Blood levels of immune cells predict survival in myeloma patients: results of an Eastern Cooperative Oncology Group phase 3 trial for newly diagnosed multiple myeloma patients.

Previously, it was reported that patients with multiple myeloma (MM) who have higher baseline levels of blood CD4(+) or CD19(+) cells have longer survival. This article extends the analysis of immune cell levels and survival in a large cohort (N = 504) of patients with MM entered on Eastern Cooperative Oncology Group (ECOG) phase 3 trial (9486). Newly diagnosed patients with MM received 2 cycles of vincristine, bischloroethylnitrosourea, melphalan, cytoxan, prednisone (VBMCP) and were treated on one of 3 randomized arms: VBMCP with either interferon or high-dose cyclophosphamide, or VBMCP alone. Blood immune cell levels were studied at trial entry (baseline), after 2 cycles of chemotherapy, after 2 years of therapy, and at relapse. Baseline CD3(+), CD4(+), CD8(+), CD19(+), and CD4(+) subset cell levels were all positively associated with survival (P =.0087 to P <.0001). A multivariate analysis incorporating CD4(+) and CD19(+) cell levels defined 3 separate groups of patients with MM to survival outcome. Higher CD19(+) blood levels were positively associated with MM-patient survival at entry to the study, at year 2, and at relapse (P <.0001 at all 3 timepoints). Patients with MM had evidence of immune cell reconstitution after 2 years of therapy, but the rate and extent of recovery was greater for CD8(+), which was greater than CD4(+), which was greater than CD19(+). This latter data affirms the positive relationship between the quantitative status of the blood immune system in MM and survival. In addition, the importance of the CD19(+) blood cells to survival is evident throughout the course of MM. Therapeutic efforts to maintain an intact immune system may be crucial in maximizing chemotherapeutic and/or immunotherapy efforts in this disease.     

Mutational activation of N- and K-ras oncogenes in plasma cell dyscrasias

The frequency of N- and K-ras oncogene mutations was investigated in plasma cell dyscrasias. Genomic DNAs from 128 patients were selected for this study: 30 monoclonal gammopathies of undetermined significance, 8 solitary plasmacytomas, 77 multiple myelomas (MM), and 13 plasma cell leukemias (PCL). A two-step experimental approach was devised. All samples were screened for mutations by single-strand conformation polymorphism analysis. DNA fragments displaying an altered electrophoretic mobility were further studied by direct sequencing to confirm and characterize the nature of the mutations. Ras mutations are not randomly distributed because they are detectable only in MM (9%) and PCL (30.7%). N-ras codons 12, 13, and 61 and K-ras codon 12 were found to be mutated, but N-ras codon 61 mutation was the most frequent finding (63.6%). In conclusion, ras mutations were found in PCL, and in a subset of MM characterized by advanced-stage disease and adverse prognostic parameters. Furthermore, based on our findings, it is possible to speculate that ras mutations represent a late molecular lesion in the process of multistep carcinogenesis.     

Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation

Minimal residual disease (MRD) assessment is standard in many hematologic malignancies but is considered investigational in multiple myeloma (MM). We report a prospective analysis of the prognostic importance of MRD detection by multiparameter flow cytometry (MFC) in 295 newly diagnosed MM patients uniformly treated in the GEM2000 protocol VBMCP/VBAD induction plus autologous stem cell transplantation [ASCT]). MRD status by MFC was determined at day 100 after ASCT. Progression-free survival (PFS; median 71 vs 37 months, P < .001) and overall survival (OS; median not reached vs 89 months, P = .002) were longer in patients who were MRD negative versus MRD positive at day 100 after ASCT. Similar prognostic differentiation was seen in 147 patients who achieved immunofixation-negative complete response after ASCT. Moreover, MRD(-) immunofixation-negative (IFx(-)) patients and MRD(-) IFx(+) patients had significantly longer PFS than MRD(+) IFx(-) patients. Multivariate analysis identified MRD status by MFC at day 100 after ASCT as the most important independent prognostic factor for PFS (HR = 3.64, P = .002) and OS (HR = 2.02, P = .02). Our findings demonstrate the clinical importance of MRD evaluation by MFC, and illustrate the need for further refinement of MM re-sponse criteria. This trial is registered at http://clinicaltrials.gov under identifier NCT00560053.     

Infrequent point mutations in codons 12 and 61 of ras oncogenes in human hepatocellular carcinomas.

