Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

A transforming MET mutation discovered in non-small cell lung cancer using microarray-based resequencing.

We have designed resequencing microarrays to test the performance of this platform when interrogating a large number of exons (164 total) from genes associated with cancer. To evaluate false positive and negative rates, dideoxy sequencing was done for 335,420 bases interrogated by the arrays. From the array data, calls could be made for approximately 97.5% of the bases, and false positive rates were very low with only a single mutation reported from the array dataset for which the corresponding dideoxy trace had a clean wildtype sequence. For the nucleotide positions where array calls were made, false negative rates were 1.41% for heterozygous mutations. All the homozygous mutations were detected, but 8.11% were erroneously reported as heterozygous changes from the reference sequence by the array analysis software. In addition, 20 non-small cell lung cancer (NSCLC) samples were analyzed using the arrays, and both somatic and germline mutations were found. The most interesting findings were two MET mutations that have recently been implemented in NSCLC. Large scale MALDI-TOF genotyping indicated that one of these mutations (T1010I) might represent a true cancer-causing genotype, whereas the other (N375S) appears to be a common germline polymorphism.

Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification.

Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10,000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500,000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.

Partial depletion of intracellular ATP mediates the stress-survival function of the PCPH oncoprotein.

Promotion of cellular resistance to stressful stimuli, including ionizing radiation and chemotherapeutic drugs, contributes to the transforming activity of the PCPH oncogene. The mechanism of this action, however, has remained unknown. Consistent with its intrinsic ATP diphosphohydrolase activity, expression of the PCPH oncoprotein in cultured cells has now been shown to result in partial depletion of intracellular ATP and consequent inhibition of the c-JUN NH2-terminal kinase-mediated stress signaling pathway. Supplementation of cells expressing the PCPH oncoprotein with exogenous ATP restored both stress-response signaling and sensitivity to cisplatin-induced apoptosis. In contrast, overexpression of the wild-type PCPH protein had a minimal effect on stress-induced signaling and on the cellular ATP content and did not protect cells from apoptosis. These results suggest that the PCPH oncoprotein confers resistance to stressors by reducing the cellular ATP concentration to levels below those required for optimal stress-induced signaling and apoptosis. Treatment with adenosine or nucleoside analogues may thus enhance the response to radiation or chemotherapy of tumors that express the PCPH oncogene.

An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays.

Changes in DNA copy number contribute to cancer pathogenesis. We now show that high-density single nucleotide polymorphism (SNP) arrays can detect copy number alterations. By hybridizing genomic representations of breast and lung carcinoma cell line and lung tumor DNA to SNP arrays, and measuring locus-specific hybridization intensity, we detected both known and novel genomic amplifications and homozygous deletions in these cancer samples. Moreover, by combining genotyping with SNP quantitation, we could distinguish loss of heterozygosity events caused by hemizygous deletion from those that occur by copy-neutral events. The simultaneous measurement of DNA copy number changes and loss of heterozygosity events by SNP arrays should strengthen our ability to discover cancer-causing genes and to refine cancer diagnosis.

Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia.

The SH2 domain-containing protein-tyrosine phosphatase PTPN11 (Shp2) is required for normal development and is an essential component of signaling pathways initiated by growth factors, cytokines, and extracellular matrix. In many of these pathways, Shp2 acts upstream of Ras. About 50% of patients with Noonan syndrome have germ-line PTPN11 gain of function mutations. Associations between Noonan syndrome and an increased risk of some malignancies, notably leukemia and neuroblastoma, have been reported, and recent data indicate that somatic PTPN11 mutations occur in children with sporadic juvenile myelomonocytic leukemia, myelodysplasic syndrome, B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia (AML). Juvenile myelomonocytic leukemia patients without PTPN11 mutations have either homozygotic NF-1 deletion or activating RAS mutations. Given the role of Shp2 in Ras activation and the frequent mutation of RAS in human tumors, these data raise the possibility that PTPN11 mutations play a broader role in cancer. We asked whether PTPN11 mutations occur in other malignancies in which activating RAS mutations occur at low but significant frequency. Sequencing of PTPN11 from 13 different human neoplasms including breast, lung, gastric, and neuroblastoma tumors and adult AML and acute lymphoblastic leukemia revealed 11 missense mutations. Five are known mutations predicted to result in an activated form of Shp2, whereas six are new mutations. Biochemical analysis confirmed that several of the new mutations result in increased Shp2 activity. Our data demonstrate that mutations in PTPN11 occur at low frequency in several human cancers, especially neuroblastoma and AML, and suggest that Shp2 may be a novel target for antineoplastic therapy.

EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.

Receptor tyrosine kinase genes were sequenced in non-small cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations of the epidermal growth factor receptor gene EGFR were found in 15of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinib-insensitive tumors or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib.

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML.

The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)-independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC(50) 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to small-molecule FLT3 inhibitors such as PKC412.