Analysis of cell free DNA to predict outcome to bevacizumab therapy in colorectal cancer patients.

To predict outcome to combination bevacizumab (BVZ) therapy, we employed cell-free DNA (cfDNA) to determine chromosomal instability (CIN), nucleosome footprints (NF) and methylation profiles in metastatic colorectal cancer (mCRC) patients. Low-coverage whole-genome sequencing (LC-WGS) was performed on matched tumor and plasma samples, collected from 74 mCRC patients from the AC-ANGIOPREDICT Phase II trial (NCT01822444), and analysed for CIN and NFs. A validation cohort of plasma samples from the University Medical Center Mannheim (UMM) was similarly profiled. 61 AC-ANGIOPREDICT plasma samples collected before and following BVZ treatment were selected for targeted methylation sequencing. Using cfDNA CIN profiles, AC-ANGIOPREDICT samples were subtyped with 92.3% accuracy into low and high CIN clusters, with good concordance observed between matched plasma and tumor. Improved survival was observed in CIN-high patients. Plasma-based CIN clustering was validated in the UMM cohort. Methylation profiling identified differences in CIN-low vs. CIN high (AUC = 0.87). Moreover, significant methylation score decreases following BVZ was associated with improved outcome (p = 0.013). Analysis of CIN, NFs and methylation profiles from cfDNA in plasma samples facilitates stratification into CIN clusters which inform patient response to treatment.

A clinical seminar on Spinoza and Bion: a conversation between Miller AND Koritar.

The present transcript follows an online discussion held on April 3, 2022, between Ian Miller, author of Clinical Spinoza: Integrating His Philosophy with Contemporary Therapeutic Practice (2022), and Endre Koritar.

BF2-Azadipyrromethene Fluorophores for Intraoperative Vital Structure Identification.

A series of mono- and bis-polyethylene glycol (PEG)-substituted BF2-azadipyrromethene fluorophores have been synthesized with emissions in the near-infrared region (700-800 nm) for the purpose of fluorescence guided intraoperative imaging; chiefly ureter imaging. The Bis-PEGylation of fluorophores resulted in higher aqueous fluorescence quantum yields, with PEG chain lengths of 2.9 to 4.6 kDa being optimal. Fluorescence ureter identification was possible in a rodent model with the preference for renal excretion notable through comparative fluorescence intensities from the ureters, kidneys and liver. Ureteral identification was also successfully performed in a larger animal porcine model under abdominal surgical conditions. Three tested doses of 0.5, 0.25 and 0.1 mg/kg all successfully identified fluorescent ureters within 20 min of administration which was sustained up to 120 min. 3-D emission heat map imaging allowed the spatial and temporal changes in intensity due to the distinctive peristaltic waves of urine being transferred from the kidneys to the bladder to be identified. As the emission of these fluorophores could be spectrally distinguished from the clinically-used perfusion dye indocyanine green, it is envisaged that their combined use could be a step towards intraoperative colour coding of different tissues.

Second Thoughts: Pseudo-reality Between Hypnosis and Spectacle.

"Second Thoughts: Pseudo-Reality Between Hypnosis and Spectacle" expands the discussion of Endre Koritar's and Robert Prince's presentations in the 2021 International Sándor Ferenczi Network webinar series, Listening with Ferenczi (Koritar, 2022a; Prince, 2022). Beginning with a segue from Prince's reference to Thomas Mann's "Mario and the Magician," the present paper expands focus from the dynamics of leadership to the grooming of followership along the pathway of image as the American national addiction to pseudo-reality, a fascinating and omnipotently expected, always disappointing entitlement (Boorstin, 1961). Political spectacle, ascendant in American executive, legislative, and judicial performance since 2016, functions as image-supercharged, with its guarantee of gratification powerfully overcoming disappointment.

Refining Glioblastoma Surgery through the Use of Intra-Operative Fluorescence Imaging Agents.

