Cardiac safety of dual anti-HER2 blockade with pertuzumab plus trastuzumab in early HER2-positive breast cancer in the APHINITY trial.

BACKGROUND: Trastuzumab increases the incidence of cardiac events (CEs) in patients with breast cancer (BC). Dual blockade with pertuzumab (P) and trastuzumab (T) improves BC outcomes and is the standard of care for high-risk human epidermal growth factor receptor 2 (HER2)-positive early BC patients. We analyzed the cardiac safety of P and T in the phase III APHINITY trial. PATIENTS AND METHODS: Left ventricular ejection fraction (LVEF) ≥ 55% was required at study entry. LVEF assessment was carried out every 3 months during treatment, every 6 months up to month 36, and yearly up to 10 years. Primary CE was defined as heart failure class III/IV and a significant decrease in LVEF (defined as ≥10% from baseline and to <50%), or cardiac death. Secondary CE was defined as a confirmed significant decrease in LVEF, or CEs confirmed by the cardiac advisory board. RESULTS: The safety analysis population consisted of 4769 patients. With 74 months of median follow-up, CEs were observed in 159 patients (3.3%): 83 (3.5%) in P + T and 76 (3.2%) in T arms, respectively. Most CEs occurred during anti-HER2 therapy (123; 77.4%) and were asymptomatic or mildly symptomatic decreases in LVEF (133; 83.6%). There were two cardiac deaths in each arm (0.1%). Cardiac risk factors indicated were age > 65 years, body mass index ≥ 25 kg/m2, baseline LVEF between 55% and <60%, and use of an anthracycline-containing chemotherapy regimen. Acute recovery from a CE based on subsequent LVEF values was observed in 127/155 patients (81.9%). CONCLUSIONS: Dual blockade with P + T does not increase the risk of CEs compared with T alone. The use of anthracycline-based chemotherapy increases the risk of a CE; hence, non-anthracycline chemotherapy may be considered, particularly in patients with cardiovascular risk factors.

Generic boundaries in the Ophiostomatales reconsidered and revised.

