Contrast Clearance Analysis (CCA) to Assess Viable Tumour following Stereotactic Radiosurgery (SRS) to Brain Metastasis in Non-Small Cell Lung Cancer (NSCLC).

BACKGROUND AND OBJECTIVE: Brain metastases are common in lung cancer and increasingly treated using targeted radiotherapy techniques such as stereotactic radiosurgery (SRS). Using MRI, post-SRS changes may be difficult to distinguish from progressive brain metastasis. Contrast clearance analysis (CCA) uses T1-weighted MRI images to assess the clearance of gadolinium and can be thus used to assess vascularity and active tumours. DESIGN AND METHODS: We retrospectively assessed CCAs in 62 patients with non-small cell lung cancer (NSCLC) undergoing 104 CCA scans in a single centre. RESULTS: The initial CCA suggested the aetiology of equivocal changes on standard MRI in 80.6% of patients. In all patients whose initial CCA showed post-SRS changes and who underwent serial CCAs, the initial diagnosis was upheld with the serial imaging. In only two cases of a presumed progressive tumour on the initial CCA, subsequent treatment for radionecrosis was instigated; a retrospective review and re-evaluation of the CCAs show that progression was reported where a thin rim of rapid contrast clearance was seen, and this finding has been subsequently recognised as a feature of post-treatment change on CCAs. The lack of concordance with CCA findings in those who underwent surgical resection was also found to be due to the over-reporting of the thin blue rim as disease in the early cases of CCA use and, in three cases, potentially related to timelines longer than 7 days prior to surgery, both factors being unknown during the early implementation phase of CCA at our centre but subsequently learned. CONCLUSIONS: Our single-centre experience shows CCA to be feasible and useful in patients with NSCLC in cases of diagnostic uncertainty in MRI. It has helped guide treatment in the majority of patients, with subsequent outcomes following the implementation of the treatment based on the results, suggesting correct classification. Recommendations from our experience of the implementation include the careful consideration of the thin rim of the rapid contrast clearance and the timing of the CCA prior to surgery for suspected brain metastasis progression.

Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins).

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.

The lung cancers: staging and response, CT, 18F-FDG PET/CT, MRI, DWI: review and new perspectives.

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer deaths in both sexes combined. Recent years have seen major advances in the diagnostic and treatment options for patients with non-small-cell lung cancer (NSCLC), including the routine use of 2-deoxy-2[18F]-fluoro-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) in staging and response evaluation, minimally invasive endoscopic biopsy, targeted radiotherapy, minimally invasive surgery, and molecular and immunotherapies. In this review, the central roles of CT and 18F-FDG PET/CT in staging and response in both NSCLC and malignant pleural mesothelioma (MPM) are critically assessed. The Tumour Node Metastases (TNM-8) staging systems for NSCLC and MPM are presented with critical appraisal of the strengths and pitfalls of imaging. Overviews of the Response Evaluation Criteria in Solid Tumours (RECIST 1.1) for NSCLC and the modified RECIST criteria for MPM are provided, together with discussion of the benefits and limitations of these anatomical-based tools. Metabolic response assessment (not evaluated by RECIST 1.1) will be explored. We introduce the Positron Emission Tomography Response Criteria in Solid Tumours (PERCIST 1.0) to include its advantages and challenges. The limitations of both anatomical and metabolic assessment criteria when applied to NSCLC treated with immunotherapy and the important concept of pseudoprogression are addressed with reference to immune RECIST (iRECIST). Separate consideration is given to the diagnosis and follow up of solitary pulmonary nodules with reference to the British Thoracic Society guidelines and Fleischner guidelines and use of the Brock (CT-based) and Herder (addition of 18F-FDG PET/CT) models for assessing malignant potential. We discuss how these models inform decisions by the multidisciplinary team, including referral of suspicious nodules for non-surgical management in patients unsuitable for surgery. We briefly outline current lung screening systems being used in the UK, Europe and North America. Emerging roles for MRI in lung cancer imaging are reviewed. The use of whole-body MRI in diagnosing and staging NSCLC is discussed with reference to the recent multicentre Streamline L trial. The potential use of diffusion-weighted MRI to distinguish tumour from radiotherapy-induced lung toxicity is discussed. We briefly summarise the new PET-CT radiotracers being developed to evaluate specific aspects of cancer biology, other than glucose uptake. Finally, we describe how CT, MRI and 18F-FDG PET/CT are moving from primarily diagnostic tools for lung cancer towards having utility in prognostication and personalised medicine with the agency of artificial intelligence.

