A prospective cohort study assessing clinical referral management & workforce allocation within a UK regional medical genetics service

Abstract Ensuring patient access to genomic information in the face of increasing demand requires clinicians to develop innovative ways of working. This paper presents the first empirical prospective observational cohort study of UK multi-disciplinary genetic service delivery. It describes and explores collaborative working practices including the utilisation and role of clinical geneticists and non-medical genetic counsellors. Six hundred and fifty new patients referred to a regional genetics service were tracked through 850 clinical contacts until discharge. Referral decisions regarding allocation of lead health professional assigned to the case were monitored, including the use of initial clinical contact guidelines. Significant differences were found in the cases led by genetic counsellors and those led by clinical geneticists. Around a sixth, 16.8% (109/650) of referrals were dealt with by a letter back to the referrer or re-directed to another service provider and 14.8% (80/541) of the remaining patients chose not to schedule an appointment. Of the remaining 461 patients, genetic counsellors were allocated as lead health professional for 46.2% (213/461). A further 61 patients did not attend. Of those who did, 86% (345/400) were discharged after one or two appointments. Genetic counsellors contributed to 95% (784/825) of total patient contacts. They provided 93.7% (395/432) of initial contacts and 26.8% (106/395) of patients were discharged at that point. The information from this study informed a planned service re-design. More research is needed to assess the effectiveness and efficiency of different models of collaborative multi-disciplinary working within genetics services. Keywords (MeSH terms) Genetic Services, Genetic Counseling, Interdisciplinary Communication, Cohort Studies, Delivery of Healthcare, Referral and Consultation

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity in vitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.This work was funded by the American Association for Cancer Research, Lustgarten Foundation, and Stand Up to Cancer as a Pancreatic Cancer Collective New Therapies Challenge grant (grant no. SU2C-AACR-PCC-01-18)

SMAD6 variants in craniosynostosis: genotype and phenotype evaluation

Purpose: Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism near BMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantl

Pathogenic variants in the paired-related homeobox 1 gene (PRRX1) cause craniosynostosis with incomplete penetrance

Purpose Studies previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the pre-osteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function variants in PRRX1 associated with craniosynostosis. Methods Trio-based genome, exome or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. Results Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9/1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, seven additional individuals (four families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multi-suture synostosis was present in 11/17 (65% of the cases). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. Conclusion This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis

Amino-acid PET versus MRI guided re-irradiation in patients with recurrent glioblastoma multiforme (GLIAA) – protocol of a randomized phase II trial (NOA 10/ARO 2013-1)

Background: The higher specificity of amino-acid positron emission tomography (AA-PET) in the diagnosis of gliomas, as well as in the differentiation between recurrence and treatment-related alterations, in comparison to contrast enhancement in T1-weighted MRI was demonstrated in many studies and is the rationale for their implementation into radiation oncology treatment planning. Several clinical trials have demonstrated the significant differences between AA-PET and standard MRI concerning the definition of the gross tumor volume (GTV). A small single-center non-randomized prospective study in patients with recurrent high grade gliomas treated with stereotactic fractionated radiotherapy (SFRT) showed a significant improvement in survival when AA-PET was integrated in target volume delineation, in comparison to patients treated based on CT/MRI alone. Methods: This protocol describes a prospective, open label, randomized, multi-center phase II trial designed to test if radiotherapy target volume delineation based on FET-PET leads to improvement in progression free survival (PFS) in patients with recurrent glioblastoma (GBM) treated with re-irradiation, compared to target volume delineation based on T1Gd-MRI. The target sample size is 200 randomized patients with a 1:1 allocation ratio to both arms. The primary endpoint (PFS) is determined by serial MRI scans, supplemented by AA-PET-scans and/or biopsy/surgery if suspicious of progression. Secondary endpoints include overall survival (OS), locally controlled survival (time to local progression or death), volumetric assessment of GTV delineated by either method, topography of progression in relation to MRIor PET-derived target volumes, rate of long term survivors (> 1 year), localization of necrosis after re-irradiation, quality of life (QoL) assessed by the EORTC QLQ-C15 PAL questionnaire, evaluation of safety of FET-application in AA-PET imaging and toxicity of re-irradiation. Discussion: This is a protocol of a randomized phase II trial designed to test a new strategy of radiotherapy target volume delineation for improving the outcome of patients with recurrent GBM. Moreover, the trial will help to develop a standardized methodology for the integration of AA-PET and other imaging biomarkers in radiation treatment planning. Trial registration: The GLIAA trial is registered with ClinicalTrials.gov (NCT01252459, registration date 02.12.2010), German Clinical Trials Registry (DRKS00000634, registration date 10.10.2014), and European Clinical Trials Database (EudraCT-No. 2012-001121-27, registration date 27.02.2012)