DNA from human hepatocellular carcinomas (HCC) were analysed for the presence of mutations in codons 12 and 61 of the K-ras, H-ras and N-ras genes. The relevant ras sequences were amplified in vitro using the polymerase chain reaction and point mutations detected by selective hybridisation using mutation-specific synthetic oligonucleotides. In one of the 19 HCCs a mutation in codon 61 of the K-ras gene was detected, whilst in 3/19 HCCs a mutation was found in codon 61 of the N-ras gene. The mutations were all heterozygous A-T transversions and were found in HCCs arising in patients with underlying cirrhosis. In two of these patients where the corresponding normal tissue was available only the wild-type ras gene was detected, indicating that oncogenic activation of the ras gene was a consequence of somatic mutation. In another patient the same mutation in codon 61 of the N-ras gene was found in cirrhotic liver tissue and in all four patients the same mutation was also detected in formalin-fixed, paraffin-embedded liver biopsy HCC tissue obtained at diagnosis. These results indicate that mutational activation of the ras genes at codon 61 is an infrequent but possibly early event in the development of HCC in Britain.     

Mutations of the ras proto-oncogenes in childhood monosomy 7

ras gene mutations are the most frequent molecular changes found in the preleukemic syndromes of adults and may play a role in initiating these diseases and in their progression to acute leukemia. However, little is known about the incidence or importance of these genetic mutations in childhood myeloproliferative states (MPS). The bone marrow (BM) monosomy 7 syndrome accounts for a large percentage of childhood MPS. Although the duration of the MPS is quite variable, children with monosomy 7 eventually develop acute myeloid leukemia (AML). We investigated 20 children (13 with MPS, 7 with AML) with BM monosomy 7 or 7q- for the presence of ras gene mutations using the polymerase chain reaction and hybridization with mutation-specific oligonucleotides. Mutations of N-ras and K-ras were detected in three children. Two patients carrying a ras mutation were in the myeloproliferative phase, and one had acute leukemia. All three patients with ras mutations either died of their disease or relapsed after BM transplantation as compared with 8 of 17 without ras mutations. However, this difference is not statistically significant (P = .14, not significant). We conclude that ras mutations are observed in childhood monosomy 7, though less frequently than in adult MDS, and may play a limited role in the progression of this disease to acute leukemia. More patients are needed to address the prognostic role of ras mutations in this rare disease.     

Clinical significance of K-ras and c-erbB-2 mutations in pancreatic adenocarcinoma and chronic pancreatitis

Background: The differentiation of chronic pancreatitis (CP) from pancreatic adenocarcinoma (PA) remains the great challenge for clinicians. The purpose of this study was to compare the prevalence of K-ras and c-erbB-2 mutations in PA and CP in order to evaluate their usefulness in differential diagnosis of those diseases. Methods: The study included 49 patients who underwent Whipple resection or distal pancreatectomy for pancreatic adenocarcinoma (26 subjects) or chronic pancreatitis (23 subjects). DNA from pancreatic tissue was analyzed for K-ras codon 12 and c-erbB-2 mutations with PCR amplifications. Results: The K-ras gene mutation has been shown in 20 (76.9%) PA cases and in 8 (34.8%) CP cases (p<0.01). Prevalence of c-erbB-2 amplification in patients with PA was 17 (65.3%), which was not different from CP, 16 (56.5%) (p=0.58). There was a significant correlation between K-ras mutation and lymph node metastases (p=0.025) as well as between K-ras mutation and G3 tumor differentiation (p=0.037). Overall median survival in patients with PA was 9.5 mo. There was no relationship between presence of K-ras (p=0.58) or c-erbB-2 (p=0.17) mutation and survival time in PA patients. Conclusion: Those results may indicate that both K-ras and c-erbB-2 play a role in pancreatic carcinogenesis, however only K-ras may provide an additional tool in differential diagnosis of CP and PC.     

