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Additional Longitudinal Analysis of Relapsed and Refractory Childhood Solid Tumors.

Table S1. Extended Cohort Annotations. Table S2. Mutational Signatures. Table S3. GISTIC Copy Number Analysis. Table S4. Somatic Variants in Relapsed/Refractory Childhood Solid Tumors (n=184 Patients with T/N WES). Table S5. Clinvar Pathogenic and Likely Pathogenic Germline Variants in Relapsed/Refractory Childhood Solid Tumors(n=184 Patients with T/N WES). Table S6. Gene Lists. Table S7. Log2(TPM+1) Values for the 2,199 Most Variable Genes Expressed Across the Cohort of Relapsed/Refractory Childhood Solid Tumors with RNA-seq (n=245). Table S8. Differential Expression Analysis of 2,199 Most Variable Genes Among Clusters. Table S9. Genes Filtered for COSMIC, Transcription Factors and Potential Drug Targets from Table S8 with log2FC greater than or equal to 2. Table S10. Differential Expression Analysis Among C3a, C3b, and C3c Subclusters. Table S11. Samples Falling in Clusters C3a-C3c and Genes that are Uniformly Highly Expressed in the Clusters. Table S12. Longitudinal Samples.

Consensus Matrix of the GSVA Enrichment Scores Computed for 50 Hallmark Gene Sets. xCell Immune Score Values by GSVA Cluster.

Significantly Differentially Expressed Transcription Factors and Drug Targets in Relapsed and Refractory Childhood Solid Tumors.

Neoantigen Landscape in Relapsed and Refractory Childhood Solid Tumors.

Most Variably Expressed Genes in Relapsed and Refractory Childhood Solid Tumors.

GISTIC Analysis of 184 Relapsed and Refractory Childhood Solid Tumors.

Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment.Significance:Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment. Significance: Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

Stimulation of platelet G protein–coupled receptors results in the cleavage of phosphatidylinositol 4,5-trisphosphate (PIP2) into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol by phospholipase C (PLCβ). It also results in the phosphorylation of PIP2 by the γ isoform of phosphatidylinositol 3-kinase (PI3Kγ) to synthesize phosphatidylinositol 3,4,5-trisphosphate. To understand the role of PIP2 in platelet signaling, we evaluated knock-out mice lacking 2 isoforms of PLCβ (PLCβ2 and PLCβ3) or lacking the Gβγ-activated isoform of PI3K (PI3Kγ). Both knock-out mice were unable to form stable thrombi in a carotid injury model. To provide a functional explanation, knock-out platelets were studied ex vivo. PLCβ2/β3–/– platelets failed to assemble filamentous actin, had defects in both secretion and mobilization of intracellular calcium, and were unable to form stable aggregates following low doses of agonists. Platelets lacking PI3Kγ disaggregated following low-dose adenosine diphosphate (ADP) and had a mildly impaired ability to mobilize intracellular calcium. Yet, they exhibited essentially normal actin assembly and secretion. Remarkably, both PLCβ2/β3–/– and PI3Kγ–/– platelets spread more slowly upon fibrinogen. These results suggest substantial redundancy in platelet signaling pathways. Nonetheless, the diminished ability of knock-out platelets to normally spread after adhesion and to form stable thrombi in vivo suggests that both PLCβ2/β3 and PI3Kγ play vital roles in platelet cytoskeletal dynamics.

Lesions of the basal forebrain deplete the neocortex of cholinergic fibers. Acetylcholine depletion in the somatosensory cortex of rats results in reduced stimulus-evoked activity in response to whisker stimulation. Previous studies demonstrate that embryonic basal forebrain transplants improve functional activity toward normal. It is not clear if the activity increase is due to cholinergic replacement or other factors present in the graft. In this study, we examined the possibility that nerve growth factor (NGF), a neurotrophin known as a survival factor and a specific protectant for cholinergic basal forebrain neurons, can preserve basal forebrain cells after a lesion and restore functional activity in the somatosensory cortex. We report that NGF alone is capable of restoring functional activity in the barrel cortex of animals with basal forebrain lesions, while vehicle injections of saline do not alter activity. Both high (10 microg) and low (5 microg) doses of NGF unilaterally injected into the lateral ventricle improved stimulus-evoked functional activity during bilateral whisker stimulation. The mechanism of NGF action is not clear since the restoration of functional activity in cortex was not accompanied by increased cholinergic activity as detected by acetylcholinesterase fiber staining. NGF may act directly on cortical neurons, although its site of action is not well defined.