Glioblastoma (GBM) is the most aggressive adult brain tumour with a dismal 2-year survival rate of 26-33%. Maximal safe resection plays a crucial role in improving patient progression-free survival (PFS). Neurosurgeons have the significant challenge of delineating normal tissue from brain tumour to achieve the optimal extent of resection (EOR), with 5-Aminolevulinic Acid (5-ALA) the only clinically approved intra-operative fluorophore for GBM. This review aims to highlight the requirement for improved intra-operative imaging techniques, focusing on fluorescence-guided imaging (FGS) and the use of novel dyes with the potential to overcome the limitations of current FGS. The review was performed based on articles found in PubMed an.d Google Scholar, as well as articles identified in searched bibliographies between 2001 and 2022. Key words for searches included 'Glioblastoma' + 'Fluorophore'+ 'Novel' + 'Fluorescence Guided Surgery'. Current literature has favoured the approach of using targeted fluorophores to achieve specific accumulation in the tumour microenvironment, with biological conjugates leading the way. These conjugates target specific parts overexpressed in the tumour. The positive results in breast, ovarian and colorectal tissue are promising and may, therefore, be applied to intracranial neoplasms. Therefore, this design has the potential to produce favourable results in GBM by reducing the residual tumour, which translates to decreased tumour recurrence, morbidity and ultimately, mortality in GBM patients. Several preclinical studies have shown positive results with targeted dyes in distinguishing GBM cells from normal brain parenchyma, and targeted dyes in the Near-Infrared (NIR) emission range offer promising results, which may be valuable future alternatives.

Implementing subtype-specific pre-clinical models of breast cancer to study pre-treatment aspirin effects.

BACKGORUND: Prior data suggest pre-diagnostic aspirin use impacts breast tumour biology and patient outcome. Here, we employed faithful surgical resection models of HER2+ and triple-negative breast cancer (TNBC), to study outcome and response mechanisms across breast cancer subtypes. METHOD: NOD/SCID mice were implanted with HER2+ MDA-MB-231/LN/2-4/H2N, trastuzumab-resistant HER2+ HCC1954 or a TNBC patient-derived xenograft (PDX). A daily low-dose aspirin regimen commenced until primary tumours reached ~250 mm3 and subsequently resected. MDA-MB-231/LN/2-4/H2N mice were monitored for metastasis utilising imaging. To interrogate the survival benefit of pre-treatment aspirin, 3 weeks post-resection, HCC1954/TNBC animals received standard-of-care (SOC) chemotherapy for 6 weeks. Primary tumour response to aspirin was interrogated using immunohistochemistry. RESULTS: Aspirin delayed time to metastasis in MDA-MB-231/LN/2-4/H2N xenografts and decreased growth of HER2+ /TNBC primary tumours. Lymphangiogenic factors and lymph vessels number were decreased in HER2+ tumours. However, no survival benefit was seen in aspirin pre-treated animals (HCC1954/TNBC) that further received adjuvant SOC, compared with animals treated with SOC alone. In an effort to study mechanisms responsible for the observed reduction in lymphangiogenesis in HER2+ BC we utilised an in vitro co-culture system of HCC1954 tumour cells and mesenchymal stromal cells (MSC). Aspirin abrogated the secretion of VEGF-C in MSCs and also decreased the lymph/angiogenic potential of the MSCs and HCC1954 by tubule formation assay. Furthermore, aspirin decreased the secretion of uPA in HCC1954 cells potentially diminishing its metastatic capability. CONCLUSION: Our data employing clinically relevant models demonstrate that aspirin alters breast tumour biology. However, aspirin may not represent a robust chemo-preventative agent in the HER2+ or TNBC setting.

Histories, Traumas, and Emotional Foreclosure from Manhattan to Dublin and Back.

The present paper begins with the particulars of clinical practice in Ireland. Through clinical example, it examines the emotion of shame, widely paired with blame, as a socially acceptable admission of psychological functioning, both in exercising and in denying the communication of more profound feeling. As a necessary emotional outlet, shame authorizes aggressions both large and small. Shame demands that certain acts, often seemingly random and subjective, are to be judged disgraceful in others. Shame demands that someone, everyone, endures hurt, at least through social judgement. Passing through the armoring of shame as social defense, clinical examples focus on the defensive action of foreclosure as an interpersonal act of nihilation, reducing another to no-thing, while at the same time diminishing one's own sense of inadequacy. Discerning this clinical pattern, the author generalizes from practice in a particular place to similar observable patterns, both with different populations, and in different contexts.

Implementing Systems Modelling and Molecular Imaging to Predict the Efficacy of BCL-2 Inhibition in Colorectal Cancer Patient-Derived Xenograft Models.

