Comparison of methods in the detection of ALK and ROS1 rearrangements in lung cancer.

INTRODUCTION: The use of targeted therapies toward specific oncogenic driver mutations has become a critical factor in the treatment of patients with lung cancer. It is therefore essential to utilize tests with high performance characteristics. Fluorescence in situ hybridization (FISH) is the standard method for detecting anaplastic lymphoma kinase (ALK) and ROS1 rearrangements in non-small-cell lung cancer but the utility of other methods such as immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) is unclear. METHODS: Three hundred and sixty-two lung cancer patients were tested with FISH, CISH, and IHC using three ALK antibodies (ALK1, 5A4, D5F3) and one ROS1 antibody in the detection of ALK and ROS1 rearrangements. RESULTS: There was a 97.4% concordance (298 of 306) between FISH and CISH for detection of ALK rearrangements. The ROS1 rearrangement status had a 97% (291 of 300) concordance between CISH and FISH. ALK protein expression was observed in 6 of 341 samples with the ALK1 and 5A4 antibodies and 5 of 341 samples with D5F3. All three antibodies stained each of the ALK FISH-positive samples (100% sensitivity). ROS1 protein expression was observed in 2 of 322 samples. One of three samples with a ROS1 rearrangement by FISH showed ROS1 protein expression (33.3% sensitivity). CONCLUSION: Our findings show good correlation between FISH versus CISH in the detection of ALK and ROS1 rearrangements. FISH versus IHC showed good correlation in the detection of ALK rearrangements but showed weak correlation in the detection of ROS1 rearrangements. These results suggest CISH and IHC could be complimentary detection methods to FISH in the detection of ALK and ROS1 rearrangements.

Characterization of the 19q12 amplification including CCNE1 and URI in different epithelial ovarian cancer subtypes.

BACKGROUND: CCNE1 is frequently amplified in high grade serous ovarian cancer and may serve as a target for ovarian cancer treatment. URI is closely related to CCNE1 at the 19q12 amplicon and may also contribute to the oncogenic effect. Our objective was to investigate the relevance of CCNE1 and URI gene amplification and protein expression in different histological subtypes of epithelial ovarian cancer (EOC). METHODS: A novel dual-color 19q12 in situ hybridization (ISH), covering CCNE1 and URI, and chromosome 19 as a surrogate using Ventana BenchMark XT platform was developed and applied to 148 EOCs. URI and CCNE1 amplifications were separately assessed by fluorescence in situ hybridization (FISH). Immunohistochemistry using a Cyclin E1 and a novel URI monoclonal antibody was performed. RESULTS: Amplification of 19q12 was found in 36.6%, CCNE1 in 21.7%, URI in 9.9%, and both genes simultaneously in 9% of EOC cases. High Cyclin E1 and URI protein expression were observed in 52.2% and 26.1%, respectively. Amplification of 19q12 occurred in all EOC subtypes and was associated with amplification and expression of CCNE1/Cyclin E1, URI, TP53 mutation, and advanced stage. CONCLUSION: The novel 19q12 ISH probe reliably detects both CCNE1 and URI amplifications as confirmed by FISH. The combination of 19q12 amplification with Cyclin E1 and URI protein expression may help to select patients more likely to benefit from CDK2 targeted therapies.

BCL2 antibodies targeted at different epitopes detect varying levels of protein expression and correlate with frequent gene amplification in diffuse large B-cell lymphoma.

Patients with aggressive, BCL2 protein-positive (+) diffuse large B-cell lymphoma (DLBCL) often experience rapid disease progression that is refractory to standard therapy. However, there is potential for false-negative staining of BCL2 using the standard monoclonal mouse 124 antibody that hinders the identification of these high-risk DLBCL patients. Herein, we compare 2 alternative rabbit monoclonal antibodies (E17 and SP66) to the 124 clone in staining for BCL2 in formalin-fixed, paraffin-embedded DLBCL tissues. Overall, in 2 independent DLBCL cohorts, E17 and SP66 detected BCL2 expression more frequently than 124. In the context of MYC expression, cases identified as BCL2 (+) with SP66 demonstrated the strongest correlation with worse overall survival. The 124 clone failed to detect BCL2 expression in the majority of translocation (+), amplification (+), and activated B-cell DLBCL cases in which high levels of BCL2 protein are expected. Using dual in situ hybridization as a new tool to detect BCL2 translocation and amplification, we observed similar results as previously reported for fluorescence in situ hybridization for translocation but a higher amplification frequency, indicating that BCL2 amplification may be underreported in DLBCL. Among the discrepant cases, phosphorylation of BCL2 at T69 and/or S70 was more common than in the concordant cases and may contribute to the 124 false negatives, in addition to previously associated mutations within the epitope region. The accurate detection of BCL2 expression is important in the prognosis and treatment of DLBCL particularly with new anti-BCL2 therapies.