Malignant ectomesenchymoma is a rare tumor arising from mature ganglion cells with immature myogenous elements, with only 4 pediatric intracranial cases having been previously reported. The authors report a rare case of intracranial malignant ectomesenchymoma originating from the falx cerebri in a 10-year-old boy. The patient presented with a 2-week history of headache, nausea, and blurry vision, with mild lateral gaze diplopia. A CT scan revealed a solitary 7.2 × 3.8-cm dural-based mass that extended along the falx. No metastatic disease was identified, and the lesion was grossly resected without complication. Pathological investigation identified single and small groups of cells in a myxoid background, with polygonal or spindle-shaped cells containing eccentric nuclei and prominent nucleoli. Immunohistochemical staining of some cells was positive for smooth-muscle actin, CD99, and vimentin, whereas other cells (often process forming) were positive for S100 protein, synaptophysin, and neurofilament protein. Staining was negative for CD138, CD45, α-fetoprotein, CK AE1/3, glial fibrillary acidic protein, CK7, CK20, CD31, CD34, myoD, and desmin. Normal immunopositivity was seen for INI-1. The Ki 67 immunostaining had25% reactivity. The patient was treated with a sarcoma-based chemotherapy regimen and radiation to the craniospinal axis, and was found to be without recurrence or metastatic disease at 20 months.

1. The development of cortical responses to somatic stimulation was studied in kittens 2-5 wk of age using the 2-deoxyglucose (2DG) technique. During the 2DG experiment each kitten received an innocuous intermittent vertical displacement stimulus to the forepaw. 2. The pattern of metabolic activity was substantially different in young animals compared with adults. In the individual autoradiographs of the 2-wk-old kittens stimulus-evoked 2DG uptake in primary somatosensory cortex was localized to a small spot in the upper portion of the cortex, whereas in the adult the label extended vertically through the cortical layers and appeared more column-like. Individual patches of label were substantially smaller and less dense in young animals. Over a period of several weeks the evoked activity evolved to the more extensive adult pattern. The 2DG uptake displayed a mature distribution by approximately 4-5 wk of age. During this period, the cortical architecture also evolved from an immature to a mature arrangement. 3. The evoked activity was reconstructed into two-dimensional maps; the distribution of label > or = 1.5 SD above background was considered to be stimulus related. In the adult, the pattern appeared as a strip or strips of increased metabolic activity that extended in the rostrocaudal direction for approximately 1 mm. In contrast, the activity pattern in animals 2-4 wk old was less discretely organized into "strips" and was more diffusely spread over several mms of somatosensory cortex. The two-dimensional pattern gradually coalesced into a more localized strip by approximately 4-5 wk of age. Although the pattern of label was more widespread in the young animals, the absolute distance of the spread of activity did not vary substantially, regardless of the age of the animal. 4. Other measurements regarding the distribution of activity at different ages indicate that the amount of cortex activated increases in absolute terms, although the percent of cortex activated by the stimulus decreases. The overall intensity of the 2DG uptake as measured on the two-dimensional maps increases with age, as does the variability of the 2DG uptake; a wider range of intensity values is seen in the adult. Plots created from the individual two-dimensional reconstructions allowed a measure of "patch strength" at different ages. These histograms relate the most intense region of uptake in a given map to the spatial distribution of activity spreading in the medial and lateral directions.(ABSTRACT TRUNCATED AT 400 WORDS)

The 2-deoxy-glucose (2DG) technique was used to study changes in stimulus-evoked metabolic activity in the somatosensory cortex of the squirrel monkey Saimiri sciureus after unilateral digit amputation. Two to 52 weeks after digit 2 on the left hand was removed, a somatic stimulus was applied to digit 3 bilaterally. In area 3b corresponding to the deafferented side of the brain, the area of stimulus-evoked metabolic activity was greater than that on the opposite, control side of the brain within the same animal. The extent of the topographic projection map of 2DG label in area 3b on the deafferented side of the brain was 1.92 to 4.75 times greater than that on the control side. There was no difference, however, in the topographical area of stimulus-evoked metabolic activity between the left and right somatosensory cortices in a normal, unoperated animal. These data suggest that the changes in functional organization observed using electrophysiological recordings in somatosensory cortex after peripheral denervation may have a metabolic substrate.

