Germline DNA Of Patients Research Articles

Future of Personalized Medicine in Oncology: A Systems Biology Approach

The development of cost-effective technologies able to comprehensively assess DNA, RNA, protein, and metabolites in patient tumors has fueled efforts to tailor medical care. Indeed validated molecular tests assessing tumor tissue or patient germline DNA already drive therapeutic decision making. However, many theoretical and regulatory challenges must still be overcome before fully realizing the promise of personalized molecular medicine. The masses of data generated by high-throughput technologies are challenging to manage, visualize, and convert to the knowledge required to improve patient outcomes. Systems biology integrates engineering, physics, and mathematical approaches with biologic and medical insights in an iterative process to visualize the interconnected events within a cell that determine how inputs from the environment and the network rewiring that occurs due to the genomic aberrations acquired by patient tumors determines cellular behavior and patient outcomes. A cross-disciplinary systems biology effort will be necessary to convert the information contained in multidimensional data sets into useful biomarkers that can classify patient tumors by prognosis and response to therapeutic modalities and to identify the drivers of tumor behavior that are optimal targets for therapy. An understanding of the effects of targeted therapeutics on signaling networks and homeostatic regulatory loops will be necessary to prevent inadvertent effects as well as to develop rational combinatorial therapies. Systems biology approaches identifying molecular drivers and biomarkers will lead to the implementation of smaller, shorter, cheaper, and individualized clinical trials that will increase the success rate and hasten the implementation of effective therapies into the clinical armamentarium.

Abstract 5011: Potential tumor suppressor role of PHD2 : functional study of mutations identified in germline DNA of patients with congenital polycythemia with or without paraganglioma

Abstract The Hypoxia Inducible Factor (HIF) plays a major role in tissue response to hypoxia. HIF induces expression of many genes involved in anaerobic glycolysis, angiogenesis, erythropoiesis and cell proliferation. HIF-alpha subunit is tightly regulated by a family of specific hydroxylases in normoxia. PHD (prolyl-hydroxylase domain) proteins hydroxylate HIF-alpha, thereby allowing its attachment to the VHL protein, a component of an E3 ubiquitin ligase complex involved in protein ubiquitination and degradation. Mutations in HIF regulators have been described and lead to the stabilization of HIF, activation of target genes and development of either tumors or polycythemia. Heterozygous germline mutations in the VHL gene predispose to the von Hippel-Lindau disease characterized by a panel of highly vascularized tumors (central nervous system and retinal hemangioblastomas, pheochromocytomas, renal cell carcinoma, pancreatic endocrine tumors) whereas homozygous or compound heterozygous VHL mutations are responsible for isolated polycythemia (”Chuvash polycythemia”) with high circulating erythropoietin levels. Recently, heterozygous germline mutations in the PHD2 gene have been described in few patients with such congenital polycythemia and a particular mutation (H374R) has been identified in a patient with a polycythemia and a recurrent paraganglioma1. This observation raised the question of a possible role of PHD2 loss of function in the genesis of tumors usually seen in the VHL disease. We report here four new heterozygous PHD2 germline mutations in patients with familial or apparently sporadic polycythemia. We performed a functional study of these mutants compared to the H374R mutant and we showed differences in the capacity of the PHD2 mutants to downregulate HIF-alpha. These differences, tested in various conditions, are highly reproducible and suggest that there may be a genotype/phenotype correlation for the PHD2 mutants like the VHL mutants with their different ability to regulate HIF associated with different phenotypes in the VHL disease. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5011.

Abstract 3585: Taqman genotyping assays can be used on decalcified and paraffin embedded tissue from patients with osteosarcoma