Resistance to chemotherapy often results from dysfunctional apoptosis, however multiple proteins with overlapping functions regulate this pathway. We sought to determine whether an extensively validated, deterministic apoptosis systems model, 'DR_MOMP', could be used as a stratification tool for the apoptosis sensitiser and BCL-2 antagonist, ABT-199 in patient-derived xenograft (PDX) models of colorectal cancer (CRC). Through quantitative profiling of BCL-2 family proteins, we identified two PDX models which were predicted by DR_MOMP to be sufficiently sensitive to 5-fluorouracil (5-FU)-based chemotherapy (CRC0344), or less responsive to chemotherapy but sensitised by ABT-199 (CRC0076). Treatment with ABT-199 significantly improved responses of CRC0076 PDXs to 5-FU-based chemotherapy, but showed no sensitisation in CRC0344 PDXs, as predicted from systems modelling. 18F-Fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) scans were performed to investigate possible early biomarkers of response. In CRC0076, a significant post-treatment decrease in mean standard uptake value was indeed evident only in the combination treatment group. Radiomic CT feature analysis of pre-treatment images in CRC0076 and CRC0344 PDXs identified features which could phenotypically discriminate between models, but were not predictive of treatment responses. Collectively our data indicate that systems modelling may identify metastatic (m)CRC patients benefitting from ABT-199, and that 18F-FDG-PET could independently support such predictions.

Spinoza's Announcement of Psychological Practice.

Following Freud's admission of his own 'dependence on Spinoza's doctrine', the present paper extends psychoanalytic discernment of a bright line from Baruch Spinoza's natural philosophy across the evolution of natural and social sciences to Freud's development of psychoanalysis and forward, to contemporary psychoanalytic thinking. The lens through which these developments unfold is a close reading of Spinoza's first, incomplete methodological statement, the Treatise on the Emendation of the Intellect. Tracking both the developing form and textual literary context from which this Spinozan inquiry proceeds, we recognize specific anticipations of contemporary psychoanalytic phenomena.

Aryl hydrocarbon receptor nuclear translocator-like (ARNTL/BMAL1) is associated with bevacizumab resistance in colorectal cancer via regulation of vascular endothelial growth factor A.

BACKGROUND: The identification of new biomarkers and the development of novel, targetable contexts of vulnerability are of urgent clinical need in drug-resistant metastatic colorectal cancer (mCRC). Aryl-Hydrocarbon-Receptor-Nuclear-Translocator-Like (ARNTL/BMAL1) is a circadian clock-regulated transcription factor promoting expression of genes involved in angiogenesis and tumour progression. We hypothesised that BMAL1 increases expression of the vascular endothelial growth factor A VEGFA gene and, thereby, confers resistance to anti-angiogenic therapy with bevacizumab (Beva), a clinically used antibody for neutralization of VEGFA. METHODS: PCR and immunohistochemistry were employed to assess BMAL1 expression in mice (C57BL/6 J Apcmin/+; BALB/c nu/nu xenografts) and CRC patients under combination chemotherapy with Beva. BMAL1 single nucleotide gene polymorphisms (SNPs) were analysed by DNA-microarray in clinical samples. BMAL1 functions were studied in human CRC cell lines using colorimetric growth, DNA-binding and reporter assays. FINDINGS: In murine CRCs, high BMAL1 expression correlated with poor preclinical response to Beva treatment. In CRC patients' tumours (n = 74), high BMAL1 expression was associated with clinical non-response to combination chemotherapy with Beva (*p = .0061) and reduced progression-free survival (PFS) [*p = .0223, Hazard Ratio (HR) = 1.69]. BMAL1 SNPs also correlated with shorter PFS (rs7396943, rs7938307, rs2279287) and overall survival (OS) [rs11022780, *p = .014, HR = 1.61]. Mechanistically, Nuclear-Receptor-Subfamily-1-Group-D-Member-1 (NR1D1/REVERBA) bound a - 672 bp Retinoic-Acid-Receptor-Related-Orphan-Receptor-Alpha-responsive-element (RORE) adjacent to a BMAL1 DNA-binding motif (E-box) in the VEGFA gene promoter, resulting in increased VEGFA synthesis and proliferation of human CRC cell lines. INTERPRETATION: BMAL1 was associated with Beva resistance in CRC. Inhibition of REVERBA-BMAL1 signalling may prevent resistance to anti-angiogenic therapy. FUND: This work was in part supported by the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]).

