Novel and existing mutations in the tyrosine kinase domain of the epidermal growth factor receptor are predictors of optimal resectability in malignant peritoneal mesothelioma.

Malignant peritoneal mesotheliomas (MPM) are rare tumors representing 20% of all malignant mesothelioma cases. The median survival for these tumors is less than a year, and like other peritoneal surface malignancies, this is due primarily to intra-abdominal recurrence and progression. Currently there is a paucity of information about the biology of these tumors and molecular perturbations that are involved in tumor formation. Elucidation of mutations and biological pathways active in these tumors may identify valuable prognostic markers, as well as facilitate the development of novel therapies. In this study, we investigate the predictive value of epidermal growth factor receptor (EGFR) mutations in achieving optimal resectability. Twenty-nine patients with MPM were evaluated at a single tertiary care center and their tumors were probed for point mutations in the catalytic TK domain of epidermal growth factor receptor (mut+). All specimens were examined for somatic mutations by polymerase chain reaction amplification, and all variants were confirmed by multiple independent amplifications. Twenty-five patients were treated with cytoreductive surgery with or without intraperitoneal hyperthermic chemotherapy and complete clinical data including age, sex, cytoreductive score, mutation, and survival were available for comparison of the mut+ and mut- groups. The median age was 56 years, 71% of the patients were male, and the median follow-up time was 14.5 months. Mutations were found in 31% (9 of 29) of the tumors. Seven of these mutations were novel, and one was the L858R mutation described in non-small-cell lung cancer. Of the 25 patients managed surgically, 7 had mut+ and 18 wild type (mut-) disease. Optimal resectability was achieved in 7 (100%) of 7 of mut+ group and 9 (50%) of 18 mut- (p = .026). All mut+ patients are alive with a mean follow-up time of 24 months, whereas 5 (28%) of 18 of the mut- group are dead of disease with a mean follow-up time of 7 months (p = .27). In an analysis of covariance model, only optimal resectability (p = .04) was found to be predictive of survival. EGFR-TK seems to be a common site for mutation in MPM, with mutations being identified in 31% of patients. The EGFR mutations identified included the L858R activating mutation, as well as eight novel EGFR-TK catalytic domain point mutations. These mutations were predictive of optimal resectability, which was the only variable found to be predictive of survival. With longer follow-up, mut+ may not only be predictive of survival but may represent a subset of patients whose disease may be responsive to TK-inhibitor therapy. Experiments confirming the activating properties of the novel mutations are warranted.

PIK3CA and PTEN mutations in adenoid cystic carcinoma of the breast metastatic to kidney.

Adenoid cystic carcinoma (ACC) of the breast rarely metastasizes and has been associated with excellent prognosis. We describe a patient with renal metastasis of primary breast ACC 5 years after the mastectomy. A detailed molecular genetic analysis of the primary and metastatic tumors demonstrated somatic mutations in 2 well-known cancer genes associated with regulation of PI3K/AKT signaling pathway: (1) PIK3CA, which encodes the catalytic alpha subunit of the phosphoinositide-3-kinase, and (2) PTEN, which encodes phosphatase and tensin homolog. The mutation identified in PIK3CA (Ex1+169 A>C) predicts an amino acid change from isoleucine to methionine at codon 31 (I31M) and resides in the p85-binding domain of exon 1. The mutation identified in PTEN (IVS4-3 C>T) resides in intron 4 near the splice acceptor site of exon 5 and was associated with an aberrant PTEN transcript lacking exon 5, which is necessary for protein tyrosine phosphatase function and tumor suppressor properties of PTEN. Increased promoter methylation of PTEN was present in renal metastasis, coinciding with the decrease in the level of normal PTEN transcript. These coexistent mutations/epigenetic inactivations in PI3K/AKT pathway may be responsible for the unusually aggressive course of ACC.

In-depth mutation and SNP discovery using DHPLC gene scanning.