The Ophiostomatales was erected in 1980. Since that time, several of the genera have been redefined and others have been described. There are currently 14 accepted genera in the Order. They include species that are the causal agents of plant and human diseases and common associates of insects such as bark beetles. Well known examples include the Dutch elm disease fungi and the causal agents of sporotrichosis in humans and animals. The taxonomy of the Ophiostomatales was confused for many years, mainly due to the convergent evolution of morphological characters used to delimit unrelated fungal taxa. The emergence of DNA-based methods has resolved much of this confusion. However, the delineation of some genera and the placement of various species and smaller lineages remains inconclusive. In this study we reconsidered the generic boundaries within the Ophiostomatales. A phylogenomic framework constructed from genome-wide sequence data for 31 species representing the major genera in the Order was used as a guide to delineate genera. This framework also informed our choice of the best markers from the currently most commonly used gene regions for taxonomic studies of these fungi. DNA was amplified and sequenced for more than 200 species, representing all lineages in the Order. We constructed phylogenetic trees based on the different gene regions and assembled a concatenated data set utilising a suite of phylogenetic analyses. The results supported and confirmed the delineation of nine of the 14 currently accepted genera, i.e. Aureovirgo, Ceratocystiopsis, Esteya, Fragosphaeria, Graphilbum, Hawksworthiomyces, Ophiostoma, Raffaelea and Sporothrix. The two most recently described genera, Chrysosphaeria and Intubia, were not included in the multi-locus analyses. This was due to their high sequence divergence, which was shown to result in ambiguous taxonomic placement, even though the results of phylogenomic analysis supported their inclusion in the Ophiostomatales. In addition to the currently accepted genera in the Ophiostomatales, well-supported lineages emerged that were distinct from those genera. These are described as novel genera. Two lineages included the type species of Grosmannia and Dryadomyces and these genera are thus reinstated and their circumscriptions redefined. The descriptions of all genera in the Ophiostomatales were standardised and refined where this was required and 39 new combinations have been provided for species in the newly emerging genera and one new combination has been provided for Sporothrix. The placement of Afroraffaelea could not be confirmed using the available data and the genus has been treated as incertae sedis in the Ophiostomatales. Paleoambrosia was not included in this study, due to the absence of living material available for this monotypic fossil genus. Overall, this study has provided the most comprehensive and robust phylogenies currently possible for the Ophiostomatales. It has also clarified several unresolved One Fungus-One Name nomenclatural issues relevant to the Order. Taxonomic novelties: New genera: Harringtonia Z.W. de Beer & M. Procter, Heinzbutinia Z.W. de Beer & M. Procter, Jamesreidia Z.W. de Beer & M. Procter, Masuyamyces Z.W. de Beer & M. Procter. New species: Masuyamyces massonianae M. Procter & Z.W. de Beer. New combinations: Dryadomyces montetyi (M. Morelet) M. Procter & Z.W. de Beer, Dryadomyces quercivorus (Kubono & Shin. Ito) M. Procter & Z.W. de Beer, Dryadomyces quercus-mongolicae (K.H. Kim et al.) M. Procter & Z.W. de Beer, Dryadomyces sulphureus (L.R. Batra) M. Procter & Z.W. de Beer, Graphilbum pusillum (Masuya) M. Procter & Z.W. de Beer, Grosmannia abieticolens (K. Jacobs & M.J. Wingf.) M. Procter & Z.W. de Beer, Grosmannia altior (Paciura et al.) M. Procter & Z.W. de Beer, Grosmannia betulae (Jankowiak et al.) M. Procter & Z.W. de Beer, Grosmannia curviconidia (Paciura et al.) M. Procter & Z.W. de Beer, Grosmannia euphyes (K. Jacobs & M.J. Wingf.) M. Procter & Z.W. de Beer, Grosmannia fenglinhensis (R. Chang et al.) M. Procter & Z.W. de Beer, Grosmannia gestamen (de Errasti & Z.W. de Beer) M. Procter & Z.W. de Beer, Grosmannia innermongolica (X.W. Liu et al.) M. Procter & Z.W. de Beer, Grosmannia pistaciae (Paciura et al.) M. Procter & Z.W. de Beer, Grosmannia pruni (Masuya & M.J. Wingf.) M. Procter & Z.W. de Beer, Grosmannia taigensis (Linnak. et al.) M. Procter & Z.W. de Beer, Grosmannia trypodendri (Jankowiak et al.) M. Procter & Z.W. de Beer, Harringtonia aguacate (D.R. Simmons et al.) M. Procter & Z.W. de Beer, Harringtonia brunnea (L.R. Batra) M. Procter & Z.W. de Beer, Harringtonia lauricola (T.C. Harr. et al.) Z.W. de Beer & M. Procter, Heinzbutinia grandicarpa (Kowalski & Butin) Z.W. de Beer & M. Procter, Heinzbutinia microspora (Arx) M. Procter & Z.W. de Beer, Heinzbutinia solheimii (B. Strzalka & Jankowiak) Z.W. de Beer & M. Procter, Jamesreidia coronata (Olchow. & J. Reid) M. Procter & Z.W. de Beer, Jamesreidia nigricarpa (R.W. Davidson) M. Procter & Z.W. de Beer, Jamesreidia rostrocoronata (R.W. Davidson & Eslyn) M. Procter & Z.W. de Beer, Jamesreidia tenella (R.W. Davidson) Z.W. de Beer & M. Procter, Leptographium cainii (Olchow. & J. Reid) M. Procter & Z.W. de Beer, Leptographium europioides (E.F. Wright & Cain) M. Procter & Z.W. de Beer, Leptographium galeiforme (B.K. Bakshi) M. Procter & Z.W. de Beer, Leptographium pseudoeurophioides (Olchow. & J. Reid) M. Procter & Z.W. de Beer, Leptographium radiaticola (J.J. Kim et al.) M. Procter & Z.W. de Beer, Masuyamyces acarorum (R. Chang & Z.W. de Beer) M. Procter & Z.W. de Beer, Masuyamyces ambrosius (B.K. Bakshi) M. Procter & Z.W. de Beer, Masuyamyces botuliformis (Masuya) Z.W. de Beer & M. Procter, Masuyamyces jilinensis (R. Chang et al.) M. Procter & Z.W. de Beer, Masuyamyces lotiformis (Z. Wang & Q. Lu) M. Procter & Z.W. de Beer, Masuyamyces pallidulus (Linnak. et al.) M. Procter & Z.W. de Beer, Masuyamyces saponiodorus (Linnak. et al.) M. Procter & Z.W. de Beer, Sporothrix longicollis (Massee & E.S. Salmon) M. Procter & Z.W. de Beer. Citation: de Beer W, Procter M, Wingfield MJ, Marincowitz S, Duong TA (2022). Generic boundaries in the Ophiostomatales reconsidered and revised. Studies in Mycology 101: 57-120. doi: 10.3114/sim.2022.101.02.