Although the role of acetylcholine in processing stimuli in the cerebral cortex is becoming defined, the impact of cholinergic activity on the character of cortical maps remains unclear. In the somatosensory cortex, topographic maps appear capable of lifelong modifications in response to alterations in the periphery. One factor proposed to influence this adaptational ability is the presence of acetylcholine in the cortex. The studies presented here, using the 2-deoxyglucose technique, demonstrate that the unilateral removal of a digit in cats, followed by stimulation of an adjacent digit, produces a pattern of metabolic activity in the somatosensory cortex that is dramatically expanded when compared with the opposite (normal) hemisphere. In contrast, experiments in which the somatosensory cortex was depleted of acetylcholine and the animal received a similar amputation led not to patterns of expanded metabolic activity, but rather to reductions in the evoked metabolic distribution. These studies implicate acetylcholine in normal map formation and in the maintenance of the capacity of cortical maps to adapt to changes in the periphery.

The platelet-specific chemokine platelet factor 4 (PF4) is released in large amounts at sites of vascular injury. PF4 binds to heparin with high affinity, but its in vivo biologic role has not been defined. We studied the role of PF4 in thrombosis using heterozygote and homozygote PF4 knock-out mice (mPF4+/– and mPF4–/–, respectively) and transgenic mice overexpressing human PF4 (hPF4+). None of these lines had an overt bleeding diathesis, but in a FeCl3 carotid artery thrombosis model, all showed impaired thrombus formation. This defect in thrombus formation in the mPF4–/– animals was corrected by infusing hPF4 over a narrow concentration range. The thrombotic defect in the mPF4+/– and mPF4–/– animals was particularly sensitive to infusions of the negatively charged anticoagulant heparin. However, the same amount of heparin paradoxically normalized thrombus formation in the hPF4+ animals, although these animals were anticoagulated systemically. Upon infusion of the positively charged protein, protamine sulfate, the reverse was observed with mPF4+/– and mPF4–/– animals having improved thrombosis, with the hPF4+ animals having worsened thrombus formation. These studies support an important role for PF4 in thrombosis, and show that neutralization of PF4 is an important component of heparin's anticoagulant effect. The mechanisms underlying these observations of PF4 biology and their clinical implications remain to be determined.

Activated platelets release their granule content in a concentrated fashion at sites of injury. We examined whether ectopically expressed factor VIII in developing megakaryocytes would be stored in α-granules and whether its release from circulating platelets would effectively ameliorate bleeding in a factor VIIInull mice model. Using the proximal glycoprotein 1bα promoter to drive expression of a human factor VIII cDNA construct, transgenic lines were established. One line had detectable human factor VIII that colocalizes with von Willebrand factor in platelets. These animals had platelet factor VIII levels equivalent to 3% to 9% plasma levels, although there was no concurrent plasma human factor VIII detectable. When crossed onto a factor VIIInull background, whole blood clotting time was partially corrected, equivalent to a 3% correction level. In a cuticular bleeding time study, these animals also had only a partial correction, but in an FeCl3 carotid artery, thrombosis assay correction was equivalent to a 50% to 100% level. These studies show that factor VIII can be expressed and stored in platelet α-granules. Our studies also suggest that platelet-released factor VIII is at least as potent as an equivalent plasma level and perhaps even more potent in an arterial thrombosis model. (Blood. 2003;102:4006-4013)

Arterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.

