Liquid biopsy for improving diagnosis and monitoring of CNS lymphomas: A RANO review.

BACKGROUND: The utility of liquid biopsies is well documented in several extracranial and intracranial (brain/leptomeningeal metastases, gliomas) tumors. METHODS: The RANO (Response Assessment in Neuro-Oncology) group has set up a multidisciplinary Task Force to critically review the role of blood and cerebrospinal fluid (CSF)-liquid biopsy in CNS lymphomas, with a main focus on primary central nervous system lymphomas (PCNSL). RESULTS: Several clinical applications are suggested: diagnosis of PCNSL in critical settings (elderly or frail patients, deep locations, and steroid responsiveness), definition of minimal residual disease, early indication of tumor response or relapse following treatments, and prediction of outcome. CONCLUSIONS: Thus far, no clinically validated circulating biomarkers for managing both primary and secondary CNS lymphomas exist. There is need of standardization of biofluid collection, choice of analytes, and type of technique to perform the molecular analysis. The various assays should be evaluated through well-organized central testing within clinical trials.

Circulating Tumor DNA Profiling for Detection, Risk Stratification, and Classification of Brain Lymphomas.

PURPOSE: Clinical outcomes of patients with CNS lymphomas (CNSLs) are remarkably heterogeneous, yet identification of patients at high risk for treatment failure is challenging. Furthermore, CNSL diagnosis often remains unconfirmed because of contraindications for invasive stereotactic biopsies. Therefore, improved biomarkers are needed to better stratify patients into risk groups, predict treatment response, and noninvasively identify CNSL. PATIENTS AND METHODS: We explored the value of circulating tumor DNA (ctDNA) for early outcome prediction, measurable residual disease monitoring, and surgery-free CNSL identification by applying ultrasensitive targeted next-generation sequencing to a total of 306 tumor, plasma, and CSF specimens from 136 patients with brain cancers, including 92 patients with CNSL. RESULTS: Before therapy, ctDNA was detectable in 78% of plasma and 100% of CSF samples. Patients with positive ctDNA in pretreatment plasma had significantly shorter progression-free survival (PFS, P < .0001, log-rank test) and overall survival (OS, P = .0001, log-rank test). In multivariate analyses including established clinical and radiographic risk factors, pretreatment plasma ctDNA concentrations were independently prognostic of clinical outcomes (PFS HR, 1.4; 95% CI, 1.0 to 1.9; P = .03; OS HR, 1.6; 95% CI, 1.1 to 2.2; P = .006). Moreover, measurable residual disease detection by plasma ctDNA monitoring during treatment identified patients with particularly poor prognosis following curative-intent immunochemotherapy (PFS, P = .0002; OS, P = .004, log-rank test). Finally, we developed a proof-of-principle machine learning approach for biopsy-free CNSL identification from ctDNA, showing sensitivities of 59% (CSF) and 25% (plasma) with high positive predictive value. CONCLUSION: We demonstrate robust and ultrasensitive detection of ctDNA at various disease milestones in CNSL. Our findings highlight the role of ctDNA as a noninvasive biomarker and its potential value for personalized risk stratification and treatment guidance in patients with CNSL.[Media: see text].

Integrated Intraoperative Optical Coherence Tomography during Scleral Pass in Strabismus Surgery.

We describe a novel application of integrated intraoperative OCT (iiOCT) to strabismus surgery during the scleral pass and demonstrate it to be a useful tool. A number of complications can arise from inappropriate scleral pass depth during strabismus surgery, leading to an increased risk of unwanted complications including endophthalmitis, retinal detachment, and a lost or slipped muscle. Our study demonstrated that the use of iiOCT provides easy to interpret, real-time feedback to the strabismus surgeon and may translate to safer, more consistent scleral suturing during strabismus surgery and strabismus surgical training. .

Contribution of dorsal horn CGRP-expressing interneurons to mechanical sensitivity.