Imaging in metastatic breast cancer, CT, PET/CT, MRI, WB-DWI, CCA: review and new perspectives.

BACKGROUND: Breast cancer is the most frequent cancer in women and remains the second leading cause of death in Western countries. It represents a heterogeneous group of diseases with diverse tumoral behaviour, treatment responsiveness and prognosis. While major progress in diagnosis and treatment has resulted in a decline in breast cancer-related mortality, some patients will relapse and prognosis in this cohort of patients remains poor. Treatment is determined according to tumor subtype; primarily hormone receptor status and HER2 expression. Menopausal status and site of disease relapse are also important considerations in treatment protocols. MAIN BODY: Staging and repeated evaluation of patients with metastatic breast cancer are central to the accurate assessment of disease extent at diagnosis and during treatment; guiding ongoing clinical management. Advances have been made in the diagnostic and therapeutic fields, particularly with new targeted therapies. In parallel, oncological imaging has evolved exponentially with the development of functional and anatomical imaging techniques. Consistent, reproducible and validated methods of assessing response to therapy is critical in effectively managing patients with metastatic breast cancer. CONCLUSION: Major progress has been made in oncological imaging over the last few decades. Accurate disease assessment at diagnosis and during treatment is important in the management of metastatic breast cancer. CT (and BS if appropriate) is generally widely available, relatively cheap and sufficient in many cases. However, several additional imaging modalities are emerging and can be used as adjuncts, particularly in pregnancy or other diagnostically challenging cases. Nevertheless, no single imaging technique is without limitation. The authors have evaluated the vast array of imaging techniques - individual, combined parametric and multimodal - that are available or that are emerging in the management of metastatic breast cancer. This includes WB DW-MRI, CCA, novel PET breast cancer-epitope specific radiotracers and radiogenomics.

PARP inhibition utilized in combination therapy with Olaparib-Temozolomide to achieve disease stabilization in a rare case of BRCA1-mutant, metastatic myxopapillary ependymoma.

Myxopapillary ependymoma (MPE) is a primary tumor of the central nervous system (CNS), characteristically an indolent malignancy involving the spinal conus medullaris, Filum terminale or cauda equina. We present a rare case of MPE, recurrent in the pelvic soft tissue with eventual pleural and intra-pulmonary metastasis. Refractory to repeated gross resection, adjuvant radiotherapy, platinum-based chemotherapy and temozolomide exploitation of mutant somatic BRCA1 status with the addition of a poly (ADP-ribose); polymerase inhibitor (PARPi) in a novel combination regimen with olaparib-temozolomide (OT) has achieved stable radiological disease after 10 cycles.

Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells.

While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.

Involvement of the Arp2/3 complex and Scar2 in Golgi polarity in scratch wound models.

Cell motility and cell polarity are essential for morphogenesis, immune system function, and tissue repair. Many animal cells move by crawling, and one main driving force for movement is derived from the coordinated assembly and disassembly of actin filaments. As tissue culture cells migrate to close a scratch wound, this directional extension is accompanied by Golgi apparatus reorientation, to face the leading wound edge, giving the motile cell inherent polarity aligned relative to the wound edge and to the direction of cell migration. Cellular proteins essential for actin polymerization downstream of Rho family GTPases include the Arp2/3 complex as an actin nucleator and members of the Wiskott-Aldrich Syndrome protein (WASP) family as activators of the Arp2/3 complex. We therefore analyzed the involvement of the Arp2/3 complex and WASP-family proteins in in vitro wound healing assays using NIH 3T3 fibroblasts and astrocytes. In NIH 3T3 cells, we found that actin and Arp2/3 complex contributed to cell polarity establishment. Moreover, overexpression of N-terminal fragments of Scar2 (but not N-WASP or Scar1 or Scar3) interfere with NIH 3T3 Golgi polarization but not with cell migration. In contrast, actin, Arp2/3, and WASP-family proteins did not appear to be involved in Golgi polarization in astrocytes. Our results thus indicate that the requirement for Golgi polarity establishment is cell-type specific. Furthermore, in NIH 3T3 cells, Scar2 and the Arp2/3 complex appear to be involved in the establishment and maintenance of Golgi polarity during directed migration.

Asymmetric masks for laboratory-based X-ray phase-contrast imaging with edge illumination.

