Low Level of Her-2 Locus Amplification by Fluorescent In Situ Hybridization Does Not Correlate with Her-2 Protein Overexpression by Immunohistochemistry in Barrett's Esophagus

An accurate evaluation of the Her-2 status has important prognostic and therapeutic implications in many carcinomas. The aim of the study was to correlate Her-2 locus (17q11.2) amplification and chromosome 17 gains as assessed by fluorescent in situ hybridization (FISH) with Her-2 protein overexpression by immunohistochemistry (IHC) in patients with Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). We analyzed 34 patients with Her-2 amplification and/or chromosome 17gains using FISH on brush cytology specimens. Seven patients (21%) showed high Her-2 locus amplification (Her-2: Cep17 ≥ 5 : 1), 5 (15%) showed low Her-2 locus amplification (Her-2: Cep17 ≥ 2 < 5 : 1), and 22 (65%) displayed gains of chromosome 17 only. Further, we confirmed Her-2 amplification on corresponding biopsies that were taken at the same occasion as the cytologybrushings. Then, we compared the FISH results with IHC data obtained from the corresponding biopsies and showed that low level of Her-2 amplification does not correlate with Her-2 protein overexpression (score +3/+2; P = 1), in contrast to the high amplification level (P = .001). Thus, in our population of BE and EAC patients, low level of Her-2 amplification does not result in detectable level of Her-2 protein as assessed by IHC

The novel P330L pathogenic variant of aromatic amino acid decarboxylase maps on the catalytic flexible loop underlying its crucial role

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C &gt; T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5'-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy

Trastuzumab Mediated T-Cell Response against HER-2/Neu Overexpressing Esophageal Adenocarcinoma Depends on Intact Antigen Processing Machinery

BACKGROUND: Esophageal adenocarcinoma (EAC) is a highly aggressive disease with poor prognosis, which frequently exhibits HER-2 gene amplification. Trastuzumab, the humanized antibody against HER-2, has potent growth inhibitory effects on HER-2 overexpressing cancers. One effect of trastuzumab is that it causes HER-2 receptor internalization and degradation, enhancing presentation of HER-2 epitopes on MHC-Class I molecules. This enhances the ability of HER-2 specific cytotoxic T lymphocytes (CTLs) to recognize and kill cancer cells. Novel strategies targeting the HER-2 receptor either directly by trastuzumab and/or indirectly by inducing a CTL response against HER-2 epitopes with, for instance, DC immunotherapy and consequently combining these strategies might prove to be very effective. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report that trastuzumab has potent growth inhibitory effects on two HER-2 overexpressing EAC cell lines OE33 and OE19. However, we found that trastuzumab and HER-2 specific CTLs act synergistically in inducing tumor lysis in OE33 but not in OE19. We discovered that in OE19 this deficient response is due to a down-regulation of the Transporter Associated with Antigen Processing-2 (TAP-2). TAP-2 is an important member of the Antigen Processing Machinery (APM), and is one of the essential elements for loading antigens on MHC class I molecules. Importantly, we demonstrated that by inducing re-expression of TAP-2 in OE19 with INF-γ treatment or by incubating the cells with INF-γ producing CTLs, the specific anti HER-2 CTL tumor lysis response and synergistic effect with trastuzumab can be restored. CONCLUSION: An inefficient response of HER-2 overexpressing EAC to trastuzumab and/or DC immunotherapy can be due to a down-regulated TAP-2 expression and thus a deficient APM. Future studies combining trastuzumab with IFN-γ and/or immune-therapies inducing potent anti HER-2 CTL responses could lead to an effective combinatorial strategy for successful treatment of HER-2 overexpressing but APM defective cancer

Stepwise radical endoscopic resection of the complete Barrett's esophagus with early neoplasia successfully eradicates pre-existing genetic abnormalities

OBJECTIVES: Malignant transformation of Barrett's mucosa is associated with the accumulation of genetic alterations. Stepwise radical endoscopic resection of the Barrett's segment with early neoplasia is a promising new treatment resulting in complete re-epithelialization of the esophagus with neosquamous epithelium. It is unknown whether radical resection also eradicates genetic abnormalities. The aim of this study was to prospectively evaluate whether genetic abnormalities as found in the Barrett's segment before radical resection are effectively eradicated and absent in the neosquamous epithelium. METHODS: Nine patients with early neoplasia who successfully underwent radical resection were included. Immunohistochemistry (IHC) was performed to assess p53 protein overexpression. DNA fluorescent in-situ hybridization was (DNA-FISH) performed for evaluation of numerical abnormalities of chromosomes 1 and 9, and losses of p16 and p53. Immunohistochemistry and DNA-FISH were performed on endoscopic resection specimens of the neoplasia and on follow-up biopsies of the neosquamous epithelium. RESULTS: DNA-FISH and IHC showed alterations in the pretreatment samples of all patients. All showed aneusomy of chromosome 1 and 9. Loss of p16 and p53 were seen in 6 and 8 patients. IHC showed intense p53 nuclear staining in seven patients. Post-treatment biopsies showed neosquamous epithelium with a normal diploid signal count for all DNA-FISH probes and normal IHC stainings in all patients. CONCLUSIONS: Radical resection of Barrett's esophagus with early neoplasia successfully eradicates pre-existing genetic abnormalities and results in neosquamous epithelium without these genetic abnormalitie