Primary sensory neurons are generally considered the only source of dorsal horn calcitonin gene-related peptide (CGRP), a neuropeptide critical to the transmission of pain messages. Using a tamoxifen-inducible CalcaCreER transgenic mouse, here we identified a distinct population of CGRP-expressing excitatory interneurons in lamina III of the spinal cord dorsal horn and trigeminal nucleus caudalis. These interneurons have spine-laden, dorsally directed, dendrites, and ventrally directed axons. As under resting conditions, CGRP interneurons are under tonic inhibitory control, neither innocuous nor noxious stimulation provoked significant Fos expression in these neurons. However, synchronous, electrical non-nociceptive Aß primary afferent stimulation of dorsal roots depolarized the CGRP interneurons, consistent with their receipt of a VGLUT1 innervation. On the other hand, chemogenetic activation of the neurons produced a mechanical hypersensitivity in response to von Frey stimulation, whereas their caspase-mediated ablation led to mechanical hyposensitivity. Finally, after partial peripheral nerve injury, innocuous stimulation (brush) induced significant Fos expression in the CGRP interneurons. These findings suggest that CGRP interneurons become hyperexcitable and contribute either to ascending circuits originating in deep dorsal horn or to the reflex circuits in baseline conditions, but not in the setting of nerve injury.

The ability to sense pain is critical to our survival. Normally, pain is provoked by intense heat or cold temperatures, strong force or a chemical stimulus, for example, capsaicin, the pain-provoking substance in chili peppers. However, if nerve fibers in the arms or legs are damaged, pain can occur in response to touch or pressure stimuli that are normally painless. This hypersensitivity is called mechanical allodynia. A protein called calcitonin gene-related peptide, or CGRP, has been implicated in mechanical allodynia and other chronic pain conditions, such as migraine. CGRP is found in, and released from, the neurons that receive and transmit pain messages from tissues, such as skin and muscles, to the spinal cord. However, only a few distinct groups of CGRP-expressing neurons have been identified and it is unclear if these nerve cells also contribute to mechanical allodynia. To investigate this, Löken et al. genetically engineered mice so that all nerve cells containing CGRP produced red fluorescent light when illuminated with a laser. This included a previously unexplored group of CGRP-expressing neurons found in a part of the spinal cord that is known to receive information about non-painful stimuli. Using neuroanatomical methods, Löken et al. monitored the activity of these neurons in response to various stimuli, before and after a partial nerve injury. This partial injury was induced via a surgery that cut off a few, but not all, branches of a key leg nerve. The experiments showed that in their normal state, the CGRP-expressing neurons hardly responded to mechanical stimulation. In fact, it was difficult to establish what they normally respond to. However, after a nerve injury, brushing the mice's skin evoked significant activity in these cells. Moreover, when these CGRP cells were artificially stimulated, the stimulation induced hypersensitivity to mechanical stimuli, even when the mice had no nerve damage. These results suggest that this group of neurons, which are normally suppressed, can become hyperexcitable and contribute to the development of mechanical allodynia. In summary, Löken et al. have identified a group of nerve cells in the spinal cord that process mechanical information and contribute to touch-evoked pain. Future studies will identify the nerve circuits that are targeted by CGRP released from these nerve cells. These circuits represent a new therapeutic target for managing chronic pain conditions related to nerve damage, specifically mechanical allodynia, which is the most common complaint of patients with chronic pain.

Ablation of spinal cord estrogen receptor α-expressing interneurons reduces chemically induced modalities of pain and itch.

