Large B-cell lymphoma with IRF4 rearrangement (LBCL-IRF4) predominantly affects children and young adults (CAYA) and presents as localized disease with excellent prognosis. Although most tumors harbor IRF4 rearrangements (IRF4-R), the existence of cryptic rearrangements has been suggested. Whether IRF4-R LBCL in adults represents the same biological entity remains unclear. To address this question, 35 CAYA patients diagnosed with LBCL-IRF4 and 7 adult (>40-year-old) LBCL with IRF4-R were investigated by an integrative molecular approach. Targeted sequencing structural variant analysis (SV-NGS) confirmed the IRF4-R in 84% of investigated cases. In CAYA, an enrichment (55% vs 29% in adults) of translocations involving IGH, with a breakpoint cluster 3' to IRF4 (EXOC2) was observed, including a cryptic insertion of IGHM-IGHJ5 into IRF4 locus. Novel translocations, IRF4::MIR142 in two CAYA (7%) and IRF4::BATF in one adult, were discovered. Whole-exome sequencing analysis identified unreported mutations in LBCL-IRF4 as YY1 and ZC3H12A (11% each). The high incidence of mutations affecting the B-cell receptor/NF-KB pathways in LBCL-IRF4 was confirmed in cases with diffuse component only. None of the CAYA tumors were classified by LymphGen tool. Adult IRF4-R LBCL showed higher levels of genetic complexity and a distinct mutational profile with mutations on KMT2D and DTX1 (43% each), and MYD88-L265P (29%), and were predicted as EZB or MCD (29% each). These differential genetic features were in line with different clinical presentations (extranodal involvement and advanced stage). In conclusion, IRF4-R architecture and mutational profile differ according to age, supporting that not all cases should be classified as LBCL-IRF4.

The peripheral nervous system (PNS) orchestrates organ function in health and disease. Most cancers, including pancreatic ductal adenocarcinoma (PDAC), are infiltrated by PNS neurons, and this contributes to the complex tumour microenvironment (TME)1,2. However, neuronal cell bodies reside in various PNS ganglia, far from the tumour mass. Thus, cancer-innervating or healthy-organ-innervating neurons are lacking in current tissue-sequencing datasets. To molecularly characterize pancreas- and PDAC-innervating neurons at single-cell resolution, we developed Trace-n-Seq. This method uses retrograde tracing of axons from tissues to their respective ganglia, followed by single-cell isolation and transcriptomic analysis. By characterizing more than 5,000 individual sympathetic and sensory neurons, with about 4,000 innervating PDAC or healthy pancreas, we reveal novel neuronal cell types and molecular networks that are distinct to the pancreas, pancreatitis, PDAC or melanoma metastasis. We integrate single-cell datasets of innervating neurons and the TME to establish a neuron-cancer-microenvironment interactome, delineate cancer-driven neuronal reprogramming and generate a pancreatic-cancer nerve signature. Pharmacological denervation induces a pro-inflammatory TME and increases the effectiveness of immune-checkpoint inhibitors. The taxane nab-paclitaxel causes intratumoral neuropathy, which attenuates PDAC growth and, in combination with sympathetic denervation, results in synergistic tumour regression. Our multi-dimensional data provide insights into the networks and functions of PDAC-innervating neurons, and support the inclusion of denervation in future therapies.

The nucleolus is a membraneless organelle and an excellent stress sensor. Any changes in its architecture or composition lead to nucleolar stress, resulting in cell cycle arrest and interruption of ribosomal activity, critical factors in aging and cancer. In this study, we identified and described the pivotal role of the RNA-binding protein HNRNPK in ribosome and nucleolar dynamics. We developed an in vitro model of endogenous HNRNPK overexpression and an in vivo mouse model of ubiquitous HNRNPK overexpression. These models showed disruptions in translation as the HNRNPK overexpression caused alterations in the nucleolar structure, resulting in p53-dependent nucleolar stress, cell cycle arrest, senescence, and bone marrow failure phenotype, similar to what is observed in patients with ribosomopathies. Together, our findings identify HNRNPK as a master regulator of ribosome biogenesis and nucleolar homeostasis through p53, providing what we believe to be a new perspective on the orchestration of nucleolar integrity, ribosome function and cellular senescence.

