Techniques to Render Dendritic Spines Visible in the Microscope.

A tiny detail visible on certain neurons at the limit of resolution in light microscopy went in 130 years of neuroscience research through a dazzling career from suspicious staining artifact to what we recognize today as a complex postsynaptic molecular machine: the dendritic spine.This chapter deals with techniques to make spines visible. The original technique, Golgi silver staining, is still being used today. Electron microscopy and automated field ion beam scanning electron microscopy are ultrahigh resolution techniques, albeit specialized. Other methods are intracellular injection, uptake of dyes, and recently the exploitation of genetically modified animals in which certain neurons express fluorescent protein in all their processes, including the nooks and crannies of their dendritic spines.

Current and future diagnostic and treatment strategies for patients with invasive lobular breast cancer.

BACKGROUND: Invasive lobular breast cancer (ILC) is the second most common type of breast cancer after invasive breast cancer of no special type (NST), representing up to 15% of all breast cancers. DESIGN: Latest data on ILC are presented, focusing on diagnosis, molecular make-up according to the European Society for Medical Oncology Scale for Clinical Actionability of molecular Targets (ESCAT) guidelines, treatment in the early and metastatic setting and ILC-focused clinical trials. RESULTS: At the imaging level, magnetic resonance imaging-based and novel positron emission tomography/computed tomography-based techniques can overcome the limitations of currently used imaging techniques for diagnosing ILC. At the pathology level, E-cadherin immunohistochemistry could help improving inter-pathologist agreement. The majority of patients with ILC do not seem to benefit as much from (neo-)adjuvant chemotherapy as patients with NST, although chemotherapy might be required in a subset of high-risk patients. No differences in treatment efficacy are seen for anti-human epidermal growth factor receptor 2 (HER2) therapies in the adjuvant setting and cyclin-dependent kinases 4 and 6 inhibitors in the metastatic setting. The clinical utility of the commercially available prognostic gene expression-based tests is unclear for patients with ILC. Several ESCAT alterations differ in frequency between ILC and NST. Germline BRCA1 and PALB2 alterations are less frequent in patients with ILC, while germline CDH1 (gene coding for E-cadherin) alterations are more frequent in patients with ILC. Somatic HER2 mutations are more frequent in ILC, especially in metastases (15% ILC versus 5% NST). A high tumour mutational burden, relevant for immune checkpoint inhibition, is more frequent in ILC metastases (16%) than in NST metastases (5%). Tumours with somatic inactivating CDH1 mutations may be vulnerable for treatment with ROS1 inhibitors, a concept currently investigated in early and metastatic ILC. CONCLUSION: ILC is a unique malignancy based on its pathological and biological features leading to differences in diagnosis as well as in treatment response, resistance and targets as compared to NST.

Features of durable response and treatment efficacy for capecitabine monotherapy in advanced breast cancer: real-world evidence from a large single-centre cohort.

PURPOSE: In metastatic breast cancer (MBC) population treated with capecitabine monotherapy, we investigated clinical-pathological features as possible biomarkers for the oncological outcome. METHODS: Retrospective study of consecutive MBC patients treated at University Hospitals Leuven starting capecitabine between 1999 and 2017. The primary endpoint was the durable response (DR), defined as non-progressive disease for > 52 weeks. Other main endpoints were objective response rate (ORR), time to progression (TTP) and overall survival (OS). RESULTS: We included 506 patients; mean age at primary breast cancer diagnosis was 51.2 years; 18.2% had de novo MBC; 98.8% were pre-treated with taxanes and/or anthracycline. DR was reached in 11.6%. Patients with DR, as compared to those without DR, were more likely oestrogen receptor (ER) positive (91.5% vs. 76.8%, p = 0.010) at first diagnosis, had a lower incidence of lymph node (LN) involvement (35.6% vs. 49.9%, p = 0.039) before starting capecitabine, were more likely to present with metastases limited to ≤ 2 involved sites (54.2% vs. 38.5%, p = 0.020) and time from metastasis to start of capecitabine was longer (mean 3.5 vs. 2.7 years, p = 0.020). ORR was 22%. Median TTP and OS were 28 and 58 weeks, respectively. In multivariate analysis (only performed for TTP), ER positivity (hazard ratio (HR) = 0.529, p < 0.0001), HER2 negativity (HR = 0.582, p = 0.024), absence of LN (HR = 0.751, p = 0.008) and liver involvement (HR = 0.746, p = 0.013), older age at capecitabine start (HR = 0.925, p < 0.0001) and younger age at diagnosis of MBC (HR = 0.935, p = 0.001) were significant features of longer TTP. CONCLUSION: Our data display relevant clinical-pathological features associated with DR and TTP in patients receiving capecitabine monotherapy for MBC.

