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  • Ferrostatin-1 br The aim of the

    2020-08-12


    The aim of the present study was to clarify the extent and copy number of the amplicons commonly found in breast can-cers. This work focused on the chromosome regions noted above, given that these amplicons contain known or potential targets of therapy. For this purpose, we screened an archive of formalin-fixed and paraffin-embedded (FFPE) invasive 
    breast cancer specimens using multiplex ligation-dependent probe amplification (MLPA).
    2. Materials and methods
    2.1. Patients and control cell lines
    Four hundred invasive breast cancers obtained from opera-tions at the Department of Surgery in Kanazawa University Hospital between 2010 and 2014 were used. According to the recommendation of the 13th St. Gallen International Breast Cancer Conference (2013) Expert Panel, immunohistochemical (IHC) tests for Ferrostatin-1 receptor (cut-off of 1% of positive nu-clei), progesterone receptor (cut-off of 1% of positive nuclei), and Ki-67 (cut-off of 30% of positive nuclei), together with 3
    + IHC for ERBB2 (HercepTest™) (Agilent Technology, Santa Clara, CA) or in situ hybridization tests for ERBB2 amplifica-tion were used to classify the tumors into five clinico-pathologic surrogate subtypes [12], as follows: Luminal A– like (LA), Luminal B–like/HER2-negative (LB-), Luminal B–like/HER2-positive (LB+), HER2-positive and non-luminal (HER2), and triple negative (TN).
    A total of 322 tumors, excluding 78 tumors subjected to pre-operative chemotherapies, were examined. Serial sections cut from representative FFPE cancer specimens were used for MLPA and FISH. In the surgeries of 293 patients, the MAS–
    coated slides™ (Matsunami, Osaka, Japan) were touched to the cancer tissue, dried, and fixed immediately in methacarn so-lution (methanol/acetic acid, 3:1), dehydrated in a series of eth-anol solutions, and stored in a freezer pending FISH analysis.
    DNA was subjected to MLPA using the MLPA P078-C1 Breast tumor kit (MRC-Holland, Amsterdam, the Netherlands), which contains one to four probes for each of
    22 established cancer-related genes, as shown in Table 1. The resulting PCR products were separated on an ABI-310 capillary sequencer (Applied Biosystems, Foster City, CA) and the results were interpreted using GeneMapper software (Applied Biosystems). Data analysis was performed with Cof-falyser MLPA-DAT software version 9.4 (MRC-Holland) to normalize peak values. The test was performed in duplicate, and the arithmetic mean of all the probe peaks was calculated. Average peak values were defined as follows: above 2.0, ‘am-plified’; between 1.3 and 2.0, ‘gain’; between 0.7 and 1.3, ‘nor-mal’; below 0.7, ‘lost’. Both ‘amplified’ and ‘gain’ results were considered MLPA-positive.
    The FISH protocols for FFPE tissues and imprinted cancer cells were as described previously [13]. All BAC probes were mapped to the UCSC Genome Browser assembly
    Amplicons in breast cancers
    Table 1 Comparison of MLPA and FISH
    Name of gene Chromosomal FISH probe
    MLPA
    Clinico-pathological surrogate subtype
    Total
    locus of gene
    LA
    HER2 TN
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Lost
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Gain
    Amp
    Gain
    Amp
    Gain
    Gain
    No. of examined cases
    No. of Amp-positive cases
    NOTE. Values are presented as the number of cases with gene amplification validated by FISH (numerators) divided by the numbers of cases with ‘amplified’ or ‘gain’ cases classified by MLPA (denominators).
    Abbreviations: LA, Luminal A–like; LB-, Luminal B–like/HER2-negative; LB+, Luminal B–like/HER2-positive; HER2, HER2-positive; TN, triple negative; Amp,’amplified’ by MLPA; Gain, ‘gain’ by MLPA.
    ID: hg38 or 19 (UCSC Genome Browser. http://genome.ucsc. edu/). All FISH probes used in this study are summarized in Table 1. All but one was acquired from BACPAC Resources 
    (Oakland, CA); the sole exception was CTD-2501F13, which was obtained from Thermo Fisher Scientific (Waltham, MA). The probes were labeled with SpectrumOrange™ or
    SpectrumGreen™ using a nick translation kit (Abbott Labora-tories, Abbott Park, IL). For quantification of gene amplifica-tion, DNA probes specific for the alpha satellite DNA of the centromeric regions of each chromosome 6, 7, 8, 11, 16, 17, and 20 (CEP™6, 7, 8, 11, 16, 17, and 20) [14] were purchased from Abbott Laboratories and were co-hybridized to standard-ize the chromosome number. As a DNA probe specific to the centromere region of chromosome 19 is not commercially available, a BAC probe specific for the peri-centromeric re-gion of chromosome 19 (RP11-587H3) was used instead.