Headlines

BACKGROUND AND CLINICAL SIGNIFICANCE

BRAF is a human gene encoding the protein, B-Raf, a serine/threonine-protein kinase which plays a role in regulating the MAP kinase/ERKs signaling pathway and affects cell division, differentiation, and secretion. Activating mutations of BRAF are known to be involved in several human cancers.

MUTATION SCREENING

More than 30 mutations of the BRAF gene associated with human cancers have been identified. The frequency of BRAF mutations varies widely in human cancers, from > 80% in melanomas and nevi, to as little as 0-18% in other tumors, such as 1-3% in lung cancers and 5% in colorectal cancer. In 90% of the cases, thymine is substituted with adenine at nucleotide 1799, leading to an amino acid change from valine (V) to glutamate (E) at codon 600 or “V600E” in the activation segment of the protein. This mutation has been widely observed in thyroid carcinoma, colorectal cancer, melanoma and non-small-cell lung cancer. In 2011, a large-scale next-generation sequencing effort identified V600E as a likely driver mutation in 100% of cases of hairy cell leukaemia.

The antitumor efficacy resulting from RAF pathway inhibition has led to the development of specific inhibitors of mutated B-raf protein for anticancer use. One example is Vemurafenib (RG7204), which was approved by the US Food and Drug Administration as Zelboraf for the treatment of metastatic melanoma in 2011. Other B-raf inhibitors include GDC-0879, PLX-4720, and Sorafenib Tosylate (Nexavar) targeting melanoma, colorectal and renal cancers.

BRAF ASSAY DESCRIPTION

Next-generation-sequencing-based analysis of exon 1, encompassing positions 1,330-1814 and covering 54 known mutations in the BRAF gene. Sample types for mutation analysis include:

Formalin-Fixed, Paraffin-Embedded Tumor Tissue
+ 40-80 um total section thickness, ≥ 20% tumor
+ Shipped overnight, room temperature

Fresh-Frozen Tissue
+ 5-25 mg
+ Shipped overnight, frozen, on dry ice

Blood
+ 5-10 ml Blood in EDTA purple-top tube(s)

Markers in Cancer: A Joint Meeting by ASCO, EORTC and NCI, October 11-13, 2012, Hollywood, FL

10/11/2012 from 5:30 to 6:30PM in General Poster Session A

Abstract:

Background: Gene expression profiling has been shown to be effective in analyzing postoperative tumor samples in various cancers. However, in analyzing small specimens such as core biopsies, the limited amount of available material makes multi-gene analyses difficult or impossible. Microarray-based analyses also provide limited dynamic range. We describe the development of targeted RNA-sequencing methodology which combines the power of a universal RNA amplification with NGS for an ultra-deep expression analysis of multiple target genes, enabling <100 ng of sample input for multi-gene analysis in a single tube format. Methods: The gene expression patterns of triple-negative breast cancer FFPE samples were analyzed using a 96-gene breast cancer biomarker panel across three different platforms: Affymetrix Human Gene ST 1.0 microarrays, a pre-developed OncoScore qRT-PCR panel, and targeted RNA-seq. For targeted RNA-seq analysis, the 96-gene panel was amplified using a universal, single-tube “XP-PCR” amplification strategy followed by sequence analysis using the Ion-Torrent Personal Genome Machine. Results: Targeted RNA-seq provided the most sensitivity in terms of detection rates with <100 ng FFPE RNA input and provides unlimited dynamic range with increased sequencing depth. Expression ratio compression issues typically associated with a high number of pre-amplification cycles in standard multiplex-primed methods were not observed here. Low expressing genes, undetectable by qRT-PCR analysis from 1,000 ng input FFPE RNA, were detected and eligible for expression analysis with a significant number of sequencing reads. Alternative transcription/splicing analysis is also possible from sequence analysis of the target transcripts using targeted RNA-seq. Conclusions: By combining universally primed pre-amplification and NGS in multi-gene expression analysis, targeted RNA-seq provides the most sensitive gene expression analysis methodology.

The FLT3 gene FLT3 (Fms-like tyrosine kinase 3), encodes a cytokine receptor that, in humans, is expressed on the surface of hematopoietic progenitor cells.  Signaling through FLT3 plays a role in cell survival, proliferation, differentiation, and is important for lymphocyte (B cell and T cell) development, but not for the development of other blood cells (myeloid development).  FLT3 is also a proto-oncogene which, when mutated, can induce an oncogenic cascade. Specifically, mutations in the FLT3 gene can lead to the development of leukemias, cancers of bone marrow hematopoietic progenitors. FLT3 length mutations (FLT3-LM or FLT3-ITD, internal tandem duplications) are the most common mutations associated with acute myelogenous leukemia (AML) and are prognostic indicators associated with adverse disease outcome.

The NGS-based assay covers:

+      All prognostic internal tandem duplication “ITD” mutations

+      Codon D835 mutation

+      Codon I836 deletion

Gynecologic Oncology Group 85th Semi-Annual Meeting

The status of DNA repair pathways is critical to the efficacy and toxicity of many treatment strategies that target DNA.  This includes PARP inhibitors, alkylators like temozolomide and cisplatin, antimetabolites like 5-fluorouracil, topoisomerase inhibitors like etoposide and replication inhibitors like hydroxyurea.  Since many genes are involved in the multiple DNA repair pathways, a comprehensive genotyping analysis is necessary to understand the status of these pathways.  It has been cost prohibitive to analyze all the genes due to the labor-intensive nature of analyzing single genes.  Targeted capture and massively parallel sequencing has made it economically possible to sequence many genes simultaneously to obtain comprehensive coverage and also allow for improved mutation detection sensitivity, including those genes whose mutational status has been associated with PARP inhibitor sensitivity.  Next generation sequencing enables identification of potential responders therapies through comprehensive mutational analysis to genes critical for DNA repair.