Denaturing high-performance liquid chromatography (DHPLC) is a new technology used in the discovery of genetic variations (mutations), such as single-base substitutions (or single nucleotide polymorphisms) and small deletions or insertions. These genetic variations can be routinely detected by DHPLC gene scanning at the germ-line and somatic levels. Epigenetic alterations, such as changes in DNA methylation status at defined loci, can also be assessed using DHPLC-based methodologies. The biological impact of these genetic variations depends on the location and identity of the DNA sequence alteration. The discovery of functionally relevant genetic variations can be exploited throughout the drug discovery and development processes. Examples of the application of DHPLC for sequence variant detection will be presented and discussed, with emphasis on target validation by candidate gene scanning and mutation detection in disease pathway genes, as well as the discovery of therapeutically significant mutations associated with drug metabolism and resistance.

Renal medullary carcinomas: histopathologic phenotype associated with diverse genotypes.

Chromosomal abnormalities and gene mutations have become major determinants in the classification of kidney carcinomas. Most renal medullary carcinomas develop in patients with hereditary sickle cell disease, but sporadic cases unassociated with sickle cell disease have also been described, for which underlying genetic abnormality is unknown. We evaluated 3 patients with renal medullary carcinoma (1 patient with sickle cell disease and 2 patients without sickle cell disease) for germ line and somatic mutations in genes commonly involved in pathogenesis of renal carcinomas using denaturing high-performance liquid chromatography and direct sequencing. Chromosomal abnormalities were studied by the conventional cytogenetic and SNP arrays analysis. Expression of hypoxia-inducible factor 1α was examined using immunohistochemistry. Two new mutations in the gene for fumarate hydratase were identified in 1 case of medullary renal carcinoma without sickle cell disease: a germ line mutation in exon 6 (R233H) and an acquired (somatic) mutation in exon 8 (P374S). No fumarate hydratase mutations were identified in the other 2 patients. The second sporadic case of renal medullary carcinoma harbored double somatic mutations in von Hippel-Lindau gene, and renal medullary carcinoma in the patient with sickle cell disease showed von Hippel-Lindau gene promoter methylation (epigenetic silencing). No consistent pattern of chromosomal abnormalities was found between 2 cases tested. All 3 cases showed increased hypoxia-inducible factor 1α expression. Medullary renal carcinomas from patients with or without sickle cell disease show involvement of genes important in hypoxia-induced signaling pathways. Generalized cellular hypoxia (in sickle cell disease) or pseudohypoxia (in tumors with fumarate hydratase and von Hippel-Lindau mutations or epigenetic silencing) may act alone or in concert at the level of medullary tubular epithelium to promote development of this rare type of renal carcinoma, which could then be genetically reclassified as either fumarate hydratase-associated renal carcinomas or high-grade clear cell renal cell carcinomas.

Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250K Nsp, and SNP6.0 arrays.

Specific chromosomal alterations are recognized as important prognostic factors in chronic lymphocytic leukemia (CLL). Array-based karyotyping is gaining acceptance as an alternative to the standard fluorescence in situ hybridization (FISH) panel for detecting these aberrations. This study explores the optimum single nucleotide polymorphism (SNP) array probe density for routine clinical use, presents clinical validation results for the 250K Nsp Affymetrix SNP array, and highlights clinically actionable genetic lesions missed by FISH and conventional cytogenetics. CLL samples were processed on low (10K2.0), medium (250K Nsp), and high (SNP6.0) probe density Affymetrix SNP arrays. Break point definition and detection rates for clinically relevant genetic lesions were compared. The 250K Nsp array was subsequently validated for routine clinical use and demonstrated 98.5% concordance with the standard CLL FISH panel. SNP array karyotyping detected genomic complexity and/or acquired uniparental disomy not detected by the FISH panel. In particular, a region of acquired uniparental disomy on 17p was shown to harbor two mutated copies of TP53 that would have gone undetected by FISH, conventional cytogenetics, or array comparative genomic hybridization. SNP array karyotyping allows genome-wide, high resolution detection of copy number and uniparental disomy at genomic regions with established prognostic significance in CLL, detects lesions missed by FISH, and provides insight into gene dosage at these loci.

