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CDKN2A is a key tumour suppressor gene and loss of CDKN2A can be found in many tumours. In astrocytoma grade IV, CDKN2A is deleted in more than 50% of tumours. In many instances, low-grade gliomas with homozygous loss of CDKN2A behave like high grade tumours. The available techniques for CDKN2A loss are laborious, expensive, unreliable, or unavailable in most pathology institutes. Therefore, although it is essential for accurate brain tumour diagnosis, the routine diagnosis does not include testing for CDKN2A deletion.We developed a digital polymerase chain reaction (dPCR) assay for CDKN2A loss detection. The assay is based on counting the copy number of CDKN2A gene and of a reference gene on the same chromosome. It was tested for the detection limit with regard to tumour content and minimal DNA quantity. It was then tested on 24 clinical samples with known CDKN2A status. Additionally, we tested 44 gliomas with unknown CDKN2A status.We found that the newly developed assay is reliable in tissue with more than 50% tumour content and more than 0.4 ng of DNA. The validation cohort showed complete concordance, and we were able to detect homozygous loss in 16 gliomas with unknown CDKN2A status.The method presented can give a fast, cost-effective, clinically reliable evaluation of CDKN2A loss in tissue with more than 50% tumour content. Its ability to work with old samples and with low amounts of DNA makes it the favoured assay in cases where other techniques fail.

Supplementary Methods and Data- Changes are unmarked

The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Because such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastases are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein–coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated antitumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing antitumorigenic eosinophil activities. Specifically, TNFα/IFNγ–activated eosinophils facilitated CD4+ and CD8+ T-cell infiltration and promoted antitumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt antitumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics.Significance:These findings demonstrate antitumor activities of eosinophils in the metastatic tumor microenvironment, suggesting that harnessing eosinophil activity may be a viable clinical strategy in patients with cancer.

The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Because such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastases are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein–coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated antitumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing antitumorigenic eosinophil activities. Specifically, TNFα/IFNγ–activated eosinophils facilitated CD4+ and CD8+ T-cell infiltration and promoted antitumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt antitumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics. Significance: These findings demonstrate antitumor activities of eosinophils in the metastatic tumor microenvironment, suggesting that harnessing eosinophil activity may be a viable clinical strategy in patients with cancer.

Purpose Stratification of patients with triple-negative breast cancer (TNBC) for anti-PD-L1 therapy is based on PD-L1 expression in tumor biopsies. This study sought to evaluate the risk of PD-L1 misclassification. Methods We conducted a high-resolution analysis on ten surgical specimens of TNBC. First, we determined PD-L1 expression pattern distribution via manual segmentation and measurement of 6666 microscopic clusters of positive PD-L1 immunohistochemical staining. Then, based on these results, we generated a computer model to calculate the effect of the positive PD-L1 fraction, aggregate size, and distribution of PD-L1 positive cells on the diagnostic accuracy. Results Our computer-based model showed that larger aggregates of PD-L1 positive cells and smaller biopsy size were associated with higher fraction of false results (P P P P P Conclusion Our model can be used to estimate the risk of PD-L1 misclassification in biopsies, with potential implications for treatment decisions.

Liquid biopsy has emerged as a promising non-invasive way to diagnose tumor and monitor its progression. Different types of liquid biopsies have different advantages and limitations. In the present research, we compared the use of two types of liquid biopsy, extracellular vesicle-derived DNA (EV-DNA) and cell-free DNA (cfDNA) for identifying tumor mutations in patients with colon carcinoma. Method: DNA was extracted from the tumor tissue of 33 patients diagnosed with colon carcinoma. Targeted NGS panel, based on the hotspots panel, was used to identify tumor mutations. Pre-surgery serum and plasma were taken from the patients in which mutation was found in the tumor tissue. Extracellular vesicles were isolated from the serum followed by the extraction of EV-DNA. CfDNA was extracted from the plasma. The mutations found in the tumor were used to detect the circulating tumor DNA using ultra-deep sequencing. We compared the sensitivity of mutation detection and allele frequency obtained in EV-DNA and cfDNA. Results: The sensitivity of mutation detection in EV-DNA and cfDNA was 61.90% and 66.67%, respectively. We obtained almost identical sensitivity of mutation detection in EV-DNA and cfDNA in each of the four stages of colon carcinoma. The total DNA concentration and number mutant copies were higher in cfDNA vs. EV-DNA (p value = 0.002 and 0.003, respectively). Conclusion: Both cfDNA and EV-DNA can serve as tumor biomarkers. The use of EV-DNA did not lead to improved sensitivity or better detection of tumor DNA in the circulation.

