Repair of DNA Double-strand Breaks in G1-phase Cells

The two main DNA double-strand break (DSB) repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). DNA ends are normally resected to form single-stranded overhangs to initiate HR, but resection severely limits repair by NHEJ. In G1-phase cells, NHEJ is the primary DNA DSB repair pathway, so DNA end resection must be restricted in these cells to prevent aberrant repair. Antigen receptor gene assembly occurs in G1-phase lymphocytes, using DNA DSB intermediates to generate a variable exon required for antigen binding. We show that the histone H2A variant, H2AX, which is phosphorylated by ATM kinase to generate gamma-H2AX in chromatin flanking DSBs, prevents the earliest steps of resection at RAG DSBs and other genotoxic DSBs in G1-phase lymphocytes. This gamma-H2AX function relies on its downstream factors MDC1 and RNF8 to concentrate 53BP1 at chromatin flanking the DSB, preventing the activity of resection machinery that can act in G1-phase cells. Resection in G1 is dependent on the protein CtIP, which has been previously shown to promote resection in S-G2 phases of the cell cycle. KAP-1 is a multifunctional adaptor protein that is involved in transcriptional silencing and maintenance of heterochromatin recently been implicated in repair of heterochromatic DNA DSBs. Here, we show that KAP-1 has a role in repair outside of heterochromatin, as it also promotes resection at RAG DSBs, along with CtIP. We have unexpectedly discovered that human KAP-1 blocks resection in mouse G1-phase lymphocytes in a dominant manner. The basis for this difference between human and mouse KAP-1 is a single amino acid polymorphism specific to primates located in a disordered region of the protein. Thus, we have established KAP-1 as part of the resection machinery in G1-phase cells that must be restricted by gamma-H2AX and 53BP1 to prevent resection and aberrant repair of DNA DSBs

American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer

Purpose: To develop a guideline to improve the accuracy of human epidermal growth factor receptor 2 (HER2) testing in invasive breast cancer and its utility as a predictive marker. Methods: The American Society of Clinical Oncology and the College of American Pathologists convened an expert panel, which conducted a systematic review of the literature and developed recommendations for optimal HER2 testing performance. The guideline was reviewed by selected experts and approved by the board of directors for both organizations. Results: Approximately 20% of current HER2 testing may be inaccurate. When carefully validated testing is performed, available data do not clearly demonstrate the superiority of either immunohistochemistry (IHC) or in situ hybridization (ISH) as a predictor of benefit from anti-HER2 therapy. Recommendations: The panel recommends that HER2 status should be determined for all invasive breast cancer. A testing algorithm that relies on accurate, reproducible assay performance, including newly available types of brightfield ISH, is proposed. Elements to reliably reduce assay variation (for example, specimen handling, assay exclusion, and reporting criteria) are specified. An algorithm defining positive, equivocal, and negative values for both HER2 protein expression and gene amplification is recommended: a positive HER2 result is IHC staining of 3+ (uniform, intense membrane staining of > 30% of invasive tumor cells), a fluorescent in situ hybridization (FISH) result of more than six HER2 gene copies per nucleus or a FISH ratio (HER2 gene signals to chromosome 17 signals) of more than 2.2; a negative result is an IHC staining of 0 or 1+, a FISH result of less than 4.0 HER2 gene copies per nucleus, or FISH ratio of less than 1.8. Equivocal results require additional action for final determination. It is recommended that to perform HER2 testing, laboratories show 95% concordance with another validated test for positive and negative assay values. The panel strongly recommends validation of laboratory assay or modifications, use of standardized operating procedures, and compliance with new testing criteria to be monitored with the use of stringent laboratory accreditation standards, proficiency testing, and competency assessment. The panel recommends that HER2 testing be done in a CAP-accredited laboratory or in a laboratory that meets the accreditation and proficiency testing requirements set out by this document. Copyright © 2007 by the American Society of Clinical Oncology and College of American Pathologists. All rights reserved