Efficacy of anti-HER2 therapy in metastatic breast cancer by discordance of HER2 expression between primary and metastatic breast cancer.

PURPOSE: In stage IV breast cancer, the efficacy of human epidermal growth factor receptor 2 (HER2) targeted therapies in cases with discordance in HER2 expression between primary and metastatic site is not well known. We studied progression free (PFS) and overall survival (OS) by HER2 concordance when treating women with taxane-trastuzumab (± pertuzumab) in first or second line and trastuzumab-emtansine (T-DM1) or capecitabine-lapatinib in later lines. PATIENTS AND METHODS: Retrospective monocentric study including all breast cancer patients receiving trastuzumab between January 2002 and September 2017 at the University Hospital in Leuven; we selected metastatic patients with an available HER2 status in primary and metastatic site. The Kaplan-Meier method was used for estimating PFS/OS and log-rank test for analyzing between group differences. A Cox model is used for testing difference between groups while correcting for Pertuzumab. Multivariable Cox regression is used to model overall survival as a function group, correcting for possible confounders. RESULTS: We included 74 patients; 46 had an unchanged HER2 status (positive/positive), 9 lost HER2 (positive/negative), while 19 acquired HER2 amplification (negative/positive). 25 out of 28 cases with a discordant HER2 status were positive for ER and/or PgR in the primary site. HER2 positive/negative cases had a significantly lower PFS for taxane-trastuzumab-(pertuzumab) (PFS = 5.5 months), compared to HER2 positive/positive (PFS 9 months, p = 0.01) and HER2 negative/positive (PFS 14 months, p = 0.01) patients. PFS for later line T-DM1 (n = 30) was significantly higher for the HER2 positive/positive group (PFS 6.0 months) than for the discordant groups HER2 negative/positive (PFS 1.0 month, p = 0.04) and HER2 positive/negative (PFS 1.5 month, p = 0.01). After correcting for possible confounders, the HER2 positive/negative group had a significantly worse OS compared to HER2 positive/positive (HR 0.19, 95% CI 0.08-0.44) and negative/positive (HR 0.15, 95% 0.06-0.38). CONCLUSION: Conversion of HER2 status was seen in 28 out of 74 cases and was mostly observed in hormone receptor-positive tumors. In contrast to patients with HER2 loss, patients with a positive conversion of HER2 status derived substantial benefit from first line treatment with taxane-trastuzumab-(pertuzumab). This study highlights the importance of re-biopsying the metastatic lesion and changing treatment according to the last HER2 result.

A "Catastrophic" Heparin-Induced Thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a transient, antibody-mediated thrombocytopenia syndrome that usually follows exposure to unfractioned heparin (UFH) or low-molecular-weight heparin (LMWH). In contrast to other pathological conditions which lead to thrombocytopenia and bleeding complications, HIT results in a paradoxical prothrombotic state. It is caused by antibodies directed to complexes containing UFH or LMWH and a self-platelet protein: the platelet factor 4 (PF4). The heparin-PF4 immune complex leads to activation of platelets, monocytes, and endothelial cells which release procoagulant proteins and tissue factor with subsequent blood coagulation activation. Case Report. We describe the case of a woman undergone to knee replacement and affected by urosepsis who developed a HIT after exposure to enoxaparin. The thrombotic burden was very impressive involving the arterial and venous cerebral vessel and the venous pulmonary, hepatic, and inferior legs vascular beds. The patient was successfully treated with fondaparinux without recurrent thrombosis or bleeding. The clinical scenario could be named "catastrophic HIT" like the catastrophic antiphospholipid syndrome since they have a similar pathogenetic mechanism involving both platelets and monocytes procoagulant activities and a similar clinical manifestation with a life-threatening multiple arterial and/or venous thromboses. CONCLUSION: Patients presenting with HIT could show a very impressive thrombotic burden resembling to that of the catastrophic antiphospholipid syndrome. A careful differential diagnosis should be made towards other pathological conditions which lead to thrombocytopenia to avoid an unnecessary and potentially harmful platelet transfusion. Although fondaparinux is off-label, its use in patients with HIT is simple and seems to be effective.