Morphological and molecular characterization of Prosthogonimus falconis n. sp. (Trematoda; Prosthogonimidae), found in a peregrine falcon (Falco peregrinus) (Aves: Falconidae) in the United Arab Emirates.

At a routine health check of a female peregrine falcon, 23 trematodes preliminary identified as Prosthogonimus sp. were removed from the bursa of Fabricius. Based on morphological and molecular examination, a new species, Prosthogonimus falconis, was described. The pear-shaped flukes were 4.3-6.9 mm long, with greatest width posterior to testes. Tegumental spines measuring between 17 and 21 µm long covered the whole body. Length and width ratio of oral to ventral suckers were 1:1.3. Extracaecal, multifollicular vitelline glands commenced prior to acetabulum and terminated posterior to testes. Eggs in the distal uterus measured 21 × 12 µm. Molecular analysis of internal transcribed spacer 2, cytochrome c oxidase subunit 1 and NADH dehydrogenase subunit 1 gene regions revealed that the new species described here is phylogenetically closest to Prosthogonimus cuneatus and Prosthogonimus pellucidus clusters.

A new species of Raffaelea from beetle-infested Leucaena leucocephala.

Species of Raffaelea (Ophiostomatales: Ascomycota) are obligate symbionts of ambrosia beetles, some of which pose a substantial threat to forest trees. Leucaena leucocephala is a small mimosoid tree species that is considered as an invasive weed in most of its introduced range globally. During a field expedition on the French island of Réunion, dying L. leucocephala trees were observed. Samples were taken from these trees and isolations made from symptomatic wood tissues that included beetle tunnels, but in the absence of the beetles themselves. Multiple isolates of a fungus resembling a Raffaelea species were obtained from the discoloured wood associated with the beetle tunnels. To determine their identity, microscopic examination was performed and DNA sequences for three gene regions (ITS, LSU, TUB) were obtained. Phylogenetic analyses based on these gene regions revealed that the isolates represent a new species of Raffaelea, described here as R. borbonica sp. nov. A pathogenicity test was conducted with the fungus, which was shown to cause lesions on the inoculated seedlings, but with a low level of aggressiveness.

Creatine transporter deficiency: Novel mutations and functional studies.

X-linked cerebral creatine deficiency (MIM 300036) is caused by deficiency of the creatine transporter encoded by the SLC6A8 gene. Here we report three patients with this condition from Israel. These unrelated patients were evaluated for global developmental delays and language apraxia. Borderline microcephaly was noted in one of them. Diagnosis was prompted by brain magnetic resonance imaging and spectroscopy which revealed normal white matter distribution, but absence of the creatine peak in all three patients. Biochemical testing indicated normal plasma levels of creatine and guanidinoacetate, but an increased urine creatine/creatinine ratio. The diagnosis was confirmed by demonstrating absent ([14])C-creatine transport in fibroblasts. Molecular studies indicated that the first patient is hemizygous for a single nucleotide change substituting a single amino acid (c.619 C > T, p.R207W). Expression studies in HeLa cells confirmed the causative role of the R207W substitution. The second patient had a three base pair deletion in the SLC6A8 gene (c.1222_1224delTTC, p.F408del) as well as a single base change (c.1254 + 1G > A) at a splicing site in the intron-exon junction of exon 8, the latter occurring de novo. The third patient, had a three base pair deletion (c.1006_1008delAAC, p.N336del) previously reported in other patients with creatine transporter deficiency. These three patients are the first reported cases of creatine transporter deficiency in Israel.

Sequencing analysis of insulin receptor defects and detection of two novel mutations in INSR gene.