BACKGROUNDNon-Wilms Renal Tumors (NWRT) constitute less than 10% of all renal tumors diagnosed in childhood. We studied our experience in the diagnosis and management of these tumors to compare our results with the current literature.STUDY DESIGNThis is a retrospective observational study which includes all patients aged 0-18 years with histopathological diagnosis of NWRT treated in our center during the period 2000-2022.RESULTSWe identified 10 patients with diagnosis of: cystic nephroma(3), congenital mesoblastic nephroma(1), renal cell carcinoma(2), clear cell sarcoma of the kidney (2), renal Ewing's sarcoma(1) and malignant rhabdoid tumor of the kidney(1). Sixty percent (60%) were female. The median age at diagnosis was 3.25 years (IQR 1.5-10). Median age at diagnosis excluding CN was 6 years old (IQR 2.62-10.75). Molecular alterations were detected in 60% of the cases. Hematuria (40%) and palpable abdominal mass (40%) were the most frequent presenting symptoms. In total, 62.5% patients were misdiagnosed during the preoperative period as Wilms' tumor (WT), based on imaging data. A Radical nephroureterectomy was performed in all cases and staging lymphadenectomy in 70%. We recorded a major complication in one patient, who suffered a contralateral lower renal pole infarction due to section of a polar vessel during surgery. This patient had no preoperative vascular study. The recurrence-free survival rate was 90% with a median follow-up of 6.4 years (IQR 2-13.9).DISCUSSIONRadiological imaging has fundamental importance in the diagnosis of renal tumors, especially to identify children who might benefit from initial surgical treatment or the indication of biopsy for preoperative histopathological confirmation before initiating cytotoxic treatment. However, there are no pathognomonic imaging findings that clearly differentiate between WT and other renal tumors, nor among the heterogeneous group of NWRT[3]. In our series, 62.5% of patients were misdiagnosed as WT based on imaging features.We found different molecular alterations in 60% of our patients (Table 1). None of them predispose to the development of bilateral tumors nor correlates with predisposing syndromes of metachronous tumors.Apart from its retrospective design, this study is limited by small number of cases and a long study period.CONCLUSIONSCorrect differential diagnosis of NWRT is necessary for an adequate therapeutic approach. The molecular-genetic profile is an important step in the diagnosis of NWRT that allows for the use of targeted therapies in refractory patients. Detailed anatomical study by vascular mapping minimizes the risk of iatrogenic damage during tumor resection.

CASE:A 16-year-old boy presented with a recurrent distal femur aneurysmal bone cyst accompanied by a combined sagittal knee deformity (20° of femoral antecurvatum and 26.8° of tibial recurvatum) and limb shortening. After preoperative planning, the treatment involved new intralesional curettage, phenolization, and bone allograft filling. Additional procedures included distal extension femoral osteotomy with plate fixation, and proximal tibial osteotomy with, gradually corrected through a hexapod frame. At 2-year follow-up, lower limbs exhibited normoalignment and equal length. CONCLUSION:Complex knee deformities may occur with tumoral lesions around the knee but can be effectively addressed through double osteotomy and application of a hexapod frame.

Abstract Hyperinnervation emerges as a common hallmark in the tumor microenvironment (TME) of most cancers, with clinical studies establishing a direct link between increased innervation and poor prognosis. Despite the acknowledged pivotal role of the TME, previous molecular profiling excluded tumor-infiltrating neurons due to the unique anatomical characteristics of peripheral neurons. While their axons extend into tumor masses, their DNA- and mRNA-containing cell bodies reside in adjacent ganglia, rendering them absent from virtually all large data sets analyzing tumor material. To fill this critical gap of the role of neurons for cancer growth, therapy resistance and metastasis, we developed a multi-layered approach to unravel the molecular basis of the neural influence in a.o. pancreatic (PDAC), colon cancer and melanoma including their metastasis. First, we unveiled a complex neural network within tumors and metastases through tissue clearing and 3D imaging of patient-derived xenografts, GEMMs, and human samples. This approach was complemented by the introduction of 'Trace-n-Seq,' a novel methodology integrating retrograde axonal tracing, FACS analysis, and single-cell RNA sequencing of neurons innervating both healthy organs and tumors. Expanding this technique unveiled the molecular profiles of over 3000 individual sympathetic, parasympathetic, and sensory neurons infiltrating various tumors, metastases, and pancreatitis, which were compared to neurons innervating healthy organs. Thereby we identified novel neuronal subtypes and observed tumor-induced neuronal reprogramming. Our single-cell data, when integrated with scRNA sequencing of PDAC, unveiled comprehensive neuronal interactions. An interactome analysis identified key genes in tumor/stroma-nerve signaling. To address the functional consequence of hyperinnervation of PDAC tumors, we show data demonstrating that surgical or pharmacological denervation of tumors caused a reduction in tumor size, concomitant with diminished stromal and increased immune compartments. Our data also suggest that the anti-cancer activity of paclitaxel -not oxaliplatin- was partially dependent on its ability to directly target CANs. In fact, in a neoadjuvant setting, PDAC patients treated with Gemcitabine/nab-paclitaxel, but not FOLFIRINOX, displayed a reduction in neuronal structures, underscoring the clinical significance of these observations. In summary, our comprehensive approach has elucidated the role of neurons in PDAC, melanoma, pancreatitis, and metastasis, leveraging innovative tools for studying the molecular programs of CANs at the single-cell level. These insights not only unveil novel mechanisms orchestrated by neuronal activity but also pave the way for imminent clinical trials, aiming to combat PDAC by targeting cancer-associated neurons. Citation Format: Vera Thiel, Simon Renders, Jasper Panten, Daniel Azorin, Nicolas Dross, Albrecht Stenzinger, Sebastian Schölch, Frank Winkler, Martin Sprick, Andreas Trumpp. Analysis of the landscape of cancer-associated neurons (CANs) in diverse cancers through retrograde tracing and single cell molecular profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4225.