Neuroanatomical tract-tracing techniques that did go viral.

Neuroanatomical tracing methods remain fundamental for elucidating the complexity of brain circuits. During the past decades, the technical arsenal at our disposal has been greatly enriched, with a steady supply of fresh arrivals. This paper provides a landscape view of classical and modern tools for tract-tracing purposes. Focus is placed on methods that have gone viral, i.e., became most widespread used and fully reliable. To keep an historical perspective, we start by reviewing one-dimensional, standalone transport-tracing tools; these including today's two most favorite anterograde neuroanatomical tracers such as Phaseolus vulgaris-leucoagglutinin and biotinylated dextran amine. Next, emphasis is placed on several classical tools widely used for retrograde neuroanatomical tracing purposes, where Fluoro-Gold in our opinion represents the best example. Furthermore, it is worth noting that multi-dimensional paradigms can be designed by combining different tracers or by applying a given tracer together with detecting one or more neurochemical substances, as illustrated here with several examples. Finally, it is without any doubt that we are currently witnessing the unstoppable and spectacular rise of modern molecular-genetic techniques based on the use of modified viruses as delivery vehicles for genetic material, therefore, pushing the tract-tracing field forward into a new era. In summary, here, we aim to provide neuroscientists with the advice and background required when facing a choice on which neuroanatomical tracer-or combination thereof-might be best suited for addressing a given experimental design.

Neurochemical fingerprinting of amygdalostriatal and intra-amygdaloid projections: a tracing-immunofluorescence study in the rat.

Amygdalostriatal and intra-amygdaloid fiber connectivity was studied in rats via injections of one of the tracers Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) into various amygdaloid nuclei. To determine the neurotransmitter identity of labeled fibers we combined tracer detection with immunofluorescence staining, using antibodies against vesicular transporters (VTs) associated with glutamatergic (VGluT1, VGluT2) or GABAergic (VGAT) neurotransmission. High-magnification confocal laser scanning images were screened for overlap: occurrence inside tracer labeled fibers or axon terminals of immunofluorescence signal associated with one of the VTs. Labeled amygdalostriatal fibers were seen when tracer had been injected into the magnocellular and parvicellular portions of the basal amygdaloid nucleus and the lateral amygdaloid nucleus (nuclei belonging to 'cortical type' amygdaloid nuclei). Intra-amygdaloidal projection fibers were mostly found after tracer injections in the central and medial amygdaloid nuclei ('striatal type' amygdaloid nuclei). Terminals of tracer-labeled amygdalostriatal fibers contained immunofluorescence signal associated mostly with VGluT1 and to a lesser degree with VGluT2 or VGAT. Intra-amygdaloid labeled fibers showed colocalization mostly of VGluT1, followed by VGAT. VGluT2 co-occurred in a minority of intra-amygdaloid tracer-containing fiber terminals. We conclude from our observations that both amygdalostriatal and intra-amygdaloid projections, arising from, respectively, 'cortical type' and 'striatal type' amygdaloid nuclei contain strong glutamatergic and modest GABAergic components. The glutamatergic fibers express either VGluT1 or VGluT2. The absence in large numbers of tracer labeled fibers of expression of one of the selected VTs leads us to suspect that amygdalostriatal projection fibers may contain hitherto neglected neurotransmitters in these connections, e.g., aspartate.

Mesencephalic dopamine neurons interfacing the shell of nucleus accumbens and the dorsolateral striatum in the rat.