We report on an asymmetric mask concept that enables X-ray phase-contrast imaging without requiring any movement in the system during data acquisition. The method is compatible with laboratory equipment, namely a commercial detector and a rotating anode tube. The only motion required is that of the object under investigation which is scanned through the imaging system. Two proof-of-principle optical elements were designed, fabricated and experimentally tested. Quantitative measurements on samples of known shape and composition were compared to theory with good agreement. The method is capable of measuring the attenuation, refraction and (ultra-small-angle) X-ray scattering, does not have coherence requirements and naturally adapts to all those situations in which the X-ray image is obtained by scanning a sample through the imaging system.

Inclusion of Scar/WAVE3 in a similar complex to Scar/WAVE1 and 2.

BACKGROUND: The Scar/WAVE family of proteins mediates signals to actin assembly by direct activation of the Arp2/3 complex. These proteins have been characterised as major regulators of lamellipodia formation downstream of Rac activation and as members of large protein complexes. RESULTS: We have investigated the interactions of the three human Scar/WAVE isoforms with several previously described binding partners for Scar/WAVE 1 or 2. We find that all three Scar/WAVE isoforms behave similarly and are likely to participate in the same kinds of protein complexes that regulate actin assembly. CONCLUSION: Differences between Scar/WAVE proteins are therefore likely to be at the level of tissue distribution or subtle differences in the affinity for specific binding partners.

Calpain cleavage of the B isoform of Ins(1,4,5)P3 3-kinase separates the catalytic domain from the membrane anchoring domain.

Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] is one of the key intracellular second messengers in cells and mobilizes Ca2+ stores in the ER (endoplasmic reticulum). Ins(1,4,5)P3 has a short half-life within the cell, and is rapidly metabolized through one of two pathways, one of which involves further phosphorylation of the inositol ring: Ins(1,4,5)P3 3-kinase (IP3-3K) phosphorylates Ins(1,4,5)P3, resulting in the formation of inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5)P4]. There are three known isoforms of IP3-3K, designated IP3-3KA, IP3-3KB and IP3-3KC. These have differing N-termini, but highly conserved C-termini harbouring the catalytic domain. The three IP3-3K isoforms have different subcellular locations and the B-kinase is uniquely present in both cytosolic and membrane-bound pools. As it is the N-terminus of the B-kinase that differs most from the A- and C-kinases, we have hypothesized that this portion of the protein may be responsible for membrane localization. Although there are no known membrane-targeting protein motifs within the sequence of IP3-3KB, it is found to be tightly associated with the ER membrane. Here, we show that specific regions of the N-terminus of IP3-3KB are necessary and sufficient for efficient membrane localization of the protein. We also report that, in the presence of Ca2+, the kinase domain of IP3-3KB is cleaved from the membrane-anchoring region by calpain.

Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex.

The assembly of a branched network of actin filaments provides the mechanical propulsion that drives a range of dynamic cellular processes, including cell motility. The Arp2/3 complex is a crucial component of such filament networks. Arp2/3 nucleates new actin filaments while bound to existing filaments, thus creating a branched network. In recent years, a number of proteins that activate the filament nucleation activity of Arp2/3 have been identified, most notably the WASP (Wiskott-Aldrich syndrome protein) family. WASP-family proteins activate the Arp2/3 complex, and consequently stimulate actin assembly, in response to extracellular signals. Structural studies have provided a significant refinement in our understanding of the molecular detail of how the Arp2/3 complex nucleates actin filaments. There has also been much progress towards an understanding of the complicated signalling processes that regulate WASP-family proteins. In addition, the use of gene disruption in a number of organisms has led to new insights into the specific functions of individual WASP-family members. The present review will discuss the Arp2/3 complex and its regulators, in particular the WASP-family proteins. Emphasis will be placed on recent developments in the field that have furthered our understanding of actin dynamics and cell motility.

Relationship between p53 codon 72 polymorphism and susceptibility to sunburn and skin cancer.

Upregulation of p53 protein induces either growth arrest or apoptosis in response to cellular injury This is signaled from a highly conserved p53 domain between codons 64 and 92, where a functional polymorphism results in either a proline (p53-72P) or an arginine (p53-72R) at codon 72. Preliminary studies suggest that p53-72R may be a risk factor for cervical cancer and, consistent with this, preferential mutation and retention of the p53-72R allele has also been demonstrated in other cancers of squamous cell origin. Here we examine the relationship between allelic forms of p53 and nonmelanoma skin cancer, by determining the correlation with susceptibility to sunburn, which is a known risk factor, and then by p53 sequence analysis of a large series of tumors. We found a significant positive association between p53-72R and susceptibility to sunburn, as assessed by skin phototype and minimal erythemal dose following solar-simulated radiation (p = 0.0001 for trend). We also found a significant association between p53-72R homozygosity and nonmelanoma skin cancer in renal transplant recipients (basal cell carcinoma, p < 0.01; squamous cell carcinoma, p < 0.05) but not in immunocompetent patients compared with skin type matched controls. p53 sequence data revealed mutations in 30 of 70 (42.9%) nonmelanoma skin cancers, 28 (93%) of which were in the p53-72R allele. Loss of heterozygosity occurred more frequently in p53-72RP than in p53-72RR tumors (p = 0.0001) with preferential loss of p53-72P in heterozygotes (p = 0.016), irrespective of the mutant status of the concomitant allele. Together these data infer functional differences between polymorphic forms of p53 that are likely to be relevant to skin carcinogenesis.

