worse performance status; stage IV at diagnosis; right-sided primary tumors; liver, lung, and bone metastases; at least 3 metastatic sites; and elevated CEA, whereas PIK3CAmut (PIK3CA mutated) tumors were associated with right-sided primary tumors, high CEA serum levels, and older age. High MSI was associated with right-sided primary tumors, distant lymph nodes metastasis, and lower CEA levels. CONCLUSIONS: In our study, elevated bCTCs and RASmut were associated with clinicopathologic features known to be associated with poor prognosis; whereas the poor prognosis of BRAFmut tumors in chemo-naïve metastatic CRC is not explained by associations with poor clinicopathologic prognostic factors, except right-sided primary tumors. TRIAL REGISTRATION NUMBER: VISNU 1 ClinicalTrials.gov ID: NCT01640405/ VISNU 2 ClinicalTrials.gov ID: NCT01640444.

A phase 2 study of panitumumab with irinotecan as salvage therapy in chemorefractory KRAS exon 2 wild-type metastatic colorectal cancer patients.

BACKGROUND: Targeted agents are standard treatment for RAS wild-type metastatic colorectal cancer in the first- and second-line settings. This phase 2 study determined the benefit of targeting the epidermal growth factor receptor (EGFR) with panitumumab plus irinotecan in irinotecan-refractory patients. METHODS: KRAS exon-2 wild-type patients failing prior irinotecan received panitumumab (6 mg/kg) and irinotecan (180 mg/m²) every 2 weeks. The primary endpoint was the overall response rate (ORR). Secondary endpoints included safety, progression-free survival (PFS) and overall survival (OS). KRAS exon-2 status was evaluated centrally, along with NRAS, BRAF mutations, epiregulin, amphiregulin, PTEN and EGFR copy number status, and correlated with efficacy. RESULTS: Sixty-one patients were treated. Among the 46 wild-type RAS patients, the ORR was 15.2% (seven partial responses), with median PFS of 3.8 months (95% CI 2.7-4.3) and median OS of 12.5 months (95% CI 6.7-15.9). Wild-type BRAF patients showed a 13.0% response rate. No significant correlations between response and baseline biomarker expression were identified. Common grade 3-4 adverse events were diarrhoea and rash (18.0% each), hypomagnesaemia and asthenia (8.2% each). CONCLUSIONS: The addition of panitumumab to irinotecan as salvage therapy is feasible but has limited activity in irinotecan-refractory metastatic colorectal cancer. No biomarkers predictive of response were identified.

Novel genetic mutations detected by multigene panel are associated with hereditary colorectal cancer predisposition.

Half of the high-risk colorectal cancer families that fulfill the clinical criteria for Lynch syndrome lack germline mutations in the mismatch repair (MMR) genes and remain unexplained. Genetic testing for hereditary cancers is rapidly evolving due to the introduction of multigene panels, which may identify more mutations than the old screening methods. The aim of this study is the use of a Next Generation Sequencing panel in order to find the genes involved in the cancer predisposition of these families. For this study, 98 patients from these unexplained families were tested with a multigene panel targeting 94 genes involved in cancer predisposition. The mutations found were validated by Sanger sequencing and the segregation was studied when possible. We identified 19 likely pathogenic variants in 18 patients. Out of these, 8 were found in MMR genes (5 in MLH1, 1 in MSH6 and 2 in PMS2). In addition, 11 mutations were detected in other genes, including high penetrance genes (APC, SMAD4 and TP53) and moderate penetrance genes (BRIP1, CHEK2, MUTYH, HNF1A and XPC). Mutations c.1194G>A in SMAD4, c.714_720dup in PMS2, c.2050T>G in MLH1 and c.1635_1636del in MSH6 were novel. In conclusion, the detection of new pathogenic mutations in high and moderate penetrance genes could contribute to the explanation of the heritability of colorectal cancer, changing the individual clinical management. Multigene panel testing is a more effective method to identify germline variants in cancer patients compared to single-gene approaches and should be therefore included in clinical laboratories.

Prognostic Value of BRAF, PI3K, PTEN, EGFR Copy Number, Amphiregulin and Epiregulin Status in Patients with KRAS Codon 12 Wild-Type Metastatic Colorectal Cancer Receiving First-Line Chemotherapy with Anti-EGFR Therapy.