An injury to peripheral nerves leads to skin denervation, which often is followed by increased pain sensitivity of the denervated areas and the development of neuropathic pain. Changes in innervation patterns during the reinnervation process of the denervated skin could contribute to the development of neuropathic pain. Here, we examined the changes in the innervation pattern during reinnervation and correlated them with the symptoms of neuropathic pain. Using a multispectral labeling technique&mdash, PainBow, which we developed, we characterized dorsal root ganglion (DRG) neurons innervating distinct areas of the rats&rsquo, paw. We then used spared nerve injury, causing partial denervation of the paw, and examined the changes in innervation patterns of the denervated areas during the development of allodynia and hyperalgesia. We found that, differently from normal conditions, during the development of neuropathic pain, these areas were mainly innervated by large, non-nociceptive neurons. Moreover, we found that the development of neuropathic pain is correlated with an overall decrease in the number of DRG neurons innervating these areas. Importantly, treatment with ouabain facilitated reinnervation and alleviated neuropathic pain. Our results suggest that local changes in peripheral innervation following denervation contribute to neuropathic pain development. The reversal of these changes decreases neuropathic pain.

Introduction: The prognostic significance of immunoglobulin heavy-chain variable region gene (IGHV) mutational status in chronic lymphocytic leukemia (CLL) is well established. Previous studies have shown that CLL patients with mutated IGHV (M-IGHV) have a better prognosis, manifested in a longer time-to first treatment (TTFT) and overall survival (OS). Here we present an analysis on the impact of IGHV mutational status in an Israeli cohort of patients with CLL. Methods: A total of 254 patients with CLL (diagnosed from 1991 to 2020), followed at the Tel-Aviv Sourasky Medical Center were included. The IGHV mutational status has been determined by next-generation sequencing (NGS) or cDNA Sanger. The sequences with a germline homology 98% or higher were considered unmutated, and those with a homology less than 98% as mutated CLL. IGHV subsets were analyzed using the ARResT/AssignSubsets website. All data were statistically analyzed by IBM SPSS Statistics 27 (IBM Corporation, Armonk, NY, USA), P-values were two-sided, and the significance level was determined at a Results: Out of 254 patients, 132 (52.0%) had an unmutated IGHV gene (UM-IGHV), and 122 (48.0%) were mutated (M-IGHV). At diagnosis, most patients (Table 1) were ≤65 years of age (n=157, 61.8%), males (n=160, 63.0%) and had an absolute lymphocytic count equal to or less than (≤)15.0 x10 9/L (n=115, 52.5%). Advanced Binet stage was more commonly associated with UM-IGHV (n=44, 57.9%) (P=0.033). Among 240 patients (94.1%), the most frequently used VH gene segments (Figure 1) included; VH1-69 (n=35, 14.6%), VH4-34 (n=24, 10.0%), VH1-2 (n=17, 7.1%), and VH3-23 (n=16, 6.7%). Six major B-cell receptors (BCR) subsets (Figure 2) were identified in 21/180 of patients (11.7%), which most commonly included: CLL#1 (n=6, 28.6%), CLL#4 (n=5, 23.8%), and CLL#2(n=3, 14.3%). Patients with M-IGHV had a longer time to first treatment (TTFT) (P In multivariate analyses of the entire cohort; for TTFT (Table 2A), males (P=0.002, HR=1.9, [1.3-2.9]), >65 years of age at diagnosis (P=0.008, HR=1.8, [1.2-2.7]), advanced Binet stage (P65 years of age at diagnosis (P=0.010, HR=2.6, [1.3-5.5]) and UM-IGHV mutational status (P=0.004, HR=2.9, [1.4-5.9]) were found to be significant factors for shorter OS. In multivariate analyses performed separately for each IGHV mutational status group; males (P=0.021, HR=2.1, [1.1-4.0]), age >65 years (P=0.002, HR=3.2, [1.5-6.4]) and advanced Binet stage (P65 years at diagnosis (P=0.039, HR=4.3, [1.1-17.0]) was the only independent predictor in M-IGHV patients, while no variable maintained its statistical significance in UM-IGHV cases. Conclusion: As previously studied, our cohort demonstrates that M-IGHV CLL patients have better TTFT and OS. However, the rate of BCR subsets in Israel appears to be lower than expected. A separated multivariate analysis for M-IGHV and UM-IGHV patients revealed different independent predictors for TTFT and OS to each of the IGHV mutational status groups. Further studies with larger cohorts are required to deeply study these differences and provide further clinical insight into the pathophysiology of CLL. Figure 1 Figure 1. Disclosures Joffe: AstraZeneca: Consultancy; Epizyme: Consultancy. Herishanu: AbbVie: Honoraria, Research Funding; Janssen: Honoraria; Roche: Honoraria; Medison: Honoraria.