Abstract Insight in genetic variants predicting treatment outcome and/or side effects related to cancer medication will contribute to optimize treatment for the individual patients. However, most clinical trials performed so far in cancer treatment did not routinely collect and store patient germ line DNA. Formalin-fixed paraffin-embedded tissues (FFPE) from archived clinical samples could provide a source of germ line DNA for genetic research. Specially, in cancers like osteosarcoma with a 5-year overall survival of approximately 50-60%, archived histological specimens are often the only accessible source of biological material. This material, however, generally has been decalcified and paraffin-embedded for many years. DNA from formalin-fixed and paraffin embedded (FFPE) tissues, specially, in decalcified FFPE tissues has a higher frequency of non-reproducible sequence alterations compared with germ line DNA. In our study, we investigated whether DNA obtained from these tissues can be used for reliable single nucleotide polymorphism (SNP) genotyping. Therefore, we analyzed whether the genotypes obtained from DNA of these preserved tissues, were similar as those found in freshly DNA isolated from peripheral blood cells from the same patients. We studied two SNPs in the drug transporter MDR1 (ABCB1) using Taqman® SNP genotyping assays. Genotypes of the germ line DNA derived from freshly isolated DNA of 20 surviving patients with osteosarcoma were compared with genotypes obtained from archived material from decalcified FFPE blocks of the same patients. Decalcified FFPE-derived DNA yielded smaller PCR fragments compared to DNA extracted from peripheral blood cells, with a maximum reliable size of ∼300bp. We were able to evaluate each SNP in 19 of the 20 cases included in this study. All successfully genotyped samples showed 100%-concordance between genotypes obtained from DNA of FFPE tissue and the genotypes obtained from DNA of blood. In conclusion, we have demonstrated that decalcified FFPE tissue can be used for genetic polymorphism analysis using Taqman® allelic discrimination assays. This forms an unique opportunity to combine new insights in genetic research with historical patient cohorts, for instance of patients with osteosarcoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3585.

Epidermal Growth Factor ReceptorMutation Testing in the Care of Lung Cancer Patients

As the literature about epidermal growth factor receptor (EGFR) mutations grows and screening for mutations becomes increasingly integrated into clinical care, it is important to examine how best to do somatic mutational analyses and how best to use the test results in clinical decision making. We began offering mutation screening by comprehensive direct sequence analysis of exons 18 to 24 of the tyrosine kinase domain of EGFR in August 2004 as part of clinical cancer care and protocol therapy at our institutions. All identified potential mutations are confirmed with three to five independent PCRs of the original genomic DNA sample and, if not previously noted in the literature, are compared with the patient's germ-line DNA to ensure the finding is somatic. We formally analyzed the first 100 patients to undergo EGFR sequence analysis and found that testing was feasible and significantly affected the treatment of patients with non-small cell lung cancer (NSCLC). Patients harboring EGFR mutations were significantly more likely to receive recommendations for therapy with EGFR tyrosine kinase inhibitors (i.e., gefitinib or erlotinib) than patients without mutations. However, negative EGFR test results did not prevent physicians from administering these agents to selected patients. Ideally, a standardized technique for mutation testing could be developed, with demonstrated reproducibility and validity. Clinical trials incorporating molecular diagnostics are ongoing to assess the efficacy of EGFR tyrosine kinase inhibitors as first-line therapy for metastatic NSCLC and as adjuvant therapy for early-stage resected NSCLC. It is likely that mutation testing and other molecular analyses will be most useful in these two clinical situations.

RESPONSE: Re: Association Between Nonrandom X-Chromosome Inactivation and BRCA1 Mutation in Germline DNA of Patients With Ovarian Cancer.

RESPONSE: Re: Association Between Nonrandom X-Chromosome Inactivation and BRCA1 Mutation in Germline DNA of Patients With Ovarian Cancer.

Association Between Nonrandom X-Chromosome Inactivation and BRCA1 Mutation in Germline DNA of Patients With Ovarian Cancer

Most human female cells contain two X chromosomes, only one of which is active. The process of X-chromosome inactivation, which occurs early in development, is usually random, producing tissues with equal mixtures of cells having active X chromosomes of either maternal or paternal origin. However, nonrandom inactivation may occur in a subset of females. If a tumor suppressor gene were located on the X chromosome and if females with a germline mutation in one copy of that suppressor gene experienced nonrandom X-chromosome inactivation, then some or all of the tissues of such women might lack the wild-type suppressor gene function. This scenario could represent a previously unrecognized mechanism for development of hereditary cancers. We investigated whether such a mechanism might contribute to the development of hereditary ovarian cancers. Patterns of X-chromosome inactivation were determined by means of polymerase chain reaction amplification of the CAG-nucleotide repeat of the androgen receptor (AR) gene after methylation-sensitive restriction endonuclease digestion of blood mononuclear cell DNA from patients with invasive (n = 213) or borderline (n = 44) ovarian cancer and control subjects without a personal or family history of cancer (n = 50). BRCA1 gene status was determined by means of single-strand conformational polymorphism analysis and DNA sequencing. All statistical tests were two-sided. Among individuals informative for the AR locus, nonrandom X-chromosome inactivation was found in the DNA of 53% of those with invasive cancer versus 28% of those with borderline cancer (P = .005) and 33% of healthy control subjects (P = .016). Nonrandom X-chromosome inactivation can be a heritable trait. Nine of 11 AR-informative carriers of germline BRCA1 mutations demonstrated nonrandom X-chromosome inactivation (.0002 < P < .008, for simultaneous occurrence of both). Nonrandom X-chromosome inactivation may be a predisposing factor for the development of invasive, but not borderline, ovarian cancer.