Parallel corticostriatonigral circuits have been proposed that separately process motor, cognitive, and emotional-motivational information. Functional integration requires that interactions exist between neurons participating in these circuits. This makes it imperative to study the complex anatomical substrate underlying corticostriatonigral circuits. It has previously been proposed that dopaminergic neurons in the ventral mesencephalon may play a role in this circuit interaction. Therefore, we studied in rats convergence of basal ganglia circuits by depositing an anterograde neuroanatomical tracer into the ventral striatum together with a retrograde fluorescent tracer ipsilaterally in the dorsolateral striatum. In the mesencephalon, using confocal microscopy, we looked for possible appositions of anterogradely labeled fibers and retrogradely labeled neurons, "enhancing" the latter via intracellular injection of Lucifer Yellow. Tyrosine hydroxylase (TH) immunofluorescence served to identify dopaminergic neurons. In neurophysiological experiments, we combined orthodromic stimulation in the medial ventral striatum with recording from ventral mesencephalic neurons characterized by antidromic stimulation from the dorsal striatum. We observed terminal fields of anterogradely labeled fibers that overlap populations of retrogradely labeled nigrostriatal cell bodies in the substantia nigra pars compacta and lateral ventral tegmental area (VTA), with numerous close appositions between boutons of anterogradely labeled fibers and nigrostriatal, TH-immunopositive neurons. Neurophysiological stimulation in the medial ventral striatum caused inhibition of dopaminergic nigrostriatal neurons projecting to the ventrolateral striatal territory. Responding nigrostriatal neurons were located in the medial substantia nigra and adjacent VTA. Our results strongly suggest a functional link between ventromedial, emotional-motivational striatum, and the sensorimotor dorsal striatum via dopaminergic nigrostriatal neurons.

Breast cancer subtype and survival by parity and time since last birth.

BACKGROUND: Pregnancy affects breast cancer risk but how it affects the subtype and prognosis remain controversial. We studied the effect of parity and time since last birth on breast cancer subtype and outcome. PATIENTS AND METHODS: We conducted a retrospective multivariate cohort study including all premenopausal women with early breast cancer aged ≤ 50 years (N = 1306) at diagnosis at the University Hospitals Leuven (Jan. 2000-Dec. 2009). Primary study endpoints were the breast cancer subtype, disease-free survival, and distant disease-free survival by parity and time since last birth. Statistical methods used were baseline-category logits models and Cox proportional hazard models. Multivariable models were used to correct for possible confounders. RESULTS: Breast cancer subtypes did not differ between nulliparous (N = 266) and parous women (N = 1040) but subtypes differed significantly in parous women by time since last birth (p < 0.001). Tumors within 5 years of last birth were proportionally more likely triple negative and HER-2 like, even when corrected for age at diagnosis. After a mean follow-up period of 10 years, parous women had a better disease-free survival compared to nulliparous women (HR 0.733; CI 0.560-0.961; p = 0.025, HR 0.738; CI 0.559-0.974; p = 0.032 before and after correction for known prognostic factors, respectively). In parous women, a longer time since last birth was correlated with a longer disease-free survival compared to patients with a recent pregnancy (HR 0.976; CI 0.957-0.996; p = 0.018). However, after correction, this association completely disappeared (HR 1.010; CI 0.982-1.040; p = 0.480). CONCLUSION: We observed a better disease-free survival for parous than nulliparous women. The influence of recent birth on disease-free survival is probably due to tumor and patient characteristics, as recent birth is associated with more aggressive subtypes.

Body mass index, age at breast cancer diagnosis, and breast cancer subtype: a cross-sectional study.