Estrogens are presumed to underlie, at least in part, the greater pain sensitivity and chronic pain prevalence that women experience compared to men. Although previous studies revealed populations of estrogen receptor-expressing neurons in primary afferents and in superficial dorsal horn neurons, there is little to no information as to the contribution of these neurons to the generation of acute and chronic pain. Here we molecularly characterized neurons in the mouse superficial spinal cord dorsal horn that express estrogen receptor α (ERα) and explored the behavioral consequences of their ablation. We found that spinal ERα-positive neurons are largely excitatory interneurons and many coexpress substance P, a marker for a discrete subset of nociceptive, excitatory interneurons. After viral, caspase-mediated ablation of spinal ERα-expressing cells, we observed a significant decrease in the first phase of the formalin test, but in male mice only. ERα-expressing neuron-ablation also reduced pruritogen-induced scratching in both male and female mice. There were no ablation-related changes in mechanical or heat withdrawal thresholds or in capsaicin-induced nocifensive behavior. In chronic pain models, we found no change in Complete Freund's adjuvant-induced thermal or mechanical hypersensitivity, or in partial sciatic nerve injury-induced mechanical allodynia. We conclude that ERα labels a subpopulation of excitatory interneurons that are specifically involved in chemically evoked persistent pain and pruritogen-induced itch.

Intake of ergosterol increases the vitamin D concentrations in serum and liver of mice.

Factors that can modify the bioavailability of orally administered vitamin D are not yet widely known. Ergosterol is a common fungal sterol found in food which has a chemical structure comparable to that of vitamin D. This study aimed to investigate the effect of ergosterol on vitamin D metabolism. Therefore, 36 male wild type-mice were randomly subdivided into three groups (n = 12) and received a diet containing 25 µg vitamin D3 and either 0 mg (control), 2 mg or 7 mg ergosterol per kg diet for 6 weeks. To elucidate the impact of ergosterol on hepatic hydroxylation of vitamin D, human hepatoma cells (HepG2) were treated with different concentrations of ergosterol. Concentrations of vitamin D3 and 25-hydroxyvitamin D3 (25(OH)D3) in cells, livers and kidneys of mice and additionally 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in serum were quantified by LC-MS/MS. The concentration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in serum was analyzed by commercially-available enzyme immuno assay. The concentrations of cholesterol and triglycerides were analyzed in livers of mice by photometric assays. Analyses revealed that mice receiving 7 mg/kg ergosterol with their diet had 1.3-, 1.7- and 1.5-times higher concentrations of vitamin D3 in serum, liver and kidney, respectively, than control mice (P < 0.05), whereas no significant effects were observed in mice fed 2 mg/kg ergosterol. The hydroxylation of vitamin D remained unaffected by dietary ergosterol, since the concentration of 25-hydroxyvitamin D3 in serum and tissues and the concentrations of 1,25(OH)2D3 and 24,25(OH)2D3 in serum were not different between the three groups of mice. The lipid concentrations in liver were also not affected by dietary ergosterol. Data from the cell culture studies showed that ergosterol did not influence the conversion of vitamin D3 to 25(OH)D3. To conclude, ergosterol appears to be a modulator of vitamin D3 concentrations in the body of mice, without modulating the hydroxylation of vitamin D3 in liver.

Primary Afferent-Derived BDNF Contributes Minimally to the Processing of Pain and Itch.

BDNF is a critical contributor to neuronal growth, development, learning, and memory. Although extensively studied in the brain, BDNF is also expressed by primary afferent sensory neurons in the peripheral nervous system. Unfortunately, anatomical and functional studies of primary afferent-derived BDNF have been limited by the availability of appropriate molecular tools. Here, we used targeted, inducible molecular approaches to characterize the expression pattern of primary afferent BDNF and the extent to which it contributes to a variety of pain and itch behaviors. Using a BDNF-LacZ reporter mouse, we found that BDNF is expressed primarily by myelinated primary afferents and has limited overlap with the major peptidergic and non-peptidergic subclasses of nociceptors and pruritoceptors. We also observed extensive neuronal, but not glial, expression in the spinal cord dorsal horn. In addition, because BDNF null mice are not viable and even Cre-mediated deletion of BDNF from sensory neurons could have developmental consequences, here we deleted BDNF selectively from sensory neurons, in the adult, using an advillin-Cre-ER line crossed to floxed BDNF mice. We found that BDNF deletion in the adult altered few itch or acute and chronic pain behaviors, beyond sexually dimorphic phenotypes in the tail immersion, histamine, and formalin tests. Based on the anatomical distribution of sensory neuron-derived BDNF and its limited contribution to pain and itch processing, we suggest that future studies of primary afferent-derived BDNF should examine behaviors evoked by activation of myelinated primary afferents.