Durability of cell line xenograft resection models to interrogate tumor micro-environment targeting agents.

Angiogenesis is a key tumor microenvironment (TME) event underpinning tumor growth and metastasis. Nevertheless, the relatively poor performance of anti-angiogenic therapies in clinical trials compared to pre-clinical studies implies that classical subcutaneous xenograft models have limited predictive potential in this setting. To address this issue, we established orthotopic surgical resection models of breast cancer, which replicate the phenotype of clinical post-resection micro-metastasis. To demonstrate the power and precision of these models, we recapitulated the BETH adjuvant trial (NCT00625898) where the addition of bevacizumab (BVZ) to chemotherapy plus trastuzumab (Trast) failed to provide additional benefit. SCID mice were orthotopically implanted with bioluminescent Her2+ MDA-MB-231 or HCC1954 cells and tumors resected c.5 weeks later. Following resection, mice were treated with 10 mg/kg Trast +5 mg/kg paclitaxel (PAC) IP once weekly for 6 cycles +/- weekly BVZ (5 mg/kg IP). Metastasis was monitored by imaging. Using these models our data confirms that the addition of the anti-angiogenic antibody BVZ to adjuvant Trast + chemotherapy provides no additional benefit compared with Trast + chemotherapy alone. Previous studies using non-resection subcutaneously engrafted xenografts failed to predict this outcome. Our results provide compelling evidence for the utility of cell line xenograft resection models to predict clinical outcome for TME targeting agents.

RGD-decorated cholesterol stabilized polyplexes for targeted siRNA delivery to glioblastoma cells.

The development of an effective and safe treatment for glioblastoma (GBM) represents a significant challenge in oncology today. Downregulation of key mediators of cell signal transduction by RNA interference is considered a promising treatment strategy but requires efficient, intracellular delivery of siRNA into GBM tumor cells. Here, we describe novel polymeric siRNA nanocarriers functionalized with cRGD peptide that mediates targeted and efficient reporter gene silencing in U87R invasive human GBM cells. The polymer was synthesized via RAFT copolymerization of N-(2-hydroxypropyl)-methacrylamide (HPMA) and N-acryloxysuccinimide (NAS), followed by post-polymerization modification with cholesterol for stabilization, cationic amines for siRNA complexation, and azides for copper-free click chemistry. The novel resultant cationic polymer harboring a terminal cholesterol group, self-assembled with siRNA to yield nanosized polyplexes (~ 40 nm) with good colloidal stability at physiological ionic strength. Post-modification of the preformed polyplexes with PEG-cRGD end-functionalized with bicyclo[6.1.0]nonyne (BCN) group resulted in enhanced cell uptake and increased luciferase gene silencing in U87R cells, compared to polyplexes lacking cRGD-targeting groups.

DOUBLINGS BETWEEN BEWILDERMENT AND ENLIGHTENMENT: READING FREUD WITH HEINE ON THE TROUBLED IDENTITY OF HIRSCH-HYACINTH.

The present paper examines Freud's collapse of Heine's poignantly observed multi-cultural narratives in discerning the joke's mechanism of doubling as it progresses from initial bewilderment to momentary enlightenment. In so doing, Freud opens the door to examination of the complex Jewish cultural identity he and Heine share, as represented by the fictional character, "Hirsch-Hyacinth". Hirsch-Hyacinth is a caricature of the "marginal man" in his doubled orientation between and within conflicting aspects of self, a condition reflecting oscillation between idealization, derogation, awareness and dissociation, conditioned by internalization of societal prejudice and traumatization. Freud's tightly focused demonstration of psychoanalytic method upon the Heine joke sample proceeds toward two forms of revelation. The first illustrates the universal applicability of psychoanalytic method. The second signals the individual's ongoing reckoning with the particularities of subjective psychological experience as embedded in identification with large group assumptions of social reality.

Implementing Reverse Phase Protein Array Profiling as a Sensitive Method for the Early Pre-Clinical Detection of Off-Target Toxicities Associated with Sunitinib Malate.