PURPOSE: Evidence suggests that premenopausal obesity decreases and postmenopausal obesity increases breast cancer risk. Because it is not well known whether this is subtype dependent, we studied the association between body mass index (BMI) and age at breast cancer diagnosis, or the probability of being diagnosed with a specific breast cancer phenotype, by menopausal status. METHODS: All patients with non-metastatic operable breast cancer from the University Hospital Leuven diagnosed between January 1, 2000 and December 31, 2013 were included (n = 7020) in this cross-sectional study. Linear models and logistic regression were used for statistical analysis. Allowing correction for age-related BMI-increase, we used the age-adjusted BMI score which equals the difference between a patient's BMI score and the population-average BMI score corresponding to the patient's age category. RESULTS: The quadratic relationship between the age-adjusted BMI and age at breast cancer diagnosis (p = 0.0207) interacted with menopausal status (p < 0.0001); increased age at breast cancer diagnosis was observed with above-average BMI scores in postmenopausal women, and with below-average BMI scores in premenopausal women. BMI was linearly related to the probabilities of Luminal B and HER2-like breast cancer phenotypes, but only in postmenopausal women. The relative changes in probabilities between both these subtypes mirrored each other. CONCLUSION: BMI associates differently before and after menopause with age at breast cancer diagnosis and with the probability that breast cancer belongs to a certain phenotype. The opposite effect of increasing BMI on relative frequencies of Luminal B and HER2-like breast cancers suggests a common origin.

The Osteological Collection of the University of Cagliari: From Early Neolithic to Modern Age.

The Osteological Collection described in this paper is located at the Anthropology Laboratory annexed to the Sardinian Museum of Anthropology and Ethnography of the University of Cagliari. It has been created in 1953 by Carlo Maxia and comprises a large number of skeletal remains. At present the Collection consists of 11,854 human bones and continues to grow. The remains belong to different periods, beginning from the Early Neolithic and continuing to the Modern Age. The aim of this work is to provide information on the composition of this collection after the reorganization carried out in the last years.

Consistency in recognizing microinvasion in breast carcinomas is improved by immunohistochemistry for myoepithelial markers.

Microinvasion is the smallest morphologically identifiable stage of invasion. Its presence and distinction from in situ carcinoma may have therapeutic implications, and clinical staging also requires the recognition of this phenomenon. Microinvasion is established on the basis of several morphological criteria, which may be difficult and not perfectly reproducible among pathologists. The aim of this study was to assess the consistency of diagnosing microinvasion in the breast on traditional haematoxylin and eosin (HE) stained slides and to evaluate whether immunohistochemistry (IHC) for myoepithelial markers could improve this. Digital images were generated from representative areas of 50 cases stained with HE and IHC for myoepithelial markers. Cases were specifically selected from the spectrum of in situ to microinvasive cancers. Twenty-eight dedicated breast pathologists assessed these cases at different magnifications through a web-based platform in two rounds: first HE only and after a washout period by both HE and IHC. Consistency in the recognition of microinvasion significantly improved with the use of IHC. Concordance rates increased from 0.85 to 0.96, kappa from 0.5 to 0.85, the number of cases with 100% agreement rose from 9/50 to 25/50 with IHC and the certainty of diagnosis also increased. The use of IHC markedly improves the consistency of identifying microinvasion. This corroborates previous recommendations to use IHC for myoepithelial markers to clarify cases where uncertainty exists about the presence of microinvasion. Microinvasive carcinoma is a rare entity, and seeking a second opinion may avoid overdiagnosis.

The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014.

BACKGROUND: The morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is gaining momentum as evidence strengthens for the clinical relevance of this immunological biomarker. Accumulating evidence suggests that the extent of lymphocytic infiltration in tumor tissue can be assessed as a major parameter by evaluation of hematoxylin and eosin (H&E)-stained tumor sections. TILs have been shown to provide prognostic and potentially predictive value, particularly in triple-negative and human epidermal growth factor receptor 2-overexpressing BC. DESIGN: A standardized methodology for evaluating TILs is now needed as a prerequisite for integrating this parameter in standard histopathological practice, in a research setting as well as in clinical trials. This article reviews current data on the clinical validity and utility of TILs in BC in an effort to foster better knowledge and insight in this rapidly evolving field, and to develop a standardized methodology for visual assessment on H&E sections, acknowledging the future potential of molecular/multiplexed approaches. CONCLUSIONS: The methodology provided is sufficiently detailed to offer a uniformly applied, pragmatic starting point and improve consistency and reproducibility in the measurement of TILs for future studies.

Topographic mapping between basal forebrain cholinergic neurons and the medial prefrontal cortex in mice.