RET proto-oncogene mutations in multiple endocrine neoplasia type 2 and medullary thyroid carcinoma.

Multiple endocrine neoplasia type 2 (MEN-2) is a familial cancer syndrome inherited in an autosomal dominant fashion with age-related penetrance. The main tumour type present in all manifestations of this syndrome. MEN-2A, MEN-2B and familial medullary thyroid carcinoma (FMTC), is medullary thyroid carcinoma (MTC). MTC arises from the parafollicular or C cells of the thyroid. MEN-2A is characterised by the triad of MTC, phaeochromocytoma, and parathyroid hyperplasia. MEN-2B is characterised by features similar to those of MEN-2A, except for the absence of clinically apparent parathyroid hyperplasia, and additional stigmata including a marfanoid habitus, mucosal neuromas and ganglioneuromatosis of the gastrointestinal tract. FMTC families have MTC as their only phenotype. Missense mutations affecting conserved cysteine codons adjacent to the transmembrane domain of the RET proto-oncogene have been identified in the germline DNA of patients with MEN-2A and FMTC. A single mutation at codon 918 in the tyrosine kinase domain of the RET receptor has been associated with the MEN-2B phenotype. In a small number of FMTC families, missense point mutations have also been identified in the intracellular domain of the RET protein. RET mutation analysis of MEN-2 families has allowed the identification of genotype-phenotype correlations. While 25% of all MTCs are hereditary, the great majority of MTCs, 75%, are sporadic. Various somatic RET mutations have been identified in sporadic MTCs. In a small number of hereditary MTCs with germline mutations in RET, an additional somatic missense RET mutation has been identified. The discovery of RET mutations in MEN-2 has made possible accurate DNA-based diagnosis and predictive testing. The clinical significance of somatic RET mutations has yet to be determined.

Mutations in MLH1 are more frequent than in MSH2 in sporadic colorectal cancers with microsatellite instability.

The microsatellite instability that is a feature of tumors in patients with hereditary nonpolyposis colorectal cancer (HNPCC) is a consequence of defective DNA mismatch repair. Mutations in the DNA mismatch repair genes MSH2 and MLH1 may account for up to 90% of HNPCC kindreds. Microsatellite instability is also seen in 10-16% of sporadic colorectal cancers. A limited number of MSH2 and MLH1 mutations have been described for sporadic colorectal cancers. In this study, we screened 12 primary sporadic colorectal cancers with microsatellite instability for mutations in MSH2 and MLH1 by using reverse transcription-polymerase chain reaction (RT-PCR) and single-strand-conformation-variant (SSCV) analysis. Eight mutations were identified in six tumors. One mutation in MLH1 was found to be present in the patient's germline DNA. Four tumors had somatic mutations in MLH1, and, in two of these tumors, two different mutations were identified. A single tumor had a somatic MSH2 mutation. Our observations suggest that MLH1 is mutated more frequently than MSH2 in sporadic colorectal cancers with microsatellite instability.

Mutation analysis of the gene encoding Bruton's tyrosine kinase in a family with a sporadic case of X‐linked agammaglobulinemia reveals three female carriers

Bruton's tyrosine kinase (Btk) has been identified as the protein responsible for the primary immunodeficiency X-linked agammaglobulinemia (XLA). We and others have cloned the gene for Btk and recently reported the genomic organization. Nineteen exons were positioned within the 37 kb gene. With the sequence data derived from our genomic map, we have designed a PCR based assay to directly identify mutations of the Btk gene in germline DNA of patients with XLA. In this report, the assay was used to analyze a family with a sporadic case of XLA to determine if other female relatives carry the disease. A four base-pair deletion was found in the DNA of the affected boy and was further traced through three generations. With the direct identification of the mutations responsible for XLA, we can now diagnose conclusively the disease and identify the immunologically normal female carriers. This same technique can easily be applied to prenatal diagnosis in families where the mutation can be identified.