Photosensitivity disorders: cause, effect and management.

Abnormal photosensitivity syndromes form a significant and common group of skin diseases. They include primary (idiopathic) photodermatoses such as polymorphic light eruption (PLE), chronic actinic dermatitis (CAD), actinic prurigo, hydroa vacciniforme and solar urticaria, in addition to drug- and chemical-induced photosensitivity and photo-exacerbated dermatoses. They can be extremely disabling and difficult to diagnose. PLE, characterized by a recurrent pruritic papulo-vesicular eruption of affected skin within hours of sun exposure, is best managed by restriction of ultraviolet radiation (UVR) exposure and the use of high sun protection factor (SPF) sunscreens. If these measures are insufficient, prophylactic phototherapy with PUVA, broadband UVB or narrowband UVB (TL-01) for several weeks during spring may be necessary. CAD manifests as a dermatitis of chronically sun-exposed skin. Again, UVR exposure needs to be restricted; cyclosporine, azathioprine or PUVA may also be necessary. Actinic prurigo is characterized by the presence of excoriated papules and nodules on the face and limbs, most prominent and numerous distally. Actinic prurigo is managed again by restriction of UVR and the use of high SPF sunscreens; PUVA or broadband UVB therapy, or low doses of thalidomide may be necessary. Hydroa vacciniforme causes crops of discrete erythematous macules, 2 to 3mm in size, that evolve into blisters within a couple of days of sun exposure. Treatment for this rare disease is difficult; absorbent sunscreens and restricted UVR exposure may help. Solar urticaria is characterized by acute erythema and urticarial wealing after exposure to UVR. Treatment options for solar urticaria include non-sedating antihistamines such as fexofenadine and cetirizine; other options include absorbent sunscreens, restriction of UVR at the relevant wavelength, maintenance of a non-responsive state with natural or artificial light exposure and plasmapheresis. Industrial, cosmetic and therapeutic agents can induce exogenous drug- or chemical-induced photosensitivity. The clinical pattern is highly varied, depending on the agent; treatment is based on removal of the photosensitizer along with restriction of UVR exposure. Predominantly non-photosensitive dermatoses may also be exacerbated or precipitated by UVR; exposure to UVR should be reduced and sunscreens should be advocated, along with appropriate treatment of the underlying disease.

Proof-of-concept demonstration of edge-illumination x-ray phase contrast imaging combined with tomosynthesis.

In this note we present the first proof-of-concept results on the potential effectiveness of the edge-illumination x-ray phase contrast method (in its 'coded-aperture' based lab implementation) combined with tomosynthesis. We believe that, albeit admittedly preliminary (e.g. we only present phantom work), these results deserve early publication in a note primarily for four reasons. First, we fully modelled the imaging acquisition method, and validated the simulation directly with experimental results. This shows that the implementation of the method in the new geometry is understood, and thus that it will be possible to use the model to simulate more complex scenarios in the future. Secondly, we show that a strong phase contrast signal is preserved in the reconstructed tomosynthesis slices: this was a concern, as the high spatial frequency nature of the signal makes it sensitive to any filtration-related procedure. Third, we show that, despite the non-optimized nature of the imaging prototype used, we can perform a full angular scan at acceptable dose levels and with exposure times not excessively distant from what is required by clinical practice. Finally, we discuss how the proposed phase contrast method, unlike other approaches apart from free-space propagation (which however requires a smaller focal spot, thus reducing the flux and increasing exposure times), can be easily implemented in a tomosynthesis geometry suitable for clinical use. In summary, we find that these technical results indicate a high potential for the combination of the two methods. Combining slice separation with detail enhancement provided by phase effects would substantially increase the detectability of small lesions and/or calcifications, which we aim to demonstrate in the next steps of this study.