Comethylation of p16 and MGMT genes in colorectal carcinoma： Correlation with clinicopathological features and prognostic value

AIM: To investigate the significance of p16 and O6- methylguanine-DNA methyltransferase (MGMT) genes promoter hypermethylation and K-ras mutations on colorectal tumorigenesis and progression. METHODS: p16 and MGMT methylation status was examined on 47 tumor samples, and K-ras mutational status was examined on 85 tumor samples. For methylation analysis, a methylation specific PCR (MS-PCR) method was used. RESULTS: p16 and MGMT promoter methylation was found in 51% (24/47) and 43% (20/47) of CRCs, respectively, and the K-ras mutation was found in 44% (37/85) of CRCs. Comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease within a two-year period of observation. Only 27% of patients with simultaneous p 16 and MGMT methylation showed the detectible occurrence of metastasis and/or death, compared to 67% of patients without double methylation or with no methylation (3/11 vs 22/33, P < 0.05, χ2-test). In addition, p16 and MGMT comethylation showed a trend toward an association with longer survival in patients with CRCs (35.5 ± 6.0 mo vs 23.1 ± 3.2 mo, P = 0.072, Log-rank test). Progression of the disease within a two-year period was observed in 66% of patients carrying the K-ras mutation, compared to only 19% of patients with wild type K-ras (29/44 vs 7/37, P < 0.001, χ2-test). The presence of the K-ras mutation significantly correlated to shortened overall survival (20.0 ± 1.9 mo vs 37.0 ± 1.8 mo, P < 0.001, Log-rank test). The comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease even when K-ras mutations were included in the analysis as an independent variable. CONCLUSION: Our data suggest that comethylation of promoters of p 16 and MGMT genes could have a prognostic value in patients with CRC. Specifically, concurrent methylation of both genes correlates with better prognosis.     

N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome

To evaluate the clinical significance of N-ras mutations in the myelodysplastic syndrome (MDS) archival bone marrow samples from 252 patients were studied for the presence of N-ras exon I mutations using polymerase chain reaction amplification and differential oligonucleotide hybridization. Subsequently, clinical information about these patients was obtained and analyzed. Of 220 evaluable patients, 20 (9%) had point mutation of N-ras involving codon 12. Individuals with N- ras mutation had a significantly shorter survival period than those who were N-ras negative (P = .02). An increased risk of acute myelogenous leukemia (AML) was also found in patients with N-ras mutations (P = .005). N-ras mutations were not associated with any French-American- British (FAB) subtype, with the presence of increased myeloblasts, or with chromosomal aberrations in the bone marrow. However, the presence of increased bone marrow blasts was strongly associated with poor survival rate and risk of AML (P < .001 for each). After stratifying for the percentage of blasts, N-ras mutations remained significantly associated with shorter survival period (P = .04) and increased risk of AML (P = .02). Bone marrow cytogenetic abnormalities, particularly when multiple abnormalities were present, were significantly associated with a poor prognosis (P < .001). In conclusion, N-ras mutation, although relatively infrequent in MDS, is associated with short survival period and increased probability of developing AML.     

Detection of ras gene mutations of primary hepatic malignant tumors by polymerase chain reaction and direct sequencing method

Ras genes are converted to active oncogenes by point mutations occurring in either codon 12, 13 or 61. We used polymerase chain reaction and direct sequence method for the analysis of these mutations. We examined 13 hepatocellular carcinomas, 8 cholangiocarcinomas, 2 hepatoblastomas and 1 biliary cystadenocarcinoma. Of these tumors, ras gene mutations were detected solely in cholangiocarcinomas. Cholangiocarcinoma showed a high incidence of K-ras gene mutation. Among 8 patients with cholangiocarcinoma, the mutation was detected at codon 12 in 4 and at codon 61 in 1. The incidence of K-ras gene mutation was especially high in the hilar type of cholangiocarcinoma as compared with the peripheral type.     

Central nervous system multiple myeloma – potential roles for intrathecal therapy and measurement of cerebrospinal fluid light chains

Central nervous system (CNS) multiple myeloma (MM) is exceedingly rare and portends a dismal prognosis. While immunomodulators have contributed to the improvement in survival in MM, they appear to have limited activity against CNS MM and, paradoxically, may contribute to the evolution of resistant MM clones capable of surviving within the CNS. We undertook a retrospective analysis to characterize the features of CNS MM and outcome in 17 patients from four institutions identified between 2000 and 2011. The median age was 58 years. Patients had received a median of three prior therapies and all had been exposed to at least one of the so‐called novel anti‐MM agents before the diagnosis of CNS MM. The median time to CNS disease from initial diagnosis was 36 months. Cerebrospinal fluid (CSF) light chain measurements produced discrepant results to serum light chain measurements in some patients. Treatments included systemic pharmacotherapy, intrathecal (IT) chemotherapy and/or radiotherapy (RT). The median overall survival (OS) from diagnosis of CNS MM was only 4 months. OS was significantly better in patients who received IT chemotherapy (20 months vs. 2 months, respectively; P < 0·02). We conclude that the systematic evaluation of IT therapy and diagnostic utility of CSF light chain measurements in CNS MM are warranted.     