We describe a method to map the location of axonal arbors of many individual neurons simultaneously via the spectral properties of retrogradely transported dye-labeled vesicles. We inject overlapping regions of an axon target area with three or more different colored retrograde tracers. On the basis of the combinations and intensities of the colors in the individual vesicles transported to neuronal somata, we calculate the projection sites of each neuron's axon. This neuronal positioning system (NPS) enables mapping of many axons in a simple automated way. In our experiments, NPS combined with spectral (Brainbow) labeling of the input to autonomic ganglion cells showed that the locations of ganglion cell projections to a mouse salivary gland related to the identities of their preganglionic axonal innervation. NPS could also delineate projections of many axons simultaneously in the mouse central nervous system.

In many parts of the nervous system, signals pass across multiple synaptic relays on their way to a destination, but little is known about how these relays form and the function they serve. To get some insight into this question we ask how the connectivity patterns are organized at two successive synaptic relays in a simple, cholinergic efferent pathway. We found that the organization at successive relays in the parasympathetic nervous system strongly resemble each other despite the different embryological origin and physiological properties of the pre- and postsynaptic cells. Additionally, we found a similar developmental synaptic pruning and elaboration strategy is used at both sites to generate their adult organizations. The striking parallels in adult innervation and developmental mechanisms at the relays argue that a general strategy is in operation. We discuss why from a functional standpoint this structural organization may amplify central signals while at the same time maintaining positional targeting. DOI: http://dx.doi.org/10.7554/eLife.23193.001

Presynaptic sites typically appear as varicosities (boutons) distributed along axons. Ultrastructurally, presynaptic boutons lack obvious physical barriers that separate them from the axon proper, yet activity-related and constitutive dynamics continuously promote the “reshuffling” of presynaptic components and even their dispersal into flanking axonal segments. How presynaptic sites manage to maintain their organization and individual characteristics over long durations is thus unclear. Conceivably, presynaptic tenacity might depend on the active zone (AZ), an electron-dense specialization of the presynaptic membrane, and particularly on the cytoskeletal matrix associated with the AZ (CAZ) that could act as a relatively stable “core scaffold” that conserves and dictates presynaptic organization. At present, however, little is known on the molecular dynamics of CAZ molecules, and thus, the factual basis for this hypothesis remains unclear. To examine the stability of the CAZ, we studied the molecular dynamics of the major CAZ molecule Bassoon in cultured hippocampal neurons. Fluorescence recovery after photobleaching and photoactivation experiments revealed that exchange rates of green fluorescent protein and photoactivatable green fluorescent protein-tagged Bassoon at individual presynaptic sites are very low (τ > 8 h). Exchange rates varied between boutons and were only slightly accelerated by stimulation. Interestingly, photoactivation experiments revealed that Bassoon lost from one synapse was occasionally assimilated into neighboring presynaptic sites. Our findings indicate that Bassoon is engaged in relatively stable associations within the CAZ and thus support the notion that the CAZ or some of its components might constitute a relatively stable presynaptic core scaffold.

The Aspergillus NIMA serine/threonine kinase plays a pivotal role in controlling entrance into mitosis. A major function attributed to NIMA is the induction of chromatin condensation. We show here that the founder murine NIMA-related kinase, Nek1, is larger than previously reported, and that the full-length protein conserves the structural hallmarks of NIMA. Even though Nek1 bears two classical nuclear localization signals (NLS), the endogenous protein localizes to the cytoplasm. Ectopic overexpression of various Nek1 constructs suggests that the C-terminus of Nek1 bears cytoplasmic localization signal(s). Overexpression of nuclear constructs of Nek1 resulted in abnormal chromatin condensation, with the DNA mainly confined to the periphery of the nucleus. Advanced condensation phenotype was associated with nuclear pore complex dispersal. The condensation was not accompanied by up-regulation of mitotic or apoptotic markers. A similar phenotype has been described following NIMA overexpression, strengthening the notion that the mammalian Nek1 kinase has functional similarity to NIMA.

Recent studies suggest that central nervous system synapses can persist for weeks, months, perhaps lifetimes, yet little is known as to how synapses maintain their structural and functional characteristics for so long. As a step toward a better understanding of synaptic maintenance we examined the loss, redistribution, reincorporation, and replenishment dynamics of Synapsin I and ProSAP2/Shank3, prominent presynaptic and postsynaptic matrix molecules, respectively. Fluorescence recovery after photobleaching and photoactivation experiments revealed that both molecules are continuously lost from, redistributed among, and reincorporated into synaptic structures at time-scales of minutes to hours. Exchange rates were not affected by inhibiting protein synthesis or proteasome-mediated protein degradation, were accelerated by stimulation, and greatly exceeded rates of replenishment from somatic sources. These findings indicate that the dynamics of key synaptic matrix molecules may be dominated by local protein exchange and redistribution, whereas protein synthesis and degradation serve to maintain and regulate the sizes of local, shared pools of these proteins.