Phosphatidylinositol-3-kinase and AKT1 mutations occur early in breast carcinoma.

Mutationally activated protein kinases are appealing therapeutic targets in breast carcinoma. Mutations in phosphatidylinositol-3-kinase (PI3KCA) have been described in 8-40% of invasive breast carcinomas, and AKT1 mutations have been characterized in 1-8% of breast carcinomas. However, there is little data on these mutations in breast precursor lesions. To further delineate the molecular evolution of breast tumorigenesis, samples of invasive breast carcinoma with an accompanying in situ component were macro dissected from formalin-fixed paraffin embedded tissue and screened for mutations in PIK3CA exons 7, 9, 20, and AKT1 exon 2. Laser capture micro dissection (LCM) was performed on mutation-positive carcinomas to directly compare the genotypes of separated invasive and in situ tumor cells. Among 81 cases of invasive carcinoma, there were eight mutations in PIK3CA exon 20 (7 H1047R, 1 H1047L) and four mutations in exon 9 (2 E545K, 1 E542K, 1 E545G), totaling 12/81 (14.8%). In 11 cases examined, paired LCM in situ tumor showed the identical PIK3CA mutation in invasive and in situ carcinoma. Likewise, 3 of 78 (3.8%) invasive carcinomas showed an AKT1 E17K mutation, and this mutation was identified in matching in situ carcinoma in both informative cases. Mutational status did not correlate with clinical parameters including hormone receptor status, grade, and lymph node status. The complete concordance of PIK3CA and AKT1 mutations in matched samples of invasive and in situ tumor indicates that these mutations occur early in breast cancer development and has implications with regard to therapeutics targeted to the PI3 kinase pathway.

KIT gene mutations and copy number in melanoma subtypes.

PURPOSE: We recently identified a KIT exon 11 mutation in an anorectal melanoma of a patient who had an excellent response to treatment with imatinib. To determine the frequency of KIT mutations across melanoma subtypes, we surveyed a large series of tumors. EXPERIMENTAL DESIGN: One hundred eighty-nine melanomas were screened for mutations in KIT exons 11, 13, and 17. KIT copy number was assessed by quantitative PCR. A subset of cases was evaluated for BRAF and NRAS mutations. Immunohistochemistry was done to assess KIT (CD117) expression. RESULTS: KIT mutations were detected in 23% (3 of 13) of acral melanomas, 15.6% (7 of 45) of mucosal melanomas, 7.7% (1 of 13) of conjunctival melanomas, 1.7% (1 of 58) of cutaneous melanomas, and 0% (0 of 60) of choroidal melanomas. Almost all the KIT mutations were of the type predicted to be imatinib sensitive. There was no overlap with NRAS mutations (11.1% of acral and 24.3% of mucosal tumors) or with BRAF mutations (absent in mucosal tumors). Increased KIT copy number was detected in 27.3% (3 of 11) of acral and 26.3% (10 of 38) of mucosal melanomas, but was less common among cutaneous (6.7%; 3 of 45), conjunctival (7.1%; 1 of 14), and choroidal melanomas (0 of 28). CD117 expression, present in 39% of 105 tumors representing all melanoma types, did not correlate with either KIT mutation status or KIT copy number. CONCLUSIONS: Our findings confirm that KIT mutations are most common in acral and mucosal melanomas but do not necessarily correlate with KIT copy number or CD117 expression. Screening for KIT mutations may open up new treatment options for melanoma patients.

Pediatric KIT wild-type and platelet-derived growth factor receptor alpha-wild-type gastrointestinal stromal tumors share KIT activation but not mechanisms of genetic progression with adult gastrointestinal stromal tumors.

Fewer than 15% of gastrointestinal stromal tumors (GIST) in pediatric patients harbor KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations in contrast to a mutation rate of 80% in adult GISTs. However, some therapeutic inhibitors of KIT have efficacy in pediatric GIST, suggesting that KIT may, nevertheless, play an important role in oncogenesis. In adult GIST, characteristic cytogenetic changes occur during progression to malignancy. A better understanding of mechanisms of genetic progression and KIT and PDGFRA transforming roles in pediatric GIST might facilitate treatment advances. KIT and PDGFRA mutation analysis was done in 27 pediatric GISTs. The activation status of KIT, PDGFRA, and downstream signaling intermediates was defined, and chromosomal aberrations were determined by single nucleotide polymorphism assays. Mutations in KIT or PDGFRA were identified in 11% of pediatric GISTs. KIT and the signaling intermediates AKT and mitogen-activated protein kinase were activated in pediatric GISTs. In particular, most pediatric KIT-wild-type GISTs displayed levels of KIT activation similar to levels in adult KIT-mutant GISTs. Pediatric KIT-wild-type GISTs lacked the typical cytogenetic deletions seen in adult KIT-mutant GISTs. Notably, most pediatric KIT-wild-type GISTs progress to malignancy without acquiring large-scale chromosomal aberrations, which is a phenomenon not reported previously in malignant solid tumors. KIT activation levels in pediatric KIT-wild-type GISTs are comparable with those in KIT-mutant GISTs. Therapies that inhibit KIT activation, or crucial KIT signaling intermediates, should be explored in pediatric KIT-wild-type GIST.

Allele-specific polymerase chain reaction for the imatinib-resistant KIT D816V and D816F mutations in mastocytosis and acute myelogenous leukemia.

Oncogenic mutations of the receptor tyrosine kinase KIT contribute to the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis (SM), and some cases of acute myelogenous leukemia (AML). The D816V substitution in the activation loop of KIT results in relative resistance to the kinase inhibitor imatinib (Gleevec). Because this mutation occurs in 80 to 95% of adult SM, its detection has diagnostic and predictive significance. Unfortunately, the fraction of mutation-positive cells in clinical SM samples is often below the 20 to 30% threshold needed for detection by direct DNA sequencing. We have developed an allele-specific polymerase chain reaction assay using a mutation-specific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalin-fixed, paraffin-embedded tissue. There were no amplifications among 64 KIT wild-type tumors and cell lines, whereas all D816V-mutant samples (eight AML and 11 mast cell disease) were positive. Other D816 substitutions associated with resistance to imatinib in vitro are rare in SM. Among these D816F was detectable with the assay whereas D816H, D816Y, and D816G did not amplify. Nine biopsies (bone marrow, skin, or colon) with suspected SM were negative by denaturing high performance liquid chromatography and/or DNA sequencing but positive by allele-specific polymerase chain reaction. Thus, the assay may be useful in confirming the diagnosis of SM.