K-ras codon 12 mutations in Barrett's oesophagus and adenocarcinomas of the oesophagus and oesophagogastric junction

BACKGROUND: Activation of the ras oncogene is commonly found in gastrointestinal tract cancers, but the role of ras in the development and progression of Barrett's oesophagus and associated cancers is uncertain. METHODS: The frequency of K-ras codon 12 point mutations was assessed in 52 paraffin-embedded tissues from 44 patients with oesophageal pathology. The specimens were classified pathologically as follows: adenocarcinoma of the oesophagus or oesophagogastric junction (n = 23), Barrett's high-grade dysplasia (n = 5), low-grade dysplasia (n = 14), intestinal metaplasia (n = 4), normal oesophagus (n = 5) or normal stomach (n = 1). DNA was extracted from three consecutive sections of each paraffin block and mutations at bases 1 and 2 of K-ras codon 12 were identified using a novel restriction endonuclease-mediated selective polymerase chain reaction method. RESULTS: Mutations were found in 7 of 23 (30.4%) adenocarcinomas and in 2 of 5 (40%) high-grade dysplasia specimens. No mutations were found in specimens of low-grade dysplasia, intestinal metaplasia without dysplasia, or normal oesophagus and stomach. There were no significant associations between the presence of mutations and clinicopathologic features in the patients with cancer. One patient who progressed from low-grade to high-grade dysplasia was found to have developed mutant K-ras in the course of this transformation. CONCLUSION: These results suggest that K-ras codon 12 mutations may occur frequently in patients with Barrett's oesophagus with high-grade dysplasia or adenocarcinoma of the oesophagus or oesophagogastric junction. K-ras mutation may be a late event in the Barrett's metaplasia-dysplasia-adenocarcinoma sequence.     

Clinical application of ras gene mutation for diagnosis of pancreatic adenocarcinoma

Most pancreatic adenocarcinomas are known to have ras gene (oncogene) mutations. The site of the mutations is localized in codon 12 of K-ras gene. Such high incidence and localization of the ras gene mutations have not been observed in any other human malignancies. Polymerase chain reaction and direct sequencing method enabled us to analyze DNA sequence around codon 12 of K-ras gene in small quantities of specimens obtained from needle biopsies and aspirate samples for pathological diagnosis. All the materials obtained from 12 patients with pancreatic adenocarcinoma showed the mutations, whereas those obtained from 6 patients with chronic pancreatitis showed no mutations. In several cases using the mutations of K-ras gene as a marker, this analysis supplemented conventional pathology and cytology in making the diagnosis of pancreatic adenocarcinoma. The analysis of ras-gene mutations was useful for the clinical diagnosis of pancreatic adenocarcinoma.     

Tumor angiogenesis in chronic pancreatitis and pancreatic adenocarcinoma: impact of K-ras mutations

Chronic pancreatitis (CP) is one condition in which epidemiologic studies have demonstrated a definite association with pancreatic adenocarcinoma (PAC). The pathophysiologic and molecular events that either predispose to the development of, or potentiate the growth of, PAC are unknown. Mutation of the codon 12 K-ras gene is one genetic aberration commonly associated with development of PAC. Tumor angiogenesis, or microvascular proliferation of new capillaries, is another pathophysiologic alteration associated with PAC. Although activated ras oncogenes modulate tumor angiogenesis/neovascularization in some tumors, the importance of tumor angiogenesis and the role of K-ras mutation in regulating angiogenesis in CP and PAC are unknown. The aim of this study was to elucidate the relationship between angiogenesis and K-ras mutations in CP and PAC. Tumor angiogenesis and K-ras mutations were evaluated in resected specimens from 25 CP (23 CP plus two CP with PAC) and 16 PAC patients. Tumor angiogenesis was determined using immunohistochemistry of factor VIII-related antigen (FVIIIRAg) and ras mutations were identified by enriched-nested polymerase chain reaction. The mean number of FVIIIRAg-positive blood vessels was significantly (p < 0.005) higher in PAC (23.0 +/- 7.5), CP with a mutant K-ras genome (17.7 +/- 2.8) and CP with a normal K-ras genome (6.5 +/- 3.8), compared to unaffected areas. Codon 12 K-ras mutations were detected in three of 25 CP specimens (12%) and in 15 of 16 PAC specimens (94%). In CP patients with mutant K-ras in their genome, microvessel density was significantly (p < 0.01) elevated, compared to patients with a normal K-ras genome. Statistical analyses (Spearman rank-difference correlation coefficient, Student t test, and chi2 analysis) indicated a significant association between codon 12 K-ras mutations and tumor angiogenesis in both CP and PAC. This study demonstrates a significant association between angiogenesis and K-ras mutation in both PAC and CP. At a minimum, K-ras mutation is associated with the events that increase angiogenesis and it may potentiate or promote tumor angiogenesis.     