Novel intronic germline FLCN gene mutation in a patient with multiple ipsilateral renal neoplasms.

Multiple renal tumors of diverse morphology are rare and typically seen in Birt-Hogg-Dubé syndrome. Birt-Hogg-Dubé syndrome is a rare inherited cancer syndrome caused by a germline mutation in the folliculin (FLCN) gene, but the genetic causes for histologic diversity of renal tumors in Birt-Hogg-Dubé syndrome have not been elucidated. We describe here a 64-year-old man with a novel germline mutation in the FLCN gene who presented with 3 phenotypically distinct renal tumors in the same kidney, which were histologically classified as oncocytoma (1.4 cm), oncocytic papillary carcinoma (0.5 cm), and clear cell renal carcinoma (0.8 cm). Genetic analysis of normal kidney tissue revealed a heterozygous germline FLCN mutation (intron 9, IVS9+6 C>T). Additional molecular genetic testing revealed somatic mutations and epigenetic events in genes typically associated with these specific histologic tumor types: oncocytoma harbored a second FLCN mutation (intron 12, IVS12+4 C>T), oncocytic papillary carcinoma harbored promoter methylation of FLCN, and a missense mutation in the MET gene (P246L), whereas clear cell carcinoma harbored inactivating VHL mutation (5-base pair deletion in exon 2) and VHL gene promoter methylation. In addition, chromosomal analysis of peripheral blood lymphocytes showed low level chromosome instability, not previously associated with germline mutations in the FLCN gene.

COX-2 gene polymorphisms and protein expression in renomedullary interstitial cell tumors.

Normal medullary interstitial cells regulate blood flow, water and salt absorption, and ultimately blood pressure through synthesis and secretion of prostanoids in which cycloxygenases play the rate-limiting steps. We found that most renomedullary interstitial cell tumors overexpressed cycloxygenase-2 (COX-2), with concomitant expression of microsomal prostaglandin E synthase-1 and the receptor for prostaglandin E2. Prostaglandin E2 is the major prostaglandin product of COX-2/microsomal prostaglandin E synthase-1 enzymatic pathway in medullary interstitial cells, and concomitant expression of COX-2, microsomal prostaglandin E synthase-1, and prostaglandin E2 receptor on interstitial cell tumors implies the presence of an autocrine growth loop important in pathogenesis of these tumors. Furthermore, overexpression of COX-2 protein was observed in association with homozygosity in several polymorphic sites within COX-2 gene (promoter region sites -1186 T/T and -765 G/G, intron 5 IVS5-275 T/T, and exon 10 Ex10+837 T>C), indicating their role in development of these tumors.

The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation.

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

Activity of the tyrosine kinase inhibitor PKC412 in a patient with mast cell leukemia with the D816V KIT mutation.

The majority of patients with systemic mast cell disease express the imatinib-resistant Asp816Val (D816V) mutation in the KIT receptor tyrosine kinase. Limited treatment options exist for aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). We evaluated whether PKC412, a small-molecule inhibitor of KIT with a different chemical structure from imatinib, may have therapeutic use in advanced SM with the D816V KIT mutation. We treated a patient with MCL (with an associated myelodysplastic syndrome (MDS)/myeloproliferative disorder [MPD]) based on in vitro studies demonstrating that PKC412 could inhibit D816V KIT-transformed Ba/F3 cell growth with a 50% inhibitory concentration (IC50) of 30 nM to 40 nM. The patient exhibited a partial response with significant resolution of liver function abnormalities. In addition, PKC412 treatment resulted in a significant decline in the percentage of peripheral blood mast cells and serum histamine level and was associated with a decrease in KIT phosphorylation and D816V KIT mutation frequency. The patient died after 3 months of therapy due to progression of her MDS/MPD to acute myeloid leukemia (AML). This case indicates that KIT tyrosine kinase inhibition is a feasible approach in SM, but single-agent clinical efficacy may be limited by clonal evolution in the advanced leukemic phase of this disease.