The aim of this study is to analyse the diagnostic value of bone marrow aspiration (BMA) in a retrospective cohort of patients with suspected immune thrombocytopaenia (ITP). We further measure changes in the percentage of patients who underwent this study and whether testing or not was in accordance with current guidelines at the time of diagnosis. We conducted a chart review of 243 patients with ITP who underwent follow-up in our institution between 1995 and 2022. The patients were divided into historical cohorts based on the practice guidelines of the Spanish Society of Pediatric Hematology and Oncology (SEHOP) and the American Society of Hematology (ASH) in place at the time of follow-up. For each case, time of disease presentation or initial diagnosis was defined as that which occurred in the first 72 h following disease onset. Based on data from the historical cohorts studied, we observed a lower total number of BMAs at diagnosis over time (p < 0.005). A gradual reduction was seen in the number of BMAs with the introduction of guidelines, including a progressively lower number of BMAs performed without indication (p < 0.05). Subsequent to the initial diagnosis, the procedure played a decisive role in only 2 patients (0.58

Systemic juvenile idiopathic arthritis (sJIA) is a chronic childhood inflammatory disease. SJIA accounts for approximately 5–15 per cent of all cases of JIA and has a high morbidity and mortality rate. In this disease, pulmonary complications (PC) other than pleuritis are much less frequent and not easily recognised by clinicians. Pulmonary hypertension, the most severe PC, is associated with uncontrolled disease and use of biologic therapies. We present a case of a school-age female with sJIA who died of acute cardiopulmonary instability secondary to pulmonary venous-occlusive disease demonstrated by necropsy. We describe her clinical evolution. We also undertook a narrative review of the literature about PC in sJIA to discuss the current state of the art regarding this complication. High disease activity and the use of multiple therapies include disease-modifying anti-rheumatic drugs should be a red flag for clinicians when discounting PC and pulmonary hypertension. The combination of chest X-ray, electrocardiogram and echocardiogram appear to be the best tests to achieve an early diagnosis.

BACKGROUND:Cell therapy has been proposed as part of the therapeutic arsenal to assist bone formation and remodeling in the early stages of osteonecrosis of the femoral head. The purpose of this study is to determine the effects of intraosseous inoculation of mesenchymal stem cells on bone formation and remodeling in an established experimental model of osteonecrosis of the femoral head in immature pigs. METHODS:Thirty-one 4-week-old immature Yorkshire pigs were used. Experimental osteonecrosis of the femoral head was created in the right hip of all included animals (n  =  31). The month after surgery, hip and pelvis radiographs were taken to confirm osteonecrosis of the femoral head. Four animals were excluded following surgery. Two groups were established: (A) mesenchymal stem cell-treated group (n  =  13) and (B) saline-treated group (n  =  14). One month after surgery the mesenchymal stem cell-group received an intraosseous injection of 10  ×  106 mesenchymal stem cell (5 cc) and the saline-treated group of 5 cc of physiological saline solution. Osteonecrosis of the femoral head progression was assessed by monthly X-rays (1-, 2-, 3- and 4-months post-surgery). The animals were sacrificed 1 or 3 months following the intraosseous injection. Repair tissue and osteonecrosis of the femoral head were histologically evaluated immediately after sacrifice. RESULTS:At time of sacrifice, radiographic images showed evident osteonecrosis of the femoral head with associated severe femoral head deformity in 11 of the 14 animals (78%) in the saline group and in only 2 of the 13 animals (15%) in the mesenchymal stem cell group. Histologically, the mesenchymal stem cell group showed less osteonecrosis of the femoral head and less flattening. In the saline group, there was pronounced femoral head flattening and the damaged epiphyseal trabecular bone was largely replaced with fibrovascular tissue. CONCLUSION:Intraosseous mesenchymal stem cells inoculation improved bone healing and remodeling in our immature pig osteonecrosis of the femoral head model. This work supports further investigation to determine whether mesenchymal stem cells enhance the healing process in immature osteonecrosis of the femoral head.