Cholangiocarcinoma in primary sclerosing cholangitis: K-ras mutations and Tp53 dysfunction are implicated in the neoplastic development

BACKGROUND/AIM: Cholangiocarcinoma is a feared complication of primary sclerosing cholangitis (PSC). Neoplastic bile duct strictures may be difficult to differentiate cholangiographically from the non-neoplastic bile duct irregularities characteristic of this disorder, and the diagnosis of cholangiocarcinoma may be difficult to establish with certainty, even in tissue samples. Thus, new methods which can improve the diagnostic accuracy of cholangiocarcinoma in PSC are needed. METHODS: We investigated the occurrence of K-ras codon 12 and 13 mutations, p53 protein accumulation, and Ki-67 expression in tumor tissue from PSC patients (n=33) who had developed cholangiocarcinoma, using bile duct specimens exised at liver transplantation of PSC patients without cholangiocarcinoma (n=15) as controls RESULTS: K-ras mutations were present in 11 (33%) of the cholangiocarcinoma samples and significantly more frequent in females. Nine tumors carried a codon 12 mutation, and 2 had a codon 13 mutation. The most frequent substitutions in codon 12 were GGT-->GAT (n=5) and GGT-->TGT (n=3). None of the control bile ducts had K-ras mutations. p53 protein was accumulated in 10 (31%) of the tumors, as opposed to negative findings in all the control samples. Sixteen (48%) tumors revealed either K-ras mutation or p53 accumulation. Ki-67 positivity was significantly higher in cholangiocarcinomas than in the non-neoplastic bile ducts (median 29% vs 12%, respectively; p=0.011). CONCLUSION: We conclude that K-ras mutations and p53 dysfunction are implicated in tumorigenesis of cholangiocarcinomas arising in PSC patients and that these abnormalities together with increased Ki-67 index may indicate neoplastic progression of bile ducts in these patients.     

Mutation of Ki-ras and N-ras oncogenes in myelodysplastic syndromes

Somatic mutation of the N-ras oncogene occurs frequently in de novo acute myeloid leukemia (AML). By virtue of their relation to AML, myelodysplastic syndromes (MDS) provide an in vivo model of human leukemogenesis. By using a strategy for analysis of gene mutation based on in vitro amplification of target sequences by the polymerase chain reaction (PCR) and selective oligonucleotide hybridization we analyzed the mutational status of codons 12, 13, and 61 of Ha-ras, K-ras, and N- ras in peripheral blood (PB) and/or bone marrow (BM) in 34 cases of primary MDS. Mutations at codon 12 of Ki-ras or N-ras were detected in three cases (9%): one of six cases of refractory anemia with excess blasts (RAEB) and two of nine cases of chronic myelomonocytic leukemia (CMML). The nucleotide substitution differed in each. In all cases the mutant allele was detectable in PB cells. A sustained hematologic remission was achieved after low-dose cytarabine therapy in the case of RAEB. Neither case of CMML exhibited signs of disease progression during follow-up at 7 and 12 months. In contrast, four of 31 patients without the ras mutation underwent transformation to AML within 12 months of genetic analysis. We conclude that ras mutations in MDS are heterogeneous and may develop at an early stage during the evolution of MDS. Their detection in PB cells illustrates the potential utility of ras mutation as a clonal marker in myeloid malignancy.     

