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The Japanese guidelines for the testing of KRAS mutations in colorectal cancer have been used for the past 5 years. However, new findings of RAS (KRAS/NRAS) mutations that can further predict the therapeutic effects of anti‐epidermal growth factor receptor (EGFR) antibody therapy necessitated a revision of the guidelines. The revised guidelines included the following five basic requirements for RAS mutation testing to highlight a patient group in which anti‐EGFR antibody therapy may be ineffective: First, anti‐EGFR antibody therapy may not offer survival benefit and/or tumor shrinkage to patients with expanded RAS mutations. Thus, current methods to detect KRAS exon 2 (codons 12 and 13) mutations are insufficient for selecting appropriate candidates for this therapy. Additional testing of extended KRAS/NRAS mutations is recommended. Second, repeated tests are not required for the detection; tissue materials of either primary or metastatic lesions are applicable for RAS mutation testing. Evaluating RAS mutations prior to anti‐EGFR antibody therapy is recommended. Third, direct sequencing with manual dissection or allele‐specific PCR‐based methods is currently applicable for RAS mutation testing. Fourth, thinly sliced sections of formalin‐fixed, paraffin‐embedded tissue blocks are applicable for RAS mutation testing. One section stained with H&E should be provided to histologically determine whether the tissue contains sufficient amount of tumor cells for testing. Finally, RAS mutation testing must be performed in laboratories with appropriate testing procedures and specimen management practices.

Key Points Multiple myeloma, which is located at multiple sites in the bone-marrow compartment, is a malignant plasma-cell tumour that is characterized by osteolytic bone lesions. It is a slowly proliferating tumour, typically with less than 1% of tumour cells synthesizing DNA, until late in the disease, when multiple myeloma cells are often found outside the bone marrow. A pre-malignant lesion called monoclonal gammopathy of undetermined significance (MGUS), which is present in 1% of adults, progresses to malignant multiple myeloma at a rate of 1% per year. The karyotypes of multiple myeloma are complex, and more similar to those found in epithelial tumours and the blast phase of chronic myelogenous leukaemia than to those in other haematopoietic tumours. Primary translocations — mediated by errors in B-cell-specific DNA modification processes — juxtapose one or more oncogenes and immunoglobulin transcriptional regulatory regions in ∼50% of MGUS and multiple myelomas. In contrast to other B-cell malignancies, these translocations simultaneously dysregulate a variety of oncogenes, such as the genes for cyclin D1 or D3, fibroblast growth factor receptor 3 (FGFR3) combined with the nuclear protein MMSET, and the transcription factor c-MAF. Secondary translocations that do not involve B-cell-specific processes contribute to progression by dysregulating other oncogenes. Although c- MYC is dysregulated by primary translocations in some B-cell malignancies, it is dysregulated by secondary translocations, often without involvement of an immunogloublin locus, as myeloma tumours become more proliferative at a late stage of progression. Genetic changes are similar in pre-malignant MGUS and multiple myeloma, although the latter is distinguished by the presence of activating mutations of NRAS or KRAS2 , and also a higher incidence of monosomy 13, indicating a possible tumour-suppressor gene on chromosome 13. Normal plasma cells, as well as MGUS and multiple myeloma cells, are dependent on the bone-marrow microenvironment for survival, growth and differentiation. These processes are, in part, mediated by paracrine interleukin-6 and insulin-like growth factor 1. The evolving interaction of multiple myeloma cells with the bone-marrow microenvironment is also involved in the secondary effects of malignancy, including osteolysis, anaemia and immunodeficiency. Multiple myeloma is an incurable malignancy for which the median survival has remained fixed at about 3 years for the past decade. Although MGUS can be efficiently diagnosed by a simple blood test, it is not possible to prevent progression or even predict when progression to myeloma will occur. Recent advances in understanding the molecular pathogenesis of these tumours indicate that improved approaches for prevention and treatment should be possible in the near future. Multiple myeloma is a neoplasm of terminally differentiated B cells (plasma cells) in which chromosome translocations frequently place oncogenes under the control of immunoglobulin enhancers. Unlike most haematopoietic cancers, multiple myeloma often has complex chromosomal abnormalities that are reminiscent of epithelial tumours. What causes full-blown myeloma? And can our molecular understanding of this common haematological malignancy be used to develop effective preventive and treatment strategies?