Background: Neoplasms from the ventricular system share a common location but have highly variable histogenesis. Many are slowly growing tumors that behave in a benign fashion. They can be classified as primary and secondary tumors. The most common primary tumors are ependymomas, subependymomas, subependymal giant cell astrocytomas, central neurocytomas, choroid plexus tumors, meningiomas, germinomas, pineal parenchymal tumors, papillary tumors of the pineal region, chordoid gliomas, rosette-forming glioneuronal tumors of the fourth ventricle, and craniopharyngiomas. Pilocytic astrocytomas, medulloblastomas, and atypical teratoid/rhabdoid tumors often show secondary involvement of the ventricular system. Summary: Advances in neurosurgery have facilitated access to the ventricular system increasing the number of cases in which such tumors can be biopsied. In this context, cytology has been proven to be an extremely useful diagnostic tool during intraoperative pathologic consultations. Many ventricular tumors are infrequent, and the cytologic information available is limited. In this review, we describe the cytologic features of the uncommon ventricular tumors and report on unusual findings of the more common ones. For the cytologic evaluation of brain tumors, many neuropathologists prefer formalin fixation and hematoxylin and eosin staining. In this review, we highlight the cytologic findings as seen with Diff-Quik, a very popular staining method among cytopathologists. In fact, when pathologists are unfamiliar with cytology, it is common to request the assistance of cytopathologists during the evaluation of intraoperative procedures. Key Message: Ventricular tumors of the central nervous system comprise a group of heterogeneous tumors with very different cytologic features. The cytomorphology of these tumors, including rare entities, is often very characteristic, allowing a precise recognition during intraoperative pathologic consultations. Diff-Quik is a valuable staining method that can be used alone or as a complement to hematoxylin and eosin staining. Diff-Quik allows for clear visualization of the overall architecture, cytoplasmic details, and extracellular material.

SummaryBackground and objectiveClinicopathological features of cutaneous neurofibromas presenting as large irregularly shaped congenital café‐au‐lait macules (CALM) in Neurofibromatosis type 1 (NF1) patients have not been well characterized. We aimed to analyze the histopathological findings of large “atypical” CALM in children with NF1.Patients and MethodsIn this retrospective observational study we analyzed histopathological and immunostaining features of 21 biopsy specimens from 18 large hyperpigmented macules with irregular borders with or without hypertrichosis present during the first months of life in NF1 diagnosed children.ResultsOf the 21 biopsies, ten showed a diffuse neurofibroma pattern and four exhibited characteristics of plexiform neurofibroma (PNF). In twelve specimens we observed groups of fusiform cells arranged linearly mimicking a small caliber nerve trunk with abnormal morphology. Repeated biopsies from two of these lesions performed at different ages showed transformation to a plexiform pattern. An increased interstitial cellularity was observed in 17 samples that was more evident around eccrine glands in 16 or accompanying hair follicles and vascular structures in twelve samples. All these cells had immunoreactivity for S100‐protein, CD68 and were Melan‐A positive in 15 samples.ConclusionClinicopathological findings of congenital cutaneous neurofibromas provide early diagnostic clues of NF1 with high relevance for monitoring of these patients.