Analysis of ras gene mutations in childhood myeloid leukaemia

Previous studies have shown that approximately 30% of adult acute myeloid leukaemias and 20% of adult acute lymphoid leukaemias contain point mutated ras oncogenes. In order to assess whether ras oncogenes are also involved in childhood leukaemias, we have used polymerase chain reaction (PCR) amplification and synthetic oligonucleotide probes to study the nature and frequency of ras gene mutations in childhood leukaemias, concentrating largely on the acute myeloid leukaemias (AML). Thirty-four childhood presentation AML DNAs were screened for mutations in and around codons 12, 61 and 117 of N-, K- and H-ras. Eight of these samples (24%) contained ras mutations. As in the adult disease, the gene predominantly involved was N-ras (6/8), with occasional activation of K-ras (2/6). The most common base change was a G----A transition at codon 12 or 13 (4/8). Of the patients with mutant ras, 4/8 were diagnosed as AML FAB subtype M5. Five of the 34 childhood AMLs analysed displayed abnormalities of chromosome 7. However, none of these cases contained a mutant ras gene. One AML patient was studied at relapse, 14 months after initial presentation. The presentation mutation (N61p3) was not detectable, although a new mutation (N13Cys) was readily identified. This observation extends our original finding with presentation and relapse samples of adult AML, in which it was uncommon for the relapse sample to contain the same ras mutation as the presentation DNA. In addition, two out of five patients diagnosed as juvenile CML, were found to harbour mutant ras.     

Prevalence of Ras mutations in thyroid neoplasia

OBJECTIVE: Mutations at codons 12, 13 or 61 of ras which result in constitutive activation occur frequently in human malignancies. There have been varied reports on their prevalence and hence their likely significance in the pathogenesis of primary thyroid neoplasia. To address this, we have examined a large series of benign and malignant thyroid tumours for ras mutations. DESIGN: Genomic DNA was analysed for the presence of mutations at codons 12, 13 and 61 of H-ras, K-ras and N-ras by allele-specific oligonucleotide hybridization. Direct DNA sequencing was used to confirm the mutations. PATIENTS: A total of 90 samples with benign (66) and malignant (24) thyroid disease were investigated. RESULTS: A total of 14/90 (15.5%) samples had a ras mutation. All mutations were at codon 61 of either N-ras or K-ras. The positive cases were 1/25 (4%) nodular goitre, 7/38 (18%) follicular adenoma, 4/9 (44%) follicular carcinoma, 1/1 anaplastic carcinoma, 1/1 follicular variant of papillary carcinoma, and 1 metastatic follicular carcinoma in which the primary tumour had the same mutation. CONCLUSIONS: Our data demonstrate a relatively low overall prevalence of ras mutations in thyroid neoplasia, with a predominance in follicular neoplasms. Their presence in follicular adenomas suggests that they may have an early aetiological role in the development of thyroid neoplasia.     

Unfavourable prognosis associated with K-ras gene mutation in pancreatic cancer surgical margins

BACKGROUND: Despite intent to cure surgery with negative resection margins, locoregional recurrence is common in pancreatic cancer. AIMS: To determine whether detection of K-ras gene mutation in the histologically negative surgical margins of pancreatic cancer reflects unrecognised disease. PATIENTS: Seventy patients who underwent curative resection for pancreatic ductal adenocarcinoma were evaluated. METHODS: All patients had surgical resection margins (pancreatic transection and retroperitoneal) that were histologically free of invasive cancer. DNA was extracted from these paraffin embedded surgical margins and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation at codon 12. Detection of K-ras mutation was correlated with standard clinicopathological factors. RESULTS: K-ras mutation was detected in histologically negative surgical margins of 37 of 70 (53%) patients. A significant difference in overall survival was demonstrated between patients with margins that were K-ras mutation positive compared with negative (median 15 v 55 months, respectively; p = 0.0008). By univariate and multivariate analyses, detection of K-ras mutation in the margins was a significant prognostic factor for poor survival (hazard ratio (HR) 2.8 (95% confidence interval (CI) 1.5-5.3), p = 0.0009; and HR 2.8 (95% CI 1.4-5.5), p = 0.004, respectively). CONCLUSIONS: Detection of cells harbouring K-ras mutation in histologically negative surgical margins of pancreatic cancer may represent unrecognised disease and correlates with poor disease outcome. The study demonstrates that molecular-genetic evaluation of surgical resection margins can improve pathological staging and prognostic evaluation of patients with pancreatic ductal adenocarcinoma.