The basal forebrain cholinergic innervation of the medial prefrontal cortex (mPFC) is crucial for cognitive performance. However, little is known about the organization of connectivity between the basal forebrain and the mPFC in the mouse. Using focal virus injections inducing Cre-dependent enhanced yellow fluorescent protein expression in ChAT-IRES-Cre mice, we tested the hypothesis that there is a topographic mapping between the basal forebrain cholinergic neurons and their axonal projections to the mPFC. We found that ascending cholinergic fibers to the mPFC follow four pathways and that cholinergic neurons take these routes depending on their location in the basal forebrain. In addition, a general mapping pattern was observed in which the position of cholinergic neurons measured along a rostral to caudal extent in the basal forebrain correlated with a ventral to dorsal and a rostral to caudal shift of cholinergic fiber distribution in mPFC. Finally, we found that neurons in the rostral and caudal parts of the basal forebrain differentially innervate the superficial and deep layers of the ventral regions of the mPFC. Thus, a frontocaudal organization of the cholinergic system exists in which distinct mPFC areas and cortical layers are targeted depending on the location of the cholinergic neuron in the basal forebrain.

A fourth generation of neuroanatomical tracing techniques: exploiting the offspring of genetic engineering.

The first three generations of neuroanatomical tract-tracing methods include, respectively, techniques exploiting degeneration, retrograde cellular transport and anterograde cellular transport. This paper reviews the most recent development in third-generation tracing, i.e., neurochemical fingerprinting based on BDA tracing, and continues with an emerging tracing technique called here 'selective fluorescent protein expression' that in our view belongs to an entirely new 'fourth-generation' class. Tracing techniques in this class lean on gene expression technology designed to 'label' projections exclusively originating from neurons expressing a very specific molecular phenotype. Genetically engineered mice that express cre-recombinase in a neurochemically specific neuronal population receive into a brain locus of interest an injection of an adeno-associated virus (AAV) carrying a double-floxed promoter-eYFP DNA sequence. After transfection this sequence is expressed only in neurons metabolizing recombinase protein. These particular neurons promptly start manufacturing the fluorescent protein which then accumulates and labels to full detail all the neuronal processes, including fibers and terminal arborizations. All other neurons remain optically 'dark'. The AAV is not replicated by the neurons, prohibiting intracerebral spread of 'infection'. The essence is that the fiber projections of discrete subpopulations of neurochemically specific neurons can be traced in full detail. One condition is that the transgenic mouse strain is recombinase-perfect. We illustrate selective fluorescent protein expression in parvalbumin-cre (PV-cre) mice and choline acetyltransferase-cre (ChAT-cre) mice. In addition we compare this novel tracing technique with observations in brains of native PV mice and ChAT-GFP mice. We include a note on tracing techniques using viruses.

Inhibitory interneuron classes express complementary AMPA-receptor patterns in macaque primary visual cortex.

Glutamate receptors mediate excitatory neurotransmission. A very prevalent type of glutamate receptor in the neocortex is the AMPA receptor (AMPAR). AMPARs mediate fast synaptic transmission and their functionality depends on the subunit composition. In primary visual cortex (area V1), the density and subunit composition of AMPARs differ among cortical layers and among cell types. The AMPARs expressed by the different types of inhibitory interneurons, which are crucial for network function, have not yet been characterized systematically. We investigated the distribution of AMPAR subunits in macaque V1 for three distinct subpopulations of inhibitory interneurons: parvalbumin-immunoreactive (PV-IR) interneurons, calbindin-immunoreactive (CB-IR) interneurons, and calretinin-immunoreactive (CR-IR) interneurons. We found that PV-IR cells, which have previously been identified as fast spiking, show high expression of the GluA2 and GluA3 subunits. In contrast, CB-IR and CR-IR cells, which tend to be intermediate spiking, show high expression of the GluA1 and GluA4 subunits. Thus, our data demonstrate that the expression of AMPARs divides inhibitory interneurons in macaque V1 into two categories that are compatible with existing classification methods based on calcium-binding proteins and firing behavior. Moreover, our findings suggest new approaches to target the different inhibitory interneuron classes pharmacologically in vivo.