Impact of chocolate liquor on vascular lesions in apoE-knockout mice.

Cocoa polyphenols are thought to reduce the risk of cardiovascular diseases. Thus, cocoa-containing foods may have significant health benefits. Here, we studied the impact of chocolate liquor on vascular lesion development and plaque composition in a mouse model of atherosclerosis. Apolipoprotein E (apoE)-knockout mice were assigned to two groups and fed a Western diet that contained 250 g/kg of either chocolate liquor or a polyphenol-free isoenergetic control paste for 16 weeks. In addition to fat, protein, and fibers, the chocolate liquor contained 2 g/kg of polyphenols. Compared with the control group, mice fed the chocolate liquor had larger plaque areas in the descending aorta and aortic root, which were attributed to a higher mass of vascular smooth muscle cells (VSMCs) and collagen. Vascular lipid deposits and calcification areas did not differ between the two groups. The aortic tissue level of interleukin-6 (IL-6) mRNA was 5-fold higher in the mice fed chocolate liquor than in the control mice. Chocolate-fed mice exhibited an increased hepatic saturated to polyunsaturated fatty acid ratio than the controls. Although the chocolate liquor contained 14 µg/kg of vitamin D2, the chocolate liquor-fed mice did not have measurable 25-hydroxyvitamin D2 in the serum. These mice even showed a 25% reduction in the level of 25-hydroxyvitamin D3 compared with the control mice. Overall, present data may contribute to our understanding how chocolate constituents can impact vascular lesion development.

Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

Functional Synaptic Integration of Forebrain GABAergic Precursors into the Adult Spinal Cord.

Spinal cord transplants of embryonic cortical GABAergic progenitor cells derived from the medial ganglionic eminence (MGE) can reverse mechanical hypersensitivity in the mouse models of peripheral nerve injury- and paclitaxel-induced neuropathic pain. Here, we used electrophysiology, immunohistochemistry, and electron microscopy to examine the extent to which MGE cells integrate into host circuitry and recapitulate endogenous inhibitory circuits. Whether the transplants were performed before or after nerve injury, the MGE cells developed into mature neurons and exhibited firing patterns characteristic of subpopulations of cortical and spinal cord inhibitory interneurons. Conversely, the transplanted cells preserved cortical morphological and neurochemical properties. We also observed a robust anatomical and functional synaptic integration of the transplanted cells into host circuitry in both injured and uninjured animals. The MGE cells were activated by primary afferents, including TRPV1-expressing nociceptors, and formed GABAergic, bicuculline-sensitive, synapses onto host neurons. Unexpectedly, MGE cells transplanted before injury prevented the development of mechanical hypersensitivity. Together, our findings provide direct confirmation of an extensive, functional synaptic integration of MGE cells into host spinal cord circuits. This integration underlies normalization of the dorsal horn inhibitory tone after injury and may be responsible for the prophylactic effect of preinjury transplants. SIGNIFICANCE STATEMENT: Spinal cord transplants of embryonic cortical GABAergic interneuron progenitors from the medial ganglionic eminence (MGE), can overcome the mechanical hypersensitivity produced in different neuropathic pain models in adult mice. Here, we examined the properties of transplanted MGE cells and the extent to which they integrate into spinal cord circuitry. Using electrophysiology, immunohistochemistry, and electron microscopy, we demonstrate that MGE cells, whether transplanted before or after nerve injury, develop into inhibitory neurons, are activated by nociceptive primary afferents, and form GABA-A-mediated inhibitory synapses with the host. Unexpectedly, cells transplanted into naive spinal cord prevented the development of nerve-injury-induced mechanical hypersensitivity. These results illustrate the remarkable plasticity of adult spinal cord and the potential of cell-based therapies against neuropathic pain.