Saldivar, Juan‐Sebastian

11085 Background: The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) jointly initiated an effort to establish evidence-based recommendations for the molecular analysis of lung cancers required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Methods: Three co-chairs without relevant conflicts of interest were selected, one from each of the sponsoring societies: CAP (P.T.C.), IASLC (M.L.), and AMP (N.I.L.). Writing and advisory panels were formed from additional experts from these societies. Unbiased literature searches were performed to capture articles up to February 2012, yielding 1,533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for relevance. Evidence was formally graded for each of the recommendations first formulated by the co-chairs and panel members at a public meeting. Each guideline section was assigned to at least two panelists. Successive drafts were circulated for comments to the writing panel, the advisory panel, the public (online posting), and the three professional societies. Results: We generated 37 guideline items addressing 14 areas of EGFR and ALK testing. The major, evidence-based recommendations are to test for EGFR mutations and ALK fusions in all patients with advanced stage adenocarcinoma, regardless of sex, race, or smoking history, and to prioritize EGFR and ALK testing over other molecular predictive tests. Recommendations and expert consensus opinions were generated for all other key aspects of EGFR and ALK testing in lung cancer related to oncology and pathology practice and technical issues in molecular testing. Conclusions: As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards and thereby improving patient outcomes.

Abstract Abstract 2420 Introduction: Interstitial deletion of chromosome 11q (11q-) detected by fluorescence in situ hybridization (FISH) is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL). CLL patients with 11q- often have younger age of onset, bulky lymphadenopathy, and poorer overall survival. The ataxia-telangiectasia mutated (ATM) gene, which encodes a protein kinase central to DNA damage response, is thought to be the critical gene in CLL 11q- through loss of ATM function. A subgroup of CLL patients with 11q- has been shown by other investigators to have a mutation in the remaining ATM allele, supporting a loss-of-function hypothesis. In an ongoing study, we are using SNP-based whole genome copy number variation (CNV) analysis to characterize in detail the 11q deletions in CLL patients. In addition, we have reported uniparental disomy (UPD) of 11q in CLL patients negative for FISH abnormalities. Here we report direct DNA sequence analysis of the ATM gene in CLL patients with 11q- and with UPD of 11q. Patients and Methods: We performed CNV analysis and ATM sequencing on 10 patients aged 29–67 with early-intermediate stage, untreated CLL who had high risk for disease progression based on molecular and immunophenotypic markers. Eight patients had 11q- and 2 patients had UPD of 11q. CLL cells and normal cells were separated from patient peripheral blood samples by immunomagnetic beads. CNV analysis was performed on purified genomic DNA from CLL and normal cells for each patient to distinguish acquired copy number changes in malignant cells from polymorphic CNVs in the human genome. We used the Illumina 660w-quad beadchip, a SNP-based microarray for CNV analysis. CNV data was analyzed by CNV partition and PennCNV software. DNA sequence analysis of the ATM gene was performed by the dideoxy chain termination method using an ABI 3730 DNA Analyzer. All coding regions of the ATM gene (exons 4–65) and associated splice site junctions were amplified by PCR using purified genomic DNA from CLL cells. DNA sequence of the PCR products was determined on both strands. Sequence data was analyzed by Sequencher software. Results: In the 8 CLL patients with 11q−, CNV analysis demonstrates hemizygous interstitial deletions that vary from 980 Kb to 43.2 Mb. All deletions include the region of the ATM gene at 11q22.3. Two out of these 8 patients have a mutation in the residual ATM allele. One patient (CLL010) has a point mutation in exon 61 of ATM (c.8600G>A (gly2867glu)) that likely involves the substrate-binding region of the PI3-kinase domain. One patient (CLL029) has a frameshift mutation in exon 12 of ATM (c.1401_1405delCAAGA; asp467fs) that causes a premature stop signal at codon 484, presumably resulting in a truncated protein. In the 2 CLL patients with UPD of 11q, CNV analysis demonstrates copy-neutral loss of heterozygosity that extends from either 11q12.1 (CLL008) or 11q13.1 (CLL009) to the terminus of 11q. Both patients with UPD of 11q display homozygous mutation of the ATM gene. One patient (CLL008) has a point mutation in exon 58 of ATM (c.8161G>A (asp2721asn)) that likely involves the ATP binding site in the catalytic domain. One patient (CLL009) has a frameshift mutation in exon 24 of ATM (c.3238_3247del10bp/ins11bp; asp1080fs) that creates a premature stop signal at codon 1095, presumably resulting in a truncated protein. Conclusions: We report a new finding that ATM gene mutation occurs in CLL patients with UPD of 11q. Our study further expands observations reported by others concerning the role of ATM mutations in CLL. ATM mutations that we characterized include point mutations that may interfere with kinase activity and frameshift mutations that may result in a truncated ATM protein. Our sequence analysis supports a loss-of-function mechanism for ATM in a subgroup of high-risk, untreated CLL patients. The absence of detectable ATM mutations in the majority of CLL patients with 11q- may imply the existence of hidden ATM mutations not found by our targeted exon sequencing approach or epigenetic mechanisms that result in loss of gene expression. The finding that untreated CLL patients with UPD of 11q have homozygous ATM mutation supports a clonal selection hypothesis for identical alleles of mutated ATM in the absence of mutagenic chemotherapy. Selection of a CLL subclone with mutated ATM may confer a proliferative advantage through increased cell division and/or resistance to apoptosis following DNA damage. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.