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining.

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5' ends of a bridged DSB are juxtaposed such that DNA unwinding and 3'-5' exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5' termini and exonucleolytic degradation of the 3' ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.

ATM mediates repression of DNA end-degradation in an ATP-dependent manner.

Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.

VIP is a transcriptional target of Nurr1 in dopaminergic cells.

The orphan nuclear receptor Nurr1 is required for the development of the ventral mesencephalic dopaminergic neurons. These are the same neurons that are invariantly lost in patients with Parkinson's disease. Nurr1 mRNA expression is not confined to the developing midbrain, and yet Nurr1 appears to be essential for either the maturation of progenitors into fully post-mitotic dopaminergic neurons and/or once formed, their survival. The function of Nurr1 in the transactivation of gene(s) important for neuronal development and/or maintenance is uncharacterized. To characterize potential downstream target genes of Nurr1, we sought to identify mRNAs that are differentially affected by Nurr1 expression. Using a dopaminergic cell line in which Nurr1 content was tightly regulated, differential display analysis identified transcripts altered by Nurr1 expression, including the mRNA encoding vasoactive intestinal peptide (VIP). Herein, we demonstrate that Nurr1 regulates VIP mRNA and protein levels, and transactivates the VIP promoter through Nurr1-responsive cis elements. In addition, dopaminergic cells release and utilize VIP to mediate survival when challenged with paraquat. Nurr1 regulation of VIP is also demonstrated in vivo as loss of Nurr1 function results in diminished VIP mRNA levels within the developing midbrain.

Identification of human alpha-synuclein specific single chain antibodies.

Parkinson's disease (PD) is a common neurodegenerative disease of unknown etiology. Evidence suggests a role for protein misfolding in disease pathogenesis. One pathologic feature observed in dopaminergic neurons is the intracytoplasmic eosinophilic inclusions known as Lewy bodies. One component of Lewy bodies, the presynaptic protein, alpha-synuclein forms oligomers and higher order aggregates and is proposed to be involved in dopaminergic neuronal death. In an effort to discriminate between alpha-synuclein conformational forms as well as design potential disruptors of pathogenic misfolding we panned a human phage antibody library for anti-synuclein single chain antibodies (scFvs). We identified six scFvs which recognize different conformers of alpha-synuclein in both an ELISA and Western blot analysis. These scFvs may further our understanding of alpha-synuclein's role in PD.

Flap endonuclease 1 efficiently cleaves base excision repair and DNA replication intermediates assembled into nucleosomes.

Flap Endonuclease 1 (FEN1) plays important roles both in DNA replication and in base excision repair (BER). However, in both processes FEN1 substrates are likely to be assembled into chromatin. In order to examine how FEN1 is able to work within chromatin, we prepared model nucleosome substrates containing FEN1-cleavable DNA flaps. We find that human FEN1 binds and cleaves such substrates with efficiencies similar to that displayed with naked DNA. Moreover, we demonstrate that both FEN1 and human DNA ligase I can operate successively on DNA within the same nucleosome. These results suggest that some BER steps may not require nucleosome remodeling in vivo and that FEN 1 activity during Okazaki fragment processing can occur on nucleosomal substrates.

DNA ligase I competes with FEN1 to expand repetitive DNA sequences in vitro.

Repeat sequences in various genomes undergo expansion by poorly understood mechanisms. By using an oligonucleotide system containing such repeats, we recapitulated the last steps in Okazaki fragment processing, which have been implicated in sequence expansion. A template containing either triplet or tandem repeats was annealed to a downstream primer containing complementary repeats at its 5'-end. Overlapping upstream primers, designed to strand-displace varying numbers of repeats in the downstream primer, were annealed. Human DNA ligase I joined overlapping segments of repeats generating an expansion product from the primer strands. Joining efficiency decreased with repeat length. Flap endonuclease 1 (FEN1) cleaved the displaced downstream strand and together with DNA ligase I produced non-expanded products. However, both expanded and non-expanded products formed irrespective of relative nuclease and ligase concentrations tested or enzyme addition order, suggesting the pre-existence and persistence of intermediates leading to both outcomes. FEN1 activity decreased with the length of repeat segment displaced presumably because the flap forms structures that inhibit cleavage. Increased MgCl(2) disfavored ligation of substrate intermediates that result in expansion products. Examination of expansion in vitro enables dissection of substrate and replication enzyme dynamics on repeat sequences.

Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5'-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN1 or the 5'-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3'-tail on the upstream primer adjacent to the 5'-flap. We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3'-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3'-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3'-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3'-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5'-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3'-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.