This report describes the important issues considered by the CAP committee during the development of the new checklist requirements, which address documentation, validation, quality assurance, confirmatory testing, exception logs, monitoring of upgrades, variant interpretation and reporting, incidental findings, data storage, version traceability, and data transfer confidentiality.

The cloning of the negative growth regulatory gene, p21Sdi1, has led to the convergence of the fields of cellular senescence, cell cycle regulation and tumor suppression. This gene was first cloned as an inhibitor of DNA synthesis that was overexpressed in terminally non‐dividing senescent human fibroblasts (SD11) and later as a p53 transactivated gene (WAF1) and a Cdk‐interacting protein (CIP1, p21) that inhibited cyclin‐dependent kinase activity. To identify the active region(s) of p21Sdi1, cDNA constructs encoding various deleted forms of the protein were analyzed. Amino acids 22‐71 were found to be the minimal region required for DNA synthesis inhibition. Amino acids 49‐71 were involved in binding to Cdk2, and constructs deleted in this region expressed proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amino acids 60‐76 of the p27Kip1 protein, another Cdk inhibitor. Point mutations made in p21Sdi1 in this region confirmed that amino acids common to both proteins were involved in DNA synthesis inhibition. Additionally, a chimeric protein, in which amino acids 49‐65 of p21Sdi1 were substituted with amino acids 60‐76 of p27Kip1, had almost the same DNA synthesis inhibitory activity as the wild‐type protein. The results indicate that the region of sequence similarity between p21Sdi1 and p27Kip1 encodes an inhibitory motif characteristic of this family of Cdk inhibitors.

The cyclin-dependent kinase (Cdk) inhibitor p21SDI1/WAF1/CIP1 has been found to be involved in cell senescence, cell cycle arrest, and differentiation. p21SDI1 inhibits the activity of several Cdks, in contrast to other inhibitors such as p15INK4B and p16INK4A, which act on specific cyclin-Cdk complexes. Of interest were reports that p21SDI1 also bound proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase delta, and inhibited DNA replication but not DNA repair in vitro. To better understand the function of this interaction in vivo, we first determined the region of p21SDI1 that was needed for PCNA binding. Analysis of deletion mutants of p21SDI1, which covered the majority of the protein, revealed that deletion of either amino acids 142-147 or 149-154 resulted in loss of ability to bind a glutathione S-transferase-PCNA fusion protein. Site-directed mutagenesis in this region led to the identification of the PCNA binding motif RQXXMTXFYXXXR and demonstrated that mutation of either amino acid Met-147 or Phe-150 resulted in almost complete ablation of PCNA binding. Interestingly, when we determined DNA synthesis inhibitory activity of deletion mutants or point mutants that were unable to bind Cdk2 and/or PCNA, we found that loss of binding to PCNA did not affect inhibitory activity, whereas lack of Cdk2 binding greatly reduced the same. This result suggests that the primary mechanism for inhibition of DNA synthesis by p21SDI1 occurs via inhibition of Cdk activity.

The HER2 gene is an important prognostic and therapeutic marker in newly diagnosed breast cancer. Currently, HER2 status is most frequently determined by immunohistochemical detection of HER2 protein expression on the cellular membrane surface or by fluorescence in situ hybridization analysis of HER2 gene copy number in fixed tissue using locus-specific probes for the HER2 gene and chromosome 17 centromere. However, these methods are problematic because of issues with intra-and inter-laboratory reproducibility and preanalytic variables, such as fixation time. In addition, the commonly used HER2/chromosome 17 ratio presumes that chromosome 17 polysomy is present when the centromere is amplified, even though analysis of the rest of the chromosome is not included in the assay. In this study, 97 frozen samples of invasive lobular and invasive ductal carcinoma, with known immunohistochemistry and fluorescence in situ hybridization results for HER2, were analyzed by comparative genomic hybridization to a commercially available bacterial artificial chromosome whole-genome array containing 99 probes targeted to chromosome 17 and the HER2/TOP2 amplicon. Results were 97% concordant for HER2 status, meeting the College of American Pathologists/ American Society of Clinical Oncology's validation requirements for HER2 testing. Surprisingly, not a single case of complete polysomy 17 was detected even though multiple breast cancer cases showed clear polysomies of other chromosomes. We conclude that array comparative genomic hybridization is an accurate and objective DNA-based alternative for clinical evaluation of HER2 gene copy number, and that polysomy 17 is a rare event in breast cancer.

MicroRNA (miRNA) deregulation contributes to cancer pathogenesis. However, analysis of miRNAs in diffuse large B-cell lymphoma (DLBCL) has been hindered by a focus on cell lines, limited number of miRNAs examined, and lack of copy number data. To address these restrictions, we investigated genomewide miRNA expression and copy number data in 86 DLBCLs. Permutation analysis showed that 63 miRNAs were recurrently disrupted in DLBCL, including highly expressed oncomirs not previously linked to chromosomal abnormalities. Further, using training and validation tumor groups, we defined a collection of miRNAs that robustly segregates DLBCLs into 3 subsets, which are independent of the cell-of-origin classification, extent of T-cell infiltrate, and tumor site. Instead, these unique miRNA-driven DLBCL subgroups showed markedly different MYC transcriptional activity, which explained the domi-