Diagnostic value of exome and whole genome sequencing in craniosynostosis

Background Craniosynostosis, the premature fusion of one or more cranial sutures, occurs in ~1 in 2250 births, either in isolation or as part of a syndrome. Mutations in at least 57 genes have been associated with craniosynostosis, but only a minority of these are included in routine laboratory genetic testing. Methods We used exome or whole genome sequencing to seek a genetic cause in a cohort of 40 subjects with craniosynostosis, selected by clinical or molecular geneticists as being high-priority cases, and in whom prior clinically driven genetic testing had been negative. Results We identified likely associated mutations in 15 patients (37.5%), involving 14 different genes. All genes were mutated in single families, except for IL11RA (two families). We classified the other positive diagnoses as follows: commonly mutated craniosynostosis genes with atypical presentation (EFNB1, TWIST1); other core craniosynostosis genes (CDC45, MSX2, ZIC1); genes for which mutations are only rarely associated with craniosynostosis (FBN1, HUWE1, KRAS, STAT3); and known disease genes for which a causal relationship with craniosynostosis is currently unknown (AHDC1, NTRK2). In two further families, likely novel disease genes are currently undergoing functional validation. In 5 of the 15 positive cases, the (previously unanticipated) molecular diagnosis had immediate, actionable consequences for either genetic or medical management (mutations in EFNB1, FBN1, KRAS, NTRK2, STAT3). Conclusions This substantial genetic heterogeneity, and the multiple actionable mutations identified, emphasises the benefits of exome/whole genome sequencing to identify causal mutations in craniosynostosis cases for which routine clinical testing has yielded negative results

A novel ruthenium(II)–cobaloxime supramolecular complex for photocatalytic H_2 evolution: synthesis, characterisation and mechanistic studies

We report the synthesis and characterization of novel mixed-metal binuclear ruthenium(II)–cobalt(II) photocatalysts for hydrogen evolution in acidic acetonitrile. First, 2-(2′-pyridyl)benzothiazole (pbt), 1, was reacted with RuCl_(3)·xH_(2)O to produce [Ru(pbt)_(2)Cl_2]·0.25CH_(3)COCH_3, 2, which was then reacted with 1,10-phenanthroline-5,6-dione (phendione), 3, in order to produce [Ru(pbt)_(2)(phendione)](PF_(6))_2·4H_(2)O, 4. Compound 4 was then reacted with 4-pyridinecarboxaldehyde in order to produce [Ru(pbt)_(2)(L-pyr)](PF_6)_(2)·9.5H_(2)O, 5 (where L-pyr = (4-pyridine)oxazolo[4,5-f]phenanthroline). Compound 5 was then reacted with [Co(dmgBF_2)_(2)(H_(2)O)_2] (where dmgBF_(2) = difluoroboryldimethylglyoximato) in order to produce the mixed-metal binuclear complex, [Ru(pbt)_(2)(L-pyr)Co(dmgBF_(2))_(2)(H_(2)O)](PF_(6))_2·11H_(2)O·1.5CH_(3)COCH_3, 6. [Ru(Me_(2)bpy)_2(L-pyr)Co(dmgBF_2)_(2)(OH_2)](PF_6)_(2), 7 (where Me_(2)bpy = 1,10-phenanthroline, 4,4′-dimethyl-2,2′-bipyridine) and [Ru(phen)_(2)(L-pyr)Co(dmgBF_2)_(2)(OH_2)](PF_(6))_2, 8 were also synthesised. All complexes were characterized by elemental analysis, ESI MS, HRMS, UV-visible absorption, ^(11)B, ^(19)F, and ^(59)Co NMR, ESR spectroscopy, and cyclic voltammetry, where appropriate. Photocatalytic studies carried out in acidified acetonitrile demonstrated constant hydrogen generation longer than a 42 hour period as detected by gas chromatography. Time resolved spectroscopic measurements were performed on compound 6, which proved an intramolecular electron transfer from an excited Ru(II) metal centre to the Co(II) metal centre via the bridging L-pyr ligand. This resulted in the formation of a cobalt(I)-containing species that is essential for the production of H_2 gas in the presence of H^+ ions. A proposed mechanism for the generation of hydrogen is presented

Tacaribe Virus but Not Junin Virus Infection Induces Cytokine Release from Primary Human Monocytes and Macrophages

The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation

Haploinsufficiency for ANKRD11-flanking genes makes the difference between KBG and 16q24.3 microdeletion syndromes:12 new cases

16q24 deletion involving the ANKRD11 gene, ranging from 137 kb to 2 Mb, have been associated with a microdeletion syndrome characterized by variable cognitive impairment, autism spectrum disorder, facial dysmorphisms with dental anomalies, brain abnormalities essentially affecting the corpus callosum and short stature. On the other hand, patients carrying either deletions encompassing solely ANKRD11 or its loss-of-function variants were reported in association with the KBG syndrome, characterized by a very similar phenotype, including mild-to-moderate intellectual disability, short stature and macrodontia of upper incisors, with inter and intrafamilial variability. To assess whether the haploinsufficiency of ANKRD11-flanking genes, such as ZFPM1, CDH15 and ZNF778, contributed to either the severity of the neurological impairment or was associated with other clinical features, we collected 12 new cases with a 16q24.2q24.3 deletion (de novo in 11 cases), ranging from 343 kb to 2.3 Mb. In 11 of them, the deletion involved the ANKRD11 gene, whereas in 1 case only flanking genes upstream to it were deleted. By comparing the clinical and genetic features of our patients with those previously reported, we show that the severity of the neurological phenotype and the frequency of congenital heart defects characterize the deletions that, besides ANKRD11, contain ZFPM1, CDH15 and ZNF778 as well. Moreover, the presence of thrombocytopenia and astigmatism should be taken into account to distinguish between 16q24 microdeletion syndrome and KBG syndrome. The single patient not deleted for ANKRD11, whose phenotype is characterized by milder psychomotor delay, cardiac congenital malformation, thrombocytopenia and astigmatism, confirms all this dat