Mutations in the insulin receptor gene cause the inherited insulin resistant syndromes Leprechaunism and Rabson-Mendenhall syndrome. These recessive conditions are characterized by intrauterine and post-natal growth restrictions, dysmorphic features, altered glucose homeostasis, and early demise. The insulin receptor gene (INSR) maps to the short arm of chromosome 19 and is composed of 22 exons. Here we optimize the conditions for sequencing this gene and report novel mutations in patients with severe insulin resistance. METHODS: PCR amplification of the 22 coding exons of the INSR gene was performed using M13-tailed primers. Bidirectional DNA sequencing was performed with BigDye Terminator chemistry and M13 primers and the product was analyzed on the ABI 3100 genetic analyzer. Data analysis was performed using Mutation Surveyor software comparing the sequence to a reference INSR sequence (Genbank NC_000019). RESULTS: We sequenced four patients with Leprechaunism or Rabson-Mendenhall syndromes as well as seven samples from normal individuals and confirmed previously identified mutations in the affected patients. Three of the four mutations identified in this group caused premature insertion of a stop codon. In addition, the INSR gene was sequenced in 14 clinical samples from patients with suspected insulin resistance and one novel mutation was found in an infant with a suspected diagnosis of Leprechaunism. DISCUSSION: Leprechaunism and Rabson-Mendenhall syndrome are very rare and difficult to diagnose. Diagnosis is currently based mostly on clinical criteria. Clinical availability of DNA sequencing can provide an objective way of confirming or excluding the diagnosis.

HER2 staining intensity in HER2-positive disease: relationship with FISH amplification and clinical outcome in the HERA trial of adjuvant trastuzumab.

BACKGROUND: Trastuzumab treatment improves survival of HER2-positive primary breast cancer. HER2 staining intensity varies widely in HER2-positive tumours. PATIENTS AND METHODS: We investigated whether differences in immunohistochemical (IHC) staining intensity for HER2 in HER2-positive tumors (IHC 3+ or FISH ratio ≥2.0) was associated with prognosis or benefit from trastuzumab treatment in patients randomized to 1 year or no trastuzumab in the HERceptin Adjuvant (HERA) trial. Median follow-up was 2 years. The nested case-control analysis, included 425 patients (cases) with a disease-free survival (DFS) event and two matched controls (no DFS event) per case. Tissue sections stained for HER2 were assessed for HER2 staining intensity by image analysis. RESULTS: HER2 staining intensity varied widely and correlated with HER2 gene copy number (Spearman, r = 0.498, P < 0.001) or less closely with HER2/CEP17 FISH ratio (r = 0.396, P < 0.001). We found no significant difference in DFS in the observation arm according to staining intensity (odds ratio [OR] change per 10 unit change in intensity: 1.015, 95% confidence interval [CI] 0.930-1.108) and no impact of staining intensity on benefit derived from 1-year trastuzumab (OR: 1.017, 95% CI 0.925-1.120). CONCLUSIONS: Variability in HER2 staining in HER2-positive tumours has no role in clinical management with adjuvant trastuzumab. HERA TRIAL NO: NCT00045032.

Estimating the magnitude of trastuzumab effects within patient subgroups in the HERA trial.

BACKGROUND: Trastuzumab (Herceptin(R)) improves disease-free survival (DFS) and overall survival for patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. We aimed to assess the magnitude of its clinical benefit for subpopulations defined by nodal and steroid hormone receptor status using data from the Herceptin Adjuvant (HERA) study. PATIENTS AND METHODS: HERA is an international multicenter randomized trial comparing 1 or 2 years of trastuzumab treatment with observation after standard chemotherapy in women with HER2-positive breast cancer. In total, 1703 women randomized to 1-year trastuzumab and 1698 women randomized to observation were included in these analyses. Median follow-up was 23.5 months. The primary endpoint was DFS. RESULTS: The overall hazard ratio (HR) for trastuzumab versus observation was 0.64 [95% confidence interval (CI) 0.54-0.76; P < 0.0001], ranging from 0.46 to 0.82 for subgroups. Estimated improvement in 3-year DFS in subgroups ranged from +11.3% to +0.6%. Patients with the best prognosis (those with node-negative disease and tumors 1.1-2.0 cm) had benefit similar to the overall cohort (HR 0.53, 95% CI 0.26-1.07; 3-year DFS improvement +4.6%, 95% CI -4.0% to 13.2%). CONCLUSIONS: Adjuvant trastuzumab therapy reduces the risk of relapse similarly across subgroups defined by nodal status and steroid hormone receptor status, even those at relatively low risk for relapse.

Glucokinase activator PSN-GK1 displays enhanced antihyperglycaemic and insulinotropic actions.