PURPOSE: The tyrosine kinase inhibitor (TKI) sunitinib is a multi-targeted agent approved across multiple cancer indications. Nevertheless, since approval, data has emerged to describe a worrisome side effect profile including hypertension, hand-foot syndrome, fatigue, diarrhea, mucositis, proteinuria, and (rarely) congestive heart failure. It has been hypothesized that the observed multi-parameter toxicity profile is related to "on-target" kinase inhibition in "off-target" tissues. EXPERIMENTAL DESIGN: To interrogate off-target effects in pre-clinical studies, a reverse phase protein array (RPPA) approach is employed. Mice are treated with sunitinib (40 mg kg-1 ) for 4 weeks, following which critical organs are removed. The Zeptosens RPPA platform is employed for protein expression analysis. RESULTS: Differentially expressed proteins associated with damage and/or stress are found in the majority of organs from treated animals. Proteins differentially expressed in the heart are associated with myocardial hypertrophy, ischaemia/reperfusion, and hypoxia. However, hypertrophy is not evidenced on histology. Mild proteinuria is observed; however, no changes in renal glomerular structure are visible via electron microscopy. In skin, proteins associated with cutaneous inflammation, keratinocyte hyper-proliferation, and increased inflammatory response are differentially expressed. CONCLUSIONS AND CLINICAL RELEVANCE: It is posited that pre-clinical implementation of a combined histopathological/RPPA approach provides a sensitive method to mechanistically elucidate the early manifestation of TKI on-target/organ off-target toxicities.

Beyond Anti-thinking: Psychoanalysis as Politics.

This paper discusses the political nature of psychoanalytic audacity in an era of fake news and disinformation as receptive populations accustom themselves to societal and political misrepresentations of anti-thinking. Against the aggressive rise of anti-thinking that cauterizes individual and societal registration of precarity, the ideological foundation of psychoanalytic inquiry is in the freeing of that which emotionally and ideationally, has felt to be impenetrable, making such contents and expressions available for clarification within the consensual understandings between two very different individuals. Psychoanalysis, in its dyadic pairing, its regularity of meetings, and its continuous action of recognizing what is obscure or hidden, is the heir to the Enlightenment motto, "aude sapere" the ongoing act of daring to question (Kant, 1784). Operating against defensive foreclosure, psychoanalysis conditions the toleration of painful states of mind toward contingent consideration of the causes and effects from which productive future action might be considered. The dyadic engagement of psychoanalytic participants operates as a unitary political organization in witness of the human condition, from within which what was unthinkable becomes nameable, and what is named becomes spoken in clarification of anti-thinking's foreclosures.

Copy number load predicts outcome of metastatic colorectal cancer patients receiving bevacizumab combination therapy.

Increased copy number alterations (CNAs) indicative of chromosomal instability (CIN) have been associated with poor cancer outcome. Here, we study CNAs as potential biomarkers of bevacizumab (BVZ) response in metastatic colorectal cancer (mCRC). We cluster 409 mCRCs in three subclusters characterized by different degrees of CIN. Tumors belonging to intermediate-to-high instability clusters have improved outcome following chemotherapy plus BVZ versus chemotherapy alone. In contrast, low instability tumors, which amongst others consist of POLE-mutated and microsatellite-instable tumors, derive no further benefit from BVZ. This is confirmed in 81 mCRC tumors from the phase 2 MoMa study involving BVZ. CNA clusters overlap with CRC consensus molecular subtypes (CMS); CMS2/4 xenografts correspond to intermediate-to-high instability clusters and respond to FOLFOX chemotherapy plus mouse avastin (B20), while CMS1/3 xenografts match with low instability clusters and fail to respond. Overall, we identify copy number load as a novel potential predictive biomarker of BVZ combination therapy.

READING WILLY WONKA IN THE ERA OF ANTI-THINKING.

Whether encountered as a movie or novel, Roald Dahl's Charlie and the Chocolate Factory is a childhood staple of postwar Anglophone culture. Originally published in 1964, Dahl's story of "Willie Wonka" is a morality tale for our times addressed by the present essay in relation to the precariousness, violence, intergenerational faith, and materialist fantasies reflective of contemporary life in the early twenty-first century. Compensating for the precarity of contemporary life's impoverishment as assumptions of societal stability are overthrown, this chronicle of the Bucket family details: envious desire validated by large group chosen trauma; authoritarian enslavement of inferior, colonized peoples with murderous, industrial-level human experimentation; toward gratification of the greedy fantasy of unlimited sweetness under the sway of lethal identification with the aggressor.