Superior Overall Survival in Patients with Colorectal Cancer, Regular Aspirin Use, and Combined Wild-Type PIK3CA and KRAS-Mutated Tumors.

The impact of aspirin use after the diagnosis of colorectal cancer is unknown. Among others, PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) mutational status was proposed as a molecular biomarker for the response to adjuvant aspirin therapy. However, prognostic data on aspirin use after a colorectal cancer diagnosis in relation to KRAS mutational status is limited. In a single-center retrospective study, we obtained KRAS and PIK3CA mutational status in a cohort of 153 patients with a first diagnosis of colorectal cancer receiving tumor surgery with curative intent. PIK3CA mutational status was determined by pyrosequencing, and KRAS mutational status was determined by next-generation sequencing. Clinicopathological data and survival data were assessed using patient records and reporting registers. We observed a significant 10-year overall survival benefit in patients with aspirin use and combined wild-type PIK3CA and mutated-KRAS tumors (HR = 0.38; 95% CI = 0.17-0.87; p = 0.02), but not in patients without aspirin use. Our data indicate a benefit of aspirin usage particularly for patients with combined wild-type PIK3CA and mutated-KRAS tumor characteristics.

A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing.

BACKGROUND: KRAS mutation testing is mandatory in the management of metastatic colorectal cancer prior to treatment with anti-EGFR antibodies as patients whose tumors express mutant KRAS do not benefit from these agents. Although the U.S. Food and Drug Administration has recently approved two in-vitro diagnostics kits for determination of KRAS status, there is generally no consensus on the preferred method and new tests are continuously being developed. Most of these techniques focus on the hotspot mutations at codons 12 and 13 of the KRAS gene. METHODS: We describe a two-step approach to KRAS codon 12/13 mutation testing involving high resolution melting analysis (HRM) followed by pyrosequencing using the Therascreen KRAS Pyro kit (Qiagen) of only those samples that are not clearly identified as KRAS wildtype or mutant by HRM. First, we determined KRAS status in a panel of 61 colorectal cancer samples using both methods to compare technical performance and concordance of results. Subsequently, we evaluated practicability and costs of our concept in an independent set of 120 colorectal cancer samples in a routine diagnostic setting. RESULTS: HRM and pyrosequencing appeared to be equally sensitive, allowing for clear detection of mutant alleles at a mutant allele frequency ≥12.5 %. Pyrosequencing yielded more exploitable results due to lower input requirements and a lower rate of analysis failures. KRAS codon 12/13 status was called concordantly for 98.2 % (56/57) of all samples that could be successfully analysed by both methods and 100 % (19/19) of samples that were identified mutant by HRM. Reviewing the actual effort and expenses for KRAS mutation testing in our laboratory revealed, that the selective use of pyrosequencing for only those samples that could not be analysed by HRM increased the fraction of valid results from 87.5 % for HRM alone to 99.2 % (119/120) while allowing for a net reduction of operational costs of >75 % compared to pyrosequencing alone. CONCLUSIONS: Combination of HRM and pyrosequencing in a two-step diagnostic procedure constitutes a reliable and economic analysis platform for KRAS mutation testing in colorectal cancer in a clinical setting.

Oncogenic NRAS Primes Primary Acute Myeloid Leukemia Cells for Differentiation.

RAS mutations are frequently found among acute myeloid leukemia patients (AML), generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA) and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1) in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC) driven differentiation. Taken together, our findings show that AML with inv(16) and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.

Monitoring of acute myeloid leukemia patients after allogeneic stem cell transplantation employing semi-automated CD34+ donor cell chimerism analysis.

Determination of donor chimerism profiles in blood or bone marrow from patients with allogeneic stem cell transplantation (SCT) is useful for monitoring engraftment or predicting relapse, when specific molecular markers are lacking. CD34+ donor cell chimerism (DCC) analysis in peripheral blood samples from CD34+ acute myeloid leukemia (AML) and myleodysplastic syndrome (MDS) patients proved to be a highly sensitive diagnostic tool that is useful to detect imminent relapse significantly earlier compared to total white blood cell donor cell chimerism monitoring. However, flow-cytometric enrichment of CD34+ cells requires high efforts to human resources and equipment. We present a novel semi-automated CD34+ DCC analysis procedure-employing a magnetic cell-enrichment device, DNA extraction, and short tandem repeat profiling-without the need for flow-cytometric cell sorting. Monitoring 85 patients with AML and MDS over a period of 4 years 24 relapses were detected. Semi-automated peripheral blood CD34+ DCC was diminished below 80 % in all cases of systemic relapse. Significant decrease of the CD34+ DCC value was detected 29-42 days before overt cytological relapse. Our method provides a rapid and sensitive tool for monitoring AML and MDS patients after allogeneic SCT with regard to engraftment and early detection of relapse. Here, we propose a novel semi-automated procedure for CD34+ DCC analysis after allogeneic SCT that is simple, reliable, and therefore applicable in all hematologic laboratories.

Oncogenic RAS enables DNA damage- and p53-dependent differentiation of acute myeloid leukemia cells in response to chemotherapy.

Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.

Expression profiling reveals specific gene expression signatures in gastric MALT lymphomas.

The purpose of this study is to identify genes that are involved in the etiology of Helicobacter pylori induced gastric MALT lymphoma. We compared gene expression profiles of gastric MALT lymphoma with their corresponding gastric MALT (chronic gastritis with formation of follicles and aggregates). cDNA microarrays were used to compare these two tissue types from the same patient (n = 21). Quantitative PCR and immunohistochemical staining were performed to validate the microarray results. Three hundred and fifty eight out of 11,552 genes were differentially expressed between gastric MALT lymphomas and gastric MALT. Thirty eight genes are implicated in immune response, 66 in signal transduction and 36 in cell proliferation. Interestingly, chromosome 6 was the only chromosome which was significantly over-represented with 25 genes (EASE score p = 0.01254). Several surface markers of haematopoietic cells, such as CD1c, CD40, CD44, CD53, CD83, CD86 and members of the HLA-D family were up-regulated in lymphoma tissues, indicating antigen-dependent survival of lymphoma cells. We conclude that gastric MALT lymphoma shows a specific gene expression profile, which allows the differentiation from H. pylori induced lymphoid gastritis.