AIMS/HYPOTHESIS: We evaluated the insulinotropic and antihyperglycaemic actions of glucokinase activators (GKAs), especially through acute and subchronic studies in rodent diabetes models with (2R)-2-(4-cyclopropanesulphonylphenyl)-N-(5-fluorothiazol-2-yl)-3-(tetrahydropyran-4-yl)propionamide (PSN-GK1), a novel and potent GKA. MATERIALS AND METHODS: The action of PSN-GK1 on or in the following were investigated: (1) on human liver glucokinase, insulin secretion from MIN6 cells and 2-deoxy-D: -[(3)H]glucose (2-DG) uptake into rat hepatocytes; and (2) in Zucker diabetic fatty rats and in non-diabetic C57Bl/6, diabetic db/db and ob/ob mice. RESULTS: At 5 mmol/l glucose, PSN-GK1 activated glucokinase (4.3-fold, median effective concentration [EC(50)] 130 nmol/l), increased MIN6 insulin secretion (26-fold, EC(50) 267 nmol/l) and 2-DG hepatocytic uptake (threefold, EC(50) 1 micromol/l); at higher glucose concentrations, EC(50)s and fold-effectiveness were both lower. In C57Bl/6 mice, PSN-GK1 reduced blood glucose at 1 and 10 mg/kg (by mouth), but insulin was increased significantly at only the higher dose. In hyperinsulinaemic 10-mmol/l glucose clamps, PSN-GK1 increased 2-DG incorporation into liver glycogen sixfold, directly demonstrating liver effects. PSN-GK1 improved glycaemic profiles in db/db mice and Zucker diabetic fatty rats, diabetic animal models in which GKA efficacy has not previously been described, without causing hypoglycaemia. In ob/ob mice, it dose-dependently reduced excursions in OGTTs. Moreover, after subchronic administration, no tachyphylaxis was evident and glycaemia was improved without alterations to lipid levels, liver weight, glycogen content or body weight. CONCLUSIONS/INTERPRETATION: PSN-GK1 was potently antihyperglycaemic through its effects on insulin release and hepatic glucose metabolism. It is one of the most potent GKAs described in the literature and is active in diabetic animal models where GKAs have not been reported to show efficacy to date. Ongoing human trials are investigating the potential of this novel therapeutic approach.

Actions of kainate and AMPA selective glutamate receptor ligands on nociceptive processing in the spinal cord.

Kainate receptors expressing the GluR5 subunit of glutamate receptor are present at high levels on small diameter primary afferent neurones that are considered to mediate nociceptive inputs. This suggests that GluR5 selective ligands could be novel analgesic agents. The role of kainate receptors on C fibre primary afferents has therefore been probed using three compounds that are selective for homomeric GluR5 receptors. The agonist, ATPA, and the antagonists, LY294486 and LY382884, have been tested in four models of nociception: responses evoked by noxious stimulation of the periphery have been recorded electrophysiologically (1) from hemisected spinal cords from neonatal rats in vitro, (2) from single motor units in adult rats in vivo, (3) from dorsal horn neurones in adult rats in vivo, and (4) in hotplate tests with conscious mice. In some protocols comparisons were made with the AMPA selective antagonist GYKI 53655. The agonist ATPA reduced nociceptive reflexes in vitro, but failed to have effects in vivo. In all tests, the GluR5 antagonists reduced nociceptive responses but only at doses that also affected responses to exogenous AMPA. The AMPA antagonist reduced nociceptive responses at doses causing relatively greater reductions of responses to exogenous AMPA. The results indicate that GluR5 selective ligands do reduce spinal nociceptive responses, but they are not strongly analgesic under these conditions of acute nociception.

Programs to produce high quality dichotic tapes for central auditory testing.

Dichotic stimulation, the simultaneous presentation of two different acoustic signals to the right and left ears, respectively, is used routinely in the clinical assessment of speech lateralization as well as in other central auditory testing procedures in the clinic and laboratory. At present, most researchers and clinicians depend on a few commercial sources for dichotic tapes, because there are a limited number of facilities which are able to produce such tapes. Moreover, some of the commercial tapes currently offered for sale contain important stimulus errors. In the present paper, we describe a general set of programs for the PDP 11 series of computers, which permits sequences of dichotic stimuli to be generated and output with a high degree of precision. These programs therefore allow researchers to generate any desired sequence of dichotic stimulation, for recording and taping, for use in their research. The programs operate in two stages to generate the required dichotic sequences. In the first stage, the constrained random ordering of the stimuli is generated as specified by the user. In the second stage, after the preliminary stimulus preparation has been completed (for example, using a waveform editing package; cf., D.G. Jamieson and D. A. Naugler, Comput. Biomed. Res., 1985, 18, 480), an audio tape is generated with stimuli presented dichotically, with timing and sequencing precisely as specified.