ZusammenfassungHintergrund und ZielsetzungKlinisch‐pathologische Merkmale angeborener kutaner Neurofibrome, die sich bei Patienten mit Neurofibromatose Typ 1 (NF1) als große, unregelmäßig geformte „Café‐au‐lait“‐Flecken (CALM) präsentieren, sind noch nicht gut charakterisiert. Unser Ziel war, große „atypische“ CALM bei Kindern mit NF1 histopathologisch zu analysieren.Patienten und MethodenIn dieser retrospektiven Beobachtungsstudie haben wir histopathologische und immunhistochemische Merkmale von 21 Biopsaten untersucht, die innerhalb der ersten Lebensmonate aus 18 großen hyperpigmentierten Flecken (mit oder ohne Hypertrichose) bei Kindern mit NF1 entnommen wurden.ErgebnisseVon den 21 Biopsaten zeigten zehn ein diffuses Neurofibrom, vier weitere hatten Charakteristika eines plexiformen Neurofibroms (PNF). Bei zwölf Proben fanden sich spindelförmige, linear angeordnete Zellen wie bei einem dünnen Nervenstrang mit abnormer Morphologie. Zwei dieser Läsionen wurden mit zunehmendem Alter erneut biopsiert und zeigten eine Transformation hin zu einem plexiformen Muster. In 17 Proben wurden vermehrt Zellen im interstitiellen Raum festgestellt, bei 16 waren die Zellen um ekkrine Drüsen konzentriert sowie bei zwölf Proben um Haarfollikel und Gefäßstrukturen. Alle diese Zellen exprimierten S100‐Protein und CD68, 15 Proben auch Melan‐A.SchlussfolgerungDie klinisch‐pathologischen Merkmale angeborener kutaner Neurofibrome liefern schon früh diagnostische Hinweise auf NF1 und sind hoch relevant für die Nachverfolgung der Patienten.

Pediatric large B-cell lymphomas (LBCLs) share morphological and phenotypic features with adult types but have better prognosis. The higher frequency of some subtypes such as LBCL with IRF4 rearrangement (LBCL-IRF4) in children suggests that some age-related biological differences may exist. To characterize the genetic and molecular heterogeneity of these tumors, we studied 31 diffuse LBCLs (DLBCLs), not otherwise specified (NOS); 20 LBCL-IRF4 cases; and 12 cases of high-grade B-cell lymphoma (HGBCL), NOS in patients 25 pound years using an integrated approach, including targeted gene sequencing, copynumber arrays, and gene expression profiling. Each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-kB pathway genes (CARD11, CD79B, and MYD88), losses of 17p13 and gains of chromosome 7, 11q12.3-q25, whereas DLBCL, NOS was predominantly of germinal center B-cell (GCB) subtype and carried gene mutations similar to the adult counterpart (eg, SOCS1 and KMT2D), gains of 2p16/REL, and losses of 19p13/CD70. A subset of HGBCL, NOS displayed recurrent alterations of Burkitt lymphoma-related genes such as MYC, ID3, and DDX3X and homozygous deletions of 9p21/CDKN2A, whereas other cases were genetically closer to GCB DLBCL. Factors related to unfavorable outcome were age >18 years; activated B-cell (ABC) DLBCL profile, HGBCL, NOS, high genetic complexity, 1q21-q44 gains, 2p16/REL gains/amplifications, 19p13/CD70 homozygous deletions, and TP53 and MYC mutations. In conclusion, these findings further unravel the molecular heterogeneity of pediatric and young adult LBCL, improve the classification of this group of tumors, and provide new parameters for risk stratification.

Abstract BACKGROUND Gliomas form therapy-resistant multicellular networks, using neurite-like protrusions (called Tumor Microtubes [TMs]), and display intercellular calcium transients, that drive brain tumor malignancy. METHODS A refined in vitro model was established to study calcium communication and its molecular toolkit in patient-derived glioma cells. In vitro results were then validated with longitudinal in vivo two-photon calcium imaging in awake mice. RESULTS Here we first describe the discovery of a small subpopulation of glioma cells that display intrinsically active calcium oscillations and behave like pacemakers. Accordingly, these pacemaker-like glioma cells trigger synchronized calcium activity in tumor cells that are connected with them via TMs. The application of network theory shows that glioma cell networks follow a scale-free and small-world topology, governed by a subpopulation of highly connected hub cells. Interestingly, the pacemaker cells most often act as these hubs, hence communicating with many other network members. Graph theory predicts that such network design displays a high degree of robustness, making it resistant to random damage as seen after radio- and chemotherapy – but extremely vulnerable to selective damage to key hubs. In line with this theory, laser ablation of the pacemaker-like hub cells splintered the malignant tumor network into small isolated cell clusters and led to increased cell death, whereas laser ablation of random tumor cells showed no effect. Among others, KCNN4 channels, which are upregulated in gliomas, were identified as drivers of the pacemaker-like hub cells. Suppressing their pacemaking-abilities by KCNN4 inhibition strongly reduced calcium communication and tumor growth. CONCLUSION In summary, coordinated calcium communication in glioma drives brain tumor malignancy. A specific tumor cell subpopulation was identified that acts as hubs of the multicellular network and initiates global calcium activity. The network’s vulnerability to loosing these pacemaker-like hub cells suggests a novel targeted approach against the resistant networks of gliomas.