1. The neocortical response to deprivation of somatic sensory input in young animals of different ages was compared with the same manipulation in adults. The response was measured through the use of 2-deoxyglucose (2DG) mapping. Although several features of the cortical response were similar in animals of all ages, the metabolic patterns evoked by somatic stimulation differed substantially from each other at all ages. 2. When adult cats receive a digit amputation and survive from 2 to 8 wk, the pattern of stimulus-evoked metabolic uptake expands dramatically in the somatosensory cortex contralateral to the deprived forepaw. Comparisons between the normal and experimental somatosensory cortices reveal that the distribution of activity on the experimental side was roughly an expanded version of the normal pattern. 3. Unilateral digit amputations of digit 2 were conducted on kittens 2, 4, or 6 wk old. They survived until 3–4 mo and then received a 2DG experiment, during which digit 3 was stimulated bilaterally. Evaluation of the evoked metabolic pattern indicated substantial differences from the activity elicited in adults undergoing identical manipulations. 4. The individual patches of activity that made up the metabolic pattern were similar in intensity in both hemispheres when the digit amputation was conducted at either 2, 4, or 6 wk. After a digit amputation at 2 wk, the patches were significantly narrower in the experimental hemisphere; after a digit amputation at 6 wk, the patches were significantly wider in the hemisphere receiving from the deprived forepaw. 5. Two-dimensional maps of 2DG uptake in areas 3b and 1 of the somatosensory cortex reveal that after a digit amputation at 2, 4, or 6 wk, the distribution of activity in the hemisphere receiving from the digit amputation was more dispersed and widespread than in the normal hemisphere. The dispersed pattern of uptake was not an expanded version of the normal pattern, but scattered over a wider region of somatosensory cortex. This observation is similar to the normal pattern of evoked activity seen in developing animals. 6. The total area of 2DG uptake in the somatosensory cortex contralateral to a digit amputation conducted at 2 or 4 wk was not greater than that in the normal hemisphere, even though it was more widespread. After a digit amputation at 6 wk, however, the area of evoked activity was greater in the experimental hemisphere but not of the magnitude as the same manipulation in an adult.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulus-evoked metabolic activity in the anterior parietal cortex (areas 3a, 3b, 1, and 2) occurs in the form of column-like patches. Similar patches characterize the connections to, within, and from these fields. The relation of the patches elicited metabolically to those formed by retrograde or anterograde transport, however, is not clear. If a type of projection connecting areas 3a, 3b, 1, and 2 transmits sensory information among these cortical fields, the resultant projection pattern may directly contribute to the definition of somatosensory metabolic "columns." To test this possibility, electrophysiological recordings in areas 3b and 1 of Macaca fascicularis monkeys characterized stimuli that elicited the best neuronal response at a specific cortical site. Iontophoretic injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were subsequently made into the identified cortical sites. Two days later, the animals were injected with 2-deoxyglucose (2DG) and received the somatic stimulus previously determined to best activate the neurons isolated at the injected cortical site. After injections of WGA-HRP into physiologically defined loci in area 3b, the patches of transported label within areas 3b and 1 were colocalized with evoked metabolic activity. Injections of WGA-HRP into area 1 produced anterogradely labeled terminals in areas 1 and 2 that also overlapped with patches of evoked metabolic activity, as did patches of retrogradely labeled cells located in area 3b. Patches of anterograde label found in area 3b after area 1 injections, however, were not coincident with the metabolically activated patches. These findings suggest that excitatory information is transmitted from area 3b to area 1 in a way that connects clusters of cells with similar response properties.

The role of acetylcholine (ACh) in cerebral cortical activity has recently been reevaluated. It now seems clear that this neurotransmitter increases the magnitude of cortical responses. Although substantial information has been gathered regarding the role of ACh in sensory information processing, little is known about the participation of ACh in the organization of maps in the cerebral cortex. To address this issue, we used 2 methods to manipulate the supply of ACh in the somatosensory cortex of cats: (1) unilateral neurotoxic lesions of the basal forebrain and (2) unilateral topical applications of the cholinergic antagonist, atropine. For each experimental condition, the animal received an injection of 2-deoxyglucose (2DG) while identical somatic stimuli were delivered to the right and left forepaws. In the somatosensory cortex, the 2DG uptake most often occurred in the form of patches that extended from layer II to IV. When the patches were reconstructed into 2-dimensional maps of activity throughout the somatosensory cortex, they formed strips that ran in the rostrocaudal direction. The reconstructed maps revealed that the 2DG patterns in ACh-depleted and the normal cortex were similar in their overall topographic distribution. Depletion or antagonism of ACh, however, caused the stimulus-evoked metabolic label to be reduced in dimension and density. Measurements of background activity levels were obtained by using (1) cytochrome oxidase histochemistry or (2) metabolic activity values in regions of somatosensory cortex that were not specifically stimulated. This analysis indicated that background values in the ACh-depleted hemispheres were not different from those in the normal hemispheres. The absence of ACh therefore appears to reduce the cortical response to stimulation, while background activity values do not change. These observations indicate that ACh plays a significant role in the processing of sensory information and the organization of somatosensory cortical maps.