The role of internal tandem duplication of fms -like tyrosine kinase 3 ( FLT3 /ITD), mutations at tyrosine kinase domain ( FLT3 /TKD) and N- ras mutations in the transformation of myelodysplastic syndrome (MDS) to AML was investigated in 82 MDS patients who later progressed to AML; 70 of them had paired marrow samples at diagnosis of MDS and AML available for comparative analysis. Five of the 82 patients had FLT3 /ITD at presentation. Of the 70 paired samples, seven patients acquired FLT3 /ITD during AML evolution. The incidence of FLT3 /ITD at diagnosis of MDS was significantly lower than that at AML transformation (3/70 vs 10/70, P <0.001). FLT3 /ITD(+) patients progressed to AML more rapidly than FLT3 /ITD(−) patients (2.5±0.5 vs 11.9±1.5 months, P =0.114). FLT3 /ITD(+) patients had a significantly shorter survival than FLT3 /ITD(−) patients (5.6±1.3 vs 18.0±1.7 months, P =0.0008). After AML transformation, FLT3 /ITD was also associated with an adverse prognosis. One patient had FLT3 /TKD mutation (D835Y) at both MDS and AML stages. Additional three acquired FLT3 /TKD (one each with D835 H, D835F and I836S) at AML transformation. Five of the 70 matched samples had N- ras mutation at diagnosis of MDS compared to 15 at AML transformation ( P <0.001), one lost and 11 gained N- ras mutations at AML progression. Coexistence of FLT3 /TKD and N- ras mutations was found in two AML samples. N- ras mutations had no prognostic impact either at the MDS or AML stage. Our results show that one-third of MDS patients acquire activating mutations of FLT3 or N- ras gene during AML evolution and FLT3 /ITD predicts a poor outcome in MDS.

We searched and report mutations in the BRAF and N-ras genes in 22 out of 35 (63 percent) primary sporadic melanomas. In three melanomas, mutations were concomitantly present in both genes. In all, 10 out of 12 mutations in the BRAF gene involved the ‘hot spot’ codon 600 (In all communications on mutations in the BRAF gene, the nucleotide and codon numbers have been based on the NCBI gene bank nucleotide sequence NM_004333. However, according to NCBI gene bank sequence with accession number NT_007914, there is a discrepancy of one codon (three nucleotides) in exon 1 in the sequence with accession number NM_004333. The sequence analysis of exon 1 of the BRAF gene in our laboratory has shown that the sequence derived from NT_007914 is correct ( Kumar et al., 2003) . Due to the correctness of the latter, sequence numbering of codons and nucleotides after exon 1 are changed by +1 and +3, respectively.), one tandem CT1789-90TC base change represented a novel mutation and another mutation caused a G466R amino-acid change within the glycine-rich loop in the kinase domain. Mutations in the N-ras gene in 11 melanomas were at codon 61 whereas two melanomas carried mutations in codon 12 including a tandem mutation GG>AA. We observed an inverse association between BRAF/N-ras mutations and the frequency of loss of heterozygosity (LOH) on chromosome 9 at 10 different loci. Melanomas with BRAF/N-ras mutations showed a statistically significant decreased frequency of LOH on chromosome 9 compared with cases without mutations (mean fractional allelic loss (FAL)=0.29±0.23 vs 0.72±0.33; t- test, P =0.0001). Difference in the FAL value between tumours with and without BRAF/N-ras mutations on 33 loci on five other chromosomes was not statistically significant (mean FAL 0.17±0.19 vs 0.25±0.22; t- test, P =0.24). Melanoma cases with BRAF/N-ras mutations were also associated with lower age at diagnosis than cases without mutations (mean age 80.38±7.24 vs 65.77±19.79 years; t- test, P =0.02). Our data suggest that the occurrence of BRAF/N-ras mutations compensate the requirement for the allelic loss at chromosome 9, which is one of the key events in melanoma.

Oncogenic ras genes relate to the development of human cancers. In this study, we used a plasmid-mediated short-hairpin RNA (shRNA) targeting N-ras gene to combine with clinical drug vincristine (VCR) for the treatment of human hepatoma cells. Our results showed that anti-N-Ras shRNA expression vector (pCSH1-shNR) knocked down the target mRNA and protein. Higher efficacy on growth inhibition was observed when pCSH1-shNR was combined with VCR. This synergistic effect was associated with abrogation of VCR-induced overexpressions of P-glycoprotein and multidrug resistance-associated protein 1 by pCSH1-shNR through downregulations of N-Ras and total Ras. Western blot analysis showed that pCSH1-shNR-induced downregulations of N-Ras and total Ras were potentiated by VCR. Following Ras downregulation, phosphorylations of ERK1/2 and Akt were dramatically inhibited by combinatory treatment. The data showed that pCSH1-shNR-induced inhibition of nuclear factor-κB was enhanced by VCR. In addition, the combination of pCSH1-shNR and VCR synergistically inhibited the growth of human hepatoma HepG2 in vivo . Our findings suggested that combination of gene-specific therapeutics and appropriate chemotherapeutic agents might be a promising approach for the treatment of human hepatocellular carcinoma.

