TTF-1/p63-positive poorly differentiated NSCLC: A histogenetic hypothesis from the basal reserve cell of the terminal respiratory unit

TTF-1 is expressed in the alveolar epithelium and in the basal cells of distal terminal bronchioles. It is considered the most sensitive and specific marker to define the adenocarcinoma arising from the terminal respiratory unit (TRU). TTF-1, CK7, CK5/6, p63 and p40 are useful for typifying the majority of non-small-cell lung cancers, with TTF and CK7 being typically expressed in adenocarcinomas and the latter three being expressed in squamous cell carcinoma. As tumors with coexpression of both TTF-1 and p63 in the same cells are rare, we describe different cases that coexpress them, suggesting a histogenetic hypothesis of their origin. We report 10 cases of poorly differentiated non-small-cell lung carcinoma (PD-NSCLC). Immunohistochemistry was performed by using TTF-1, p63, p40 (∆Np63), CK5/6 and CK7. EGFR and BRAF gene mutational analysis was performed by using real-time PCR. All the cases showed coexpression of p63 and TTF-1. Six of them showing CK7+ and CK5/6− immunostaining were diagnosed as “TTF-1+ p63+ adenocarcinoma”. The other cases of PD-NSCLC, despite the positivity for CK5/6, were diagnosed as “adenocarcinoma, solid variant”, in keeping with the presence of TTF-1 expression and p40 negativity. A “wild type” genotype of EGFR was evidenced in all cases. TTF1 stained positively the alveolar epithelium and the basal reserve cells of TRU, with the latter also being positive for p63. The coexpression of p63 and TTF-1 could suggest the origin from the basal reserve cells of TRU and represent the capability to differentiate towards different histogenetic lines. More aggressive clinical and morphological features could characterize these “basal-type tumors” like those in the better known “basal-like” cancer of the breast

First considerations on post processing kinematic GNSS data during a geophysical oceanographic cruise

Differential GNSS positioning on vessels is of considerable interest in various fields of application as navigation aids, precision positioning for geophysical surveys or sampling purposes especially when high resolution bathymetric surveys are conducted. However ship positioning must be considered a kinematic survey with all the associated problems. The possibility of using high-precision differential GNSS receivers in navigation is of increasing interest, also due to the very recent availability of low-cost differential receivers that may soon replace classic navigation ones based on the less accurate point positioning technique. The availability of greater plano-altimetric accuracy, however, requires an increasingly better understanding of planimetric and altimetric reference systems. In particular, the results allow preliminary considerations on the congruence between terrestrial reference systems (which the GNSS survey can easily refer to) and marine reference systems (connected to National Tidegauge Network). In spite of the fluctuations due to the physiological continuous variation of the ship's attitude, GNSS plot faithfully followed the trend of the tidal variations and highlighted the shifts between GNSS plot and the tide gauges due to the different materialization of the relative reference systems

Control of near-infrared supercontinuum bandwidth by adjusting pump pulse duration

We experimentally and numerically investigated the impact of input pump pulse duration on the near-infrared bandwidth of supercontinuum generation in a photonic crystal fiber. We continuously stretched the temporal duration of the input pump laser (centered at 1030 nm) pulses from 500 fs up to 10 ps, while keeping fixed the pump peak power. We observed that the long-wavelength edge of the supercontinuum spectrum is increased by 200 nm as the pump pulse duration grows from 500 fs to 10 ps. We provide a quantitative fit of the experimental results by means of numerical simulations. Moreover, we have explained the observed spectral broadening enhancement induced by pump pulse energy by developing an approximate yet fully analytical model for soliton energy exchange through a series of collisions in the presence of stimulated Raman scattering

Immunoistochemical expression of PD-1 and PD-L1 in bone marrow biopsies of patients with acute myeloid leukemia