INTRODUCTION: Mutational analysis of RAS is required for anti-epidermal growth factor receptor (EGFR) treatment for patients with metastatic colorectal cancer (mCRC). However, most patients with KRAS wild-type tumors still do not respond. Other molecules downstream of the EGFR may also play a role in resistance to EGFR therapies. OBJECTIVE: Our objective was to investigate the clinical importance of biomarkers in relation to response, progression-free survival, and overall survival in patients with mCRC receiving first-line treatment with anti-EGFR therapy plus chemotherapy. METHODS: We studied the EGFR pathway [EGFR, NRAS, BRAF, PIK3CA, phosphatase and tensin homolog (PTEN), amphiregulin (AREG), and epiregulin (EREG)] in 105 patients with mCRC KRAS codon 12 wild type. We analysed objective response, progression-free survival, and overall survival in molecularly defined subgroups of the patients receiving anti-EGFR therapy plus chemotherapy as first-line treatment. RESULTS: We found a significant association between RAS wild-type, BRAF wild-type, EREG, and AREG overexpression and response to anti-EGFR therapy (p = 0.003, p = 0.015, p = 0.05, and p = 0.009, respectively). Progression-free survival and overall survival were lower in patients with RAS (p = 0.36 and p ≤ 0.001, respectively) or BRAF (p = 0.003 and p = 0.002, respectively) mutant tumors. Patients with EREG and AREG messenger RNA (mRNA) expression had longer survival than those with low-expression tumors; progression-free survival and overall survival were significant for AREG (p = 0.001 and p = 0.05, respectively). Patients with EGFR amplification tumors responded better to treatment and had better survival rates, although this was not significant. PIK3CA and PTEN were not associated with either response or survival. The multivariate logistic regression model for response showed only BRAF as a significant predictor after adjustment for the other covariates (p = 0.04, odds ratio 8.3, 95 % confidence interval 0.81-86.0). CONCLUSIONS: RAS, BRAF, AREG, and EREG predict for efficacy of first-line anti-EGFR therapy in patients with mCRC.

Cancer risk and overall survival in mismatch repair proficient hereditary non-polyposis colorectal cancer, Lynch syndrome and sporadic colorectal cancer.

Mismatch repair proficient hereditary non-polyposis colorectal cancer (MSS-HNPCC) encloses a heterogeneous group of families consisting of different unknown genetic syndromes and/or aggregations cases. The lack of information about the hereditability of cancer risk in these families makes it difficult to carry out an individualized Genetic Counseling. Therefore, deep description of such families becomes important for a better classification and search for underlying susceptibility causes. The aim of this study is to describe and compare the clinical, morphological features, tumor KRAS status and overall survival in MSS-HNPCC, Lynch and sporadic colorectal cancer. A total of 37 MSS-HNPCC families, 50 Lynch families and 612 sporadic CRC were included. Clinical and morphological data were evaluated by reviewing medical and pathology reports of 55, 69 and 102 tumors respectively. KRAS/BRAF status were detected by allele specific real-time PCR. Standardized incidence ratios (SIR) were calculated among 602 MSS-HNPCC relatives and 668 Lynch relatives. Main features distinguishing MSS-HNPCC were diagnosis age (55.1 ± 12.6), preferential distal location (76%), polyp detection (45%) and familial colorectal cancer incidence (SIR = 6.6). In addition, we found increased incidences rates for kidney, stomach and uterus tumors. KRAS mutation rates were similar in the study populations (48.8 ± 5.8) but higher than those described before by Sanger sequencing. MSS-HNPCC overall survival was similar to Lynch in B Dukes' stage tumors and between Lynch and sporadic in C stage tumors. Anatomical and morphological data of MSS-HNPCC are consistent with other described populations. Our studies disclose an increased HNPCC-extracolonic tumors incidence and improved overall survival in MSS-HNPCC families.

Role of Kras status in patients with metastatic colorectal cancer receiving first-line chemotherapy plus bevacizumab: a TTD group cooperative study.

BACKGROUND: In the MACRO study, patients with metastatic colorectal cancer (mCRC) were randomised to first-line treatment with 6 cycles of capecitabine and oxaliplatin (XELOX) plus bevacizumab followed by either single-agent bevacizumab or XELOX plus bevacizumab until disease progression. An additional retrospective analysis was performed to define the prognostic value of tumour KRAS status on progression-free survival (PFS), overall survival (OS) and response rates. METHODOLOGY/PRINCIPAL FINDINGS: KRAS data (tumour KRAS status and type of mutation) were collected by questionnaire from participating centres that performed KRAS analyses. These data were then cross-referenced with efficacy data for relevant patients in the MACRO study database. KRAS status was analysed in 394 of the 480 patients (82.1%) in the MACRO study. Wild-type (WT) KRAS tumours were found in 219 patients (56%) and mutant (MT) KRAS in 175 patients (44%). Median PFS was 10.9 months for patients with WT KRAS and 9.4 months for patients with MT KRAS tumours (p=0.0038; HR: 1.40; 95% CI:1.12-1.77). The difference in OS was also significant: 26.7 months versus 18.0 months for WT versus MT KRAS, respectively (p=0.0002; HR: 1.55; 95% CI: 1.23-1.96). Univariate and multivariate analyses showed that KRAS was an independent variable for both PFS and OS. Responses were observed in 126 patients (57.5%) with WT KRAS tumours and 76 patients (43.4%) with MT KRAS tumours (p=0.0054; OR: 1.77; 95% CI: 1.18-2.64). CONCLUSIONS/SIGNIFICANCE: This analysis of the MACRO study suggests a prognostic role for tumour KRAS status in patients with mCRC treated with XELOX plus bevacizumab. For both PFS and OS, KRAS status was an independent factor in univariate and multivariate analyses.

Study of KRAS new predictive marker in a clinical laboratory.

BACKGROUND: The presence of somatic mutations in the KRAS gene has been identified as a reliable strong negative predictor for the response to targeting the epidermal growth factor receptor (EGFR), in patients with metastatic colorectal cancer and the use of anti-EGFR monoclonal antibodies such as Cetuximab and Panitumumab is now restricted to patients with no detectable KRAS mutations. Between 30 and 40 % of colorectal cancers contain a mutated KRAS oncogene. The aim of this study was to evaluate concordance between three methods to analyze KRAS mutational status in regard to clinical testing. METHODS: We analyzed KRAS mutations in codons 12 and 13 of exon 2 in one hundred formalin-fixed paraffin-embedded (FFPE) colorectal cancer samples by three different methods: Direct Sequencing and two commercial kits on allele-specific oligonucleotide hybridization (KRAS StripAssay, Vienna Lab.) and Amplification Refractory Mutation System/Scorpions (ARMS/S; TheraScreen KRAS Mutation kit DxS) based on q-PCR. RESULTS: We have found similar frequencies of KRAS mutations by TheraScreen and Strip-Assay (44 and 48 %), with a κ value of 0.90, indicating almost perfect agreement between methods. The frequency by direct sequencing was much lower (26 %) and the κ values were 0.67 (compared to TheraScreen) and 0.57 (compared to Strip-Assay) indicating low sensitivity. CONCLUSIONS: On analyzing KRAS mutation in FFPE tumor samples, direct sequencing sensitivity is too low to be used in a clinical setting. Choosing between ARMS/S; TheraScreen KRAS Mutation kit DxS and KRAS StripAssay, Vienna Lab, will depend on laboratory facilities and expertise.

Influence of KRAS p.G13D mutation in patients with metastatic colorectal cancer treated with cetuximab.

BACKGROUND: Patients with metastatic colorectal cancer (mCRC) with activating mutations at codon 12 or 13 of the KRAS gene are currently excluded from treatment with monoclonal antibodies against the epidermal growth factor receptor (EGFR), for example, cetuximab. Occasionally, some of these patients benefit from treatment with cetuximab, especially patients with a mutation at codon 13. We conducted an analysis to study the influence of the KRAS p.G13D mutation in patients with mCRC who were treated with cetuximab. MATERIALS AND METHODS: We analyzed the KRAS mutation status of 110 patients who were treated with cetuximab between September 2003 and October 2008 at Hospital Clínico, San Carlos. We compared progression-free survival, overall survival, and response rate according to KRAS mutation status. RESULTS: Patients with mutations at codon 13 compared with those with other KRAS mutations showed no statistically significant differences in progression-free survival (4.96 months [95% CI, 3.04-6.89 months] vs. 3.10 months [95% CI, 1.58-4.61 months]; hazard ratio [HR] 0.88 [95% CI, 44-1.75]; P = .72) and overall survival (8.2 months [95% CI, 4.2-12.1 months] vs. 14.6 months [95% CI, 8.0-21.2 months]; HR 0.50 [95% CI, 0.23-1.09]; P = .084). Patients with KRAS wild-type tumors have a longer progression-free survival (7.30 months [95% CI, 4.48-10.12 months]; HR 0.46 [95% CI, 0.23-0.91]; P = .025) and overall survival (19.0 months [95% CI, 10.2-27.8 months]; HR 0.32 [95% CI, 0.15-0.69]; P = .004) than patients with p.G13D-mutated tumors. Differences in the response rate were not observed between groups. CONCLUSION: Patients with mCRC and mutation at codon 13 of the KRAS gene do not appear to benefit from treatment with cetuximab. These results support the current clinical practice.

Endogenous antioxidant defence system in rat liver following mercury chloride oral intoxication.

Mercury is a highly toxic metal which induces oxidative stress. Superoxide dismutases, catalase, and glutathion peroxidase are proteins involved in the endogenous antioxidant defence system. In the present study rats were administered orally, by gavage, a single daily dose of HgCl2 for three consecutive days. In order to find a relation between the proteins involved in the antioxidant defence and mercury intoxication, parameters of liver injury, redox state of the cells, as well as intracellular protein levels and enzyme activities of Mn-dependent superoxide dismutase (MnSOD), Cu-Zn-dependent superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPx) were assayed both in blood and in liver homogenates. HgCl2 at the doses of 0.1 mg/kg produced liver damage which that was detected by a slight increase in serum alanine aminotransferase and gamma glutamyl transferase. Hepatic GSH/GSSG ratio was assayed as a parameter of oxidative stress and a significant decrease was detected, as well as significant increases in enzyme activities and protein levels of hepatic antioxidant defence systems. Changes in both MnSOD and CuZnSOD were parallel to those of liver injury and oxidative stress, while the changes detected in catalase and GPx activities were progressively increased along with the mercury intoxication. Other enzyme activities related to the glutathione redox cycle, such as glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), also increased progressively. We conclude that against low doses of mercury that produce a slight oxidative stress and liver injury, the response of the liver was to induce the synthesis and activity of the enzymes involved in the endogenous antioxidant system. The activities of all the enzymes assayed showed a rapidly induced coordinated response.

Relationship between expression of HSP70 and metallothionein and oxidative stress during mercury chloride induced acute liver injury in rats.

Mercury is a highly toxic metal which induces oxidative stress. Metallothionein and heat shock protein 70 (HSP70) are stress proteins involved in response to different stimuli. In the present study rats were administered per oral application by gavage, a single daily dose (0.1 mg/kg) of HgCl(2) for 3 consecutive days. To find a relation between these two stress proteins and mercury, parameters of liver injury, redox state of the cells, and the expression and protein levels of HSP70 and metallothionein by Northern and Western blot analysis were assayed either in blood or in liver. HgCl(2) at the doses of 0.1 mg/kg induced liver injury detected by a slight increase in serum aspartate aminotransferase and alkaline phosphatase activities and by the enhanced levels of bilirubin. Oxidative stress was detected by a significant decrease in protein-SH and an increase in thiobarbituric acid reactive substances in liver following one dose of mercury. mRNA and protein levels of both metallothionein and HSP70 increased progressively from first to third doses of mercury. We conclude that against low doses of mercury that produce a slight liver injury and oxidative stress, the liver rapidly responds by inducing the expression of metallothionein and HSP70. We suggest that metallothionein induction attenuates the decrease in protein-SH induced by the first dose of mercury, since metallothionein increases the pool of thiol groups in the cytosol eliminating oxygen radicals and inhibiting lipid peroxidation. From these results we can suggest that the changes observed in these stress proteins by the effect of mercury appear to be a response rapidly induced at transcriptional and at translational levels.