Activating mutations of the N-ras gene occur at relatively high frequency in acute myeloid leukemia and myelodysplastic syndrome. Somewhat paradoxically, ectopic expression of activated N-ras in primary hematopoietic cells and myeloid cell lines (in some cases) can lead to inhibition of proliferation. Expression of mutant N-ras in murine hematopoietic stem/progenitor cells is sufficient to induce myeloid malignancies, but these pathologies occur with long latency. This suggests that mutations that disable the growth suppressive properties of N-ras in hematopoietic cells are required for the development of frank malignancy. In the present work, the growth suppression induced by a mutant N-ras gene in U937 myeloid cells was used as the basis to screen a retroviral cDNA library for genes that prevent mutant N-ras -induced growth suppression (i.e., putative cooperating oncogenes). This screen identified the gene for the transcription factor interferon regulatory factor-2 (IRF-2), and as confirmation of the screen, overexpression of this gene in U937 cells was shown to inhibit mutant N-ras -induced growth suppression. Also recovered from the screen were two truncated clones of an uncharacterized gene (interim official symbol: PP2135 ). Overexpression of this truncated PP2135 gene in U937 cells did not appear to abrogate mutant N-ras -induced growth suppression, but rather appeared to confer an increased sensitivity of U937 cells to retroviral infection, accounting for the recovery of this gene from the genetic screen.

We applied a new strategy for the detection of N-ras gene mutations based on LightCycler technology. We designed two sets of amplimers and internal hybridization probes representing N-ras codons 12/13 and codon 61, respectively. Genomic DNAs from 134 childhood acute lymphoblastic leukemia (ALL) patients (83 common ALL, nine pre-pre-B ALL, 19 pre-B ALL, 23 T-ALL) were amplified, followed by the analysis of the melting temperatures of the PCR products on the LightCycler. PCR products exhibiting an abnormal melting characteristic were directly sequenced. Sequence analyses unravelled nucleotide substitutions at codon 12 in 10 patients, at codon 13 in three, and at codon 61 in one case. The incidence of N-rasmutations (10%) is compatible with previous reports. The LightCycler technology facilitates the rapid analysis of other genes exhibiting hot spot mutations in human malignancies.

Mutated ras genes are frequently found in human cancer. However, it has been shown that oncogenic ras inhibits growth of primary cells, through pathways involving p53 and the cell cycle inhibitors p16 super(INK4a) and p19 super(ARF). We have analysed the effect of the ectopic expression of the three mammalian ras genes on the proliferation of K562 leukemia cells, which are deficient for p53, p16 super(INK4a), p15 super(INK4b) and p19 super(ARF) genes. We have found that high expression levels of both wild-type and oncogenic H-, K- and N-ras inhibit the clonogenic growth of K562 cells. Induction of H-rasV12 expression in K562 transfectants retards growth and this effect is accompanied with an increase of p21 super(WAF1) mRNA and protein levels. Furthermore, p21 super(WAF1) promoter is activated potently by oncogenic ras and less pronounced by wild-type ras. This induction is p53-independent since a P21 super(WAF1) promoter devoid of the p53 responsive elements is still activated by Ras. Finally, inhibition of p21 super(WAF1) expression by an antisense construct partially overcomes the growth inhibitory action of oncogenic H-ras. Altogether, these results indicate that the antiproliferative effect of ras in myeloid leukemia cells is associated to the induction of p21 super(WAF1) expression and suggest the existence of p19 super(ARF) and p16 super(INK4a)-independent pathways for ras-mediated growth inhibition.

Mutations of the N-ras oncogene and p53 tumor suppressor gene were simultaneously investigated in bone marrow cells from 44 patients with myelodysplastic syndrome (MDS) or MDS-derived leukemia by single-strand conformation polymorphism (SSCP) analysis followed by direct sequencing. The mutations of the N-ras gene were detected only in two cases with MDS-derived leukemia. Three patients with MDS-derived leukemia and one with refractory anemia with excess of blasts exhibited five mutations of the p53 gene. No concomitant mutations of both genes were observed in our study, suggesting that alterations of both genes could play an important role in the progression of MDS in a non-cooperative manner.

In vitro and in vivo methods were combined to determine the function of the three Myb binding sites (NrasI, NrasII and NrasIII) within the promoter region of the mouse N-ras gene. We found that the c-Myb DNA-binding domain can bind with high affinity to NrasI and NrasII, but with a reduced affinity to NrasIII. In contrast, the full length v-Myb protein from BM2 cells only bound to the middle one of the three sites, NrasII. Both c-Myb and v-Myb functioned as repressors and reduce the basal activity of the N-ras promoter by 60%, as determined by transient transfection experiments using different regions of the N-ras promoter. This repression required a functional Myb DNA-binding domain and could not be overcome by fusion to the potent VP16 activation domain. In electrophoretic mobility shift assays, the v-Myb protein is shown to be present in different conformations depending on its binding to the NrasII or the mim-1A site. The v-Myb conformation is thus suggested to play a critical role in the regulation of v-Myb activity.