Background. Haematological and non-haematological malignancies are able to escape the host immune by the capacity to hijack the immune check-points. Several immune check-point molecules are known, such as T cell immunoglobulin mucin-3 (TIM-3), cytotoxic T-cell antigen-4 (CTLA-4), programmed death-1 (PD-1) with its ligand PD-L1 and others.1 The function of these immune check-points is to prevent the damage resulting from an excessive activation of the immune response in the setting of chronic antigenic stimulation, thus leading to autoimmune phenomena, as proved in knock-out mice models. PD-1 is normally present on activated T lymphocytes membrane, acting as a negative costimulatory receptor. PD-L1 is constitutively expressed at low levels by resting lymphocytes, antigen presenting cells and certain immunologically privileged tissues like placenta and testis. PD-L1 expression can be induced as well. In an inflammatory/infective context when T cells recognize antigens expressed by MHC-complex they start to produce inflammatory cytokines. The resulting inflammation leads to the expression of PD-L1 by hematopoietic, epithelial and endothelial cells, activating PD-1 on the surface of T-cells and therefore blocking the immune response. Previous studies have found out that PD-1 is highly expressed on T-reg cells and their binding with PD-L1 enhances suppressor T-reg functions2,3; the activation of PD-1/PD-L1 pathway reduces the lytic capacity of NK cells and B cell antibody production. In solid neoplasms PD-L1 expression by cancer cells and persistent up-regulation of PD-1 by tumourinfiltrating lymphocytes is common45. All these findings brought to the development of check-point inhibitors in the contest of solid tumors and lymphoproliferative neoplasms such as lymphoma and myeloma where the immune checkpoint blockade treatment have shown efficacy in refractory/relapsed neoplasms6. Few investigations only have been conducted on the role of PD1/PD-L1 in myeloid neoplasms, such as acute myeloid leukemia, a haematological cancer characterised by high-risk of relapse and poor prognosis. In leukemia, the bone marrow serves as a sanctuary for neoplastic cells, these cells interact with the tumour microenvironment (TME), constituted by stromal cells, endothelial cells and immune cells. The marked activation of the PD-1/PD-L1 pathway contributes to the maintenance of an immunosuppressive microenvironment. In fact, blasts are able, through the production of immunoinhibitory factors, to suppress the function of immunosurveillance and immuno-elimination of the tumor by the effector T cells. The effector T cells are "exhausted" in their capacity to secrete granzyme B, perforine and interferon gamma, and there is an upregulation of the T-reg functions, in addition to the presence of myeloid-derived suppressor cells7. PD-L1 expression and his link with PD-1 on activated lymphocytes results in an impaired antitumoral activity in murine models8. Zhang et al. investigated the role of PD-1/PD-L1 engagement in murine AML showing that PD-1-/-mice generated augmented antitumoral response in comparison with wild type mice. Similar results were obtained using anti-PD-L1 antibodies.9 A study from Zhou et al. reported how the function of adoptively transferred AML-reactive CTLs was reduced by AML-associated Tregs and how Treg depletion followed by PD-1/PD-L1 blockade showed efficacy for AML eradication in murine models.10 Objective of the study. To assess the presence of PD-1 and PD-L1 positive cells, by immunohistochemistry, in bone marrow biopsies of patients with AML. Material and methods. Four micron thickness sections were obtained from the formalin-fixed paraffin-embedded specimens. Haematoxylin-eosin staining was performed to assess the morphologic features of bone marrow. Immunohistochemical stainings were performed using a Ventana Benchmark Ultra automated staining instrument according to the manufacturer’s recommendations, using anti-PD-1 (clone NAT105, Ventana) and anti-PD-L1 (clone 22C3, Dako) antibodies. Results. We obtained 34 bone marrow trephine biopsies from newly diagnosed AML patients, 17 males and 17 females with a 67.3 mean age. We used 10 healthy bone marrow specimens as normal controls. None out of 10 control bone marrows resulted positive for either PD-1 or PD-L1 expressing cells as expected. Eleven out of 34 AML bone marrows (32,4%) showed at least 1% of PD-L1 positive cells (fig.2 b,d,f), while 6 AML bone marrow samples (17,6%) were positive for at least 1% of PD-1+ cells (fig. 1 b). Discussion. Despite the presence of relevant preclinical data regarding the role of immune check-points, few studies to evaluate the PD1/PDL1 axis have been conducted in myeloid neoplasm. Jia et al. performed flow cytometry analysis on PBMCs and BMMCs of 22 newly diagnosed AML patients and observed a significantly increased frequency of PD-1 expressing CD8 T cells in bone marrow compared to peripheral blood, suggesting a more exhausted status of these cells in relation to the suppressivePaper environment11. Dail et al. measured PD-L1 expression in 7 AML patients using immunohistochemistry and flow cytometry and found that PD-L1 was detectable (>2% cells) in all patients.12 Yang et al. assessed 45 bone marrow biopsies of MDS, CMML and AML patients and found that leukemic blasts of 9 patients (20%) were PD-L1+, while 3 (7%) were positive for PD-1. All 4 controls tested were negative for both PD-1 and PD-L1.13 Daver et al. measured PD-1 expression on bone marrow aspirates of 74 AML patients using flow cytometry. The results showed higher PD-1 expression compared to healthy controls (n=8).14Acceped To our this is the largest study evaluating by immunohistochemistry the expression of PD-1 and PDL1 in AML bone marrows and shows a significative positivity of the activation of the PD-1/PD-L1 pathway which gives a rationale to further studies regarding the charateristics of the cells involved in the PD-1/PD-L1 pathway and the immunosuppressive microenvironment. An implementation of the PD-1/PD-L1 pathway evaluation in clinical setting could have prognostic significance since the expression of PD-L1 by AML blasts has been associated with poor-risk and intermediate-risk AML15, furthermore immune checkpoint inhibitors have shown promising results in maintenance treatment of high-risk AML16, underlining not only a prognostic but also therapeutic value of the PD1/PD-L1 evaluation

Seasonal and Feeding System Effects on Qualitative Parameters of Bovine Milk Produced in the Abruzzo Region (Italy)

The aim of this study was to examine variations in cow milk composition as a function of breeding system and seasonality. This study was carried out in 16 dairy farms located in the Abruzzo region (Central Italy), equally distributed between farms that adopt grazing in the spring and summer months, and farms where the intensive system is exploited. Milk was sampled in all seasons in each of the farms involved and was analyzed with particular attention given to the quality of the lipid and protein fractions. A lower concentration of saturated fatty acids and an increase in rumenic, vaccenic and oleic acids were registered for milk samples coming from outdoor grazing, in which was also observed the greatest presence of and caseins. The opposite result was instead observed for casein, which showed the highest values from intensive farming. Evaluations also focused on retinol, which significantly increased in concentration during summer in both breeding systems. The present results suggest positive insights into the role of the outdoor breeding system in improving the main qualitative trait of bovine milk in warm seasons

Cerebrospinal fluid levels of AFP and hCG: Validation of the analytical method and application in the diagnosis of central nervous system germ cell tumors

The determination of Human Chorionic Gonadotropin (hCG) and Alpha Fetoprotein (AFP) levels on serum and amniotic fluid plays a fundamental role in the diagnosis and follow-up of specific physiological or pathological conditions (e.g., pregnancy, threat of abortion or germ cell tumors). Recently, the quantification of hCG and AFP in other biological fluids has gained great attention to support the diagnosis, prognosis and follow-up of neoplastic diseases deriving from trophoblastic cells, such as germinomas. Most of the commercial kits for hCG and AFP assays are developed to be used on biological fluids such as serum/plasma and/or urine by manufacturing companies. The aim of this work was to evaluate the suitability of the analytical method certified for the use on serum, and/or amniotic fluid for the quantification of hCG and AFP in cerebrospinal fluid, carrying out an internal validation protocol. The data reported here show that the automated immunochemical method is fit for quantification of hCG and AFP in cerebrospinal fluid (CSF), allowing selective and specific diagnosis of secreting germ cell tumors. This is confirmed by the positive correlation between elevated levels of hCG or AFP and the diagnosis of brain tumors

Serum Anti-Thyroglobulin Autoantibodies Are Specific in Predicting the Presence of Papillary-like Nuclear Features and Lymphocytic Infiltrate in the Thyroid Gland

(1) Background: Previous studies have reported a correlation between serum anti-Thyroglobulin-antibodies (TgAb) and papillary thyroid carcinoma. The aim of our study was to evaluate whether serum TgAb and anti-thyroid-peroxidase antibody (TPO) positivity was also related to pre-neoplastic histological changes such as papillary-like nuclear features (PLNF) and with the presence of lymphocytic infiltrate (LI) in thyroid surgical specimens. (2) Methods: The study was retrospectively carried out on 70 consecutively recruited patients who underwent thyroidectomy for benign process and whose TgAb and TPOAb values were retrieved from clinical records. Histological sections of thyroid surgical samples were revised, looking for PLNF and lymphocytic infiltrate. HBME1 expression was assessed by immunohistochemistry. (3) Results: Our results showed a significant association between TgAb, PLNF, and lymphocytic infiltrate. The presence of TgAb was highly specific, but less sensitive, in predicting the presence of PLNF (sensitivity = 0.6, specificity = 0.9; positive predictive value (PPV) = 0.88; negative predictive value (NPV) = 0.63). TgAb positivity showed a good association with the presence of lymphocytic infiltrate (sensitivity = 0.62, specificity = 0.9; PPV = 0.88 and NPV = 0.68). HBME1 immunoreactivity was observed in the colloid of follicles showing PLNF and/or closely associated with LI. (4) Conclusions: The presence of PLNF and LI is associated with serum TgAb positivity. The presence of TgAb and of LI could be triggered by an altered thyroglobulin contained in the HBME1-positive colloid, and could be a first defense mechanism against PLNF that probably represent early dysplastic changes in thyrocytes

FISH testing of HER2 IHC 1+ early breast cancer with unfavorable prognostic factors

FISH testing of HER2 IHC 1+ early breast cancer with unfavorable prognostic factors Background HER2-positive tumors are associated with a poor prognosis and a shortened disease-free and overall survival as well as with other unfavorable prognostic tumor characteristics (high histological grade, high proliferative index, negative or low estrogen receptor expression, etc.). HER2-positive tumors are also responsive to treatment with trastuzumab in reducing the risk of recurrence and improving survival. The aim of this study is to assess the incidence of HER2 gene amplification in selected tumors with adverse prognostic features which scored 1+ by immunohistochemistry (IHC). Methods 75 women with infiltrating ductal carcinoma (IDC) and infiltrating lobular carcinoma (ILC) scoring 1+ by IHC were included. Forty-eight invasive breast carcinoma samples were selected according to unfavorable prognostic tumor characteristics and tested by FISH. HER2 amplification was evaluated using Vysis HER2/Cep17 probe (Path Vysion HER2 DNA Probe Kit®, Abbott Molecular, IL); ratio–based amplification was considered present when the HER2/Cep17 ratio was 2 or more and copy number-based amplification was considered present when the mean HER2 copy number was more than 6, in agreement with the ASCO/CAP/SIAPEC guidelines. Results In 2013, 331 patients with invasive breast tumors were tested by IHC; 75 cases (23%) were scored 1+ of which 62 cases (19%) of IDC and 13 cases (4%) of ILC. Forty-eight invasive breast carcinoma samples (64%) were selected according to one or more unfavorable prognostic tumor characteristics; 22 out of 48 tumors (46%) showed high histological grade (G3); 27 cases (56%) had high proliferative index (Ki-67≥30%); 32 tumor samples (67%) were node-positive; and 29 cases (60%) showed vascular invasion. FISH was performed on 31 of the 1+ patients with adverse tumor characteristics and 7 IDC out of 48 (14.6%) showed HER2 amplification. Conclusions Our preliminary retrospective data suggest that 7 patients out of 48 (14.6%) scoring 1+ by IHC show HER2 amplification, in agreement with the most recently published literature data. In order to not deny the benefit deriving from trastuzumab administration, in breast cancer patients showing IHC 1+, it is advisable to test HER2 gene amplification by FISH. Bibliografia Goldhirsch A, Gelber RD, Piccart-Gebhart MJ, de Azambuja E, Procter M, Suter TM, Jackisch C, Cameron D, Weber HA, Heinzmann D, Dal Lago L, McFadden E, Dowsett M, Untch M, Gianni L, Bell R, Köhne CH, Vindevoghel A, Andersson M, Brunt AM, Otero-Reyes D, Song S, Smith I, Leyland-Jones B, Baselga J; Herceptin Adjuvant (HERA) Trial Study Team. 2 years versus 1 year of adjuvant trastuzumab for HER2-positive breast cancer (HERA): an open-label, randomised controlled trial. Lancet. 2013 Sep 21;382(9897):1021-8. Iorfida M, Dellapasqua S, Bagnardi V, Cardillo A, Rotmensz N, Mastropasqua MG, Bottiglieri L, Goldhirsch A, Viale G, Colleoni M. HER2-negative (1+) breast cancer with unfavorable prognostic features: to FISH or not to FISH? Ann Oncol. 2012 May;23(5):1371-2. Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE Jr, Davidson NE, Tan-Chiu E, Martino S, Paik S, Kaufman PA, Swain SM, Pisansky TM, Fehrenbacher L, Kutteh LA, Vogel VG, Visscher DW, Yothers G, Jenkins RB, Brown AM, Dakhil SR, Mamounas EP, Lingle WL, Klein PM, Ingle JN, Wolmark N. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005 Oct 20;353(16):1673-84

Immunohistochemical/histochemical double staining method in the study of columnar metaplasia of the oesophagus

Intestinal metaplasia in Barrett\u2019s oesopha- gus (BO) represents an important risk factor for oesophageal adenocarcinoma. Instead, few and controversial data are reported about the progression risk of columnar-lined oesophagus without intestinal metaplasia (CLO), posing an issue about its clinical management. The aim was to evaluate if some immunophenotyp- ic changes were present in CLO independently of the presence of the goblet cells. We studied a series of oesophageal biopsies from patients with endoscopic finding of columnar metapla- sia, by performing some immunohistochemical stainings (CK7, p53, AuroraA) combined with histochemistry (Alcian-blue and Alcian/PAS), with the aim of simultaneously assess the his- tochemical features in cells that shows an aber- rant expression of such antigens. We evidenced a cytoplasmic expression of CK7 and a nuclear expression of Aurora A and p53, both in goblet cells of BO and in non-goblet cells of CLO, some of which showing mild dysplasia. These find- ings suggest that some immunophenotypic changes are present in CLO and they can pre- cede the appearance of the goblet cells or can be present independently of them, confirming the conception of BO as the condition charac- terized by any extention of columnar epitheli- um. This is the first study in which a combined immunohistochemical